A Novel Whole Gene Deletion of BCKDHB by Alu-Mediated Non-allelic Recombination in a Chinese Patient With Maple Syrup Urine Disease.

PubMed

Liu, Gang; Ma, Dingyuan; Hu, Ping; Wang, Wen; Luo, Chunyu; Wang, Yan; Sun, Yun; Zhang, Jingjing; Jiang, Tao; Xu, Zhengfeng

2018-01-01

Maple syrup urine disease (MSUD) is an autosomal recessive inherited metabolic disorder caused by mutations in the BCKDHA, BCKDHB, DBT , and DLD genes. Among the wide range of disease-causing mutations in BCKDHB , only one large deletion has been associated with MSUD. Compound heterozygous mutations in BCKDHB were identified in a Chinese patient with typical MSUD using next-generation sequencing, quantitative PCR, and array comparative genomic hybridization. One allele presented a missense mutation (c.391G > A), while the other allele had a large deletion; both were inherited from the patient's unaffected parents. The deletion breakpoints were characterized using long-range PCR and sequencing. A novel 383,556 bp deletion (chr6: g.80811266_81194921del) was determined, which encompassed the entire BCKDHB gene. The junction site of the deletion was localized within a homologous sequence in two AluYa5 elements. Hence, Alu-mediated non-allelic homologous recombination is speculated as the mutational event underlying the large deletion. In summary, this study reports a recombination mechanism in the BCKDHB gene causing a whole gene deletion in a newborn with MSUD.

[Markerless DNA deletion based on Red recombination and in vivo I-Sec I endonuclease cleavage in Escherichia coli chromosome].

PubMed

Zhu, Meiqin; Yu, Jian; Zhou, Changlin; Fang, Hongqing

2016-01-01

Red-based recombineering has been widely used in Escherichia coli genome modification through electroporating PCR fragments into electrocompetent cells to replace target sequences. Some mutations in the PCR fragments may be brought into the homologous regions near the target. To solve this problem in markeless gene deletion we developed a novel method characterized with two-step recombination and a donor plasmid. First, generated by PCR a linear DNA cassette which comprises a I-Sec I site-containing marker gene and homologous arms was electroporated into cells for marker-substitution deletion of the target sequence. Second, after a donor plasmid carrying the I-Sec I site-containing fusion homologous arm was chemically transformed into the marker-containing cells, the fusion arms and the marker was simultaneously cleaved by I-Sec I endonuclease and the marker-free deletion was stimulated by double-strand break-mediated intermolecular recombination. Eleven nonessential regions in E. coli DH1 genome were sequentially deleted by our method, resulting in a 10.59% reduced genome size. These precise deletions were also verified by PCR sequencing and genome resequencing. Though no change in the growth rate on the minimal medium, we found the genome-reduced strains have some alteration in the acid resistance and for the synthesis of lycopene.

Novel factor VIII variants with a modified furin cleavage site improve the efficacy of gene therapy for hemophilia A.

PubMed

Nguyen, G N; George, L A; Siner, J I; Davidson, R J; Zander, C B; Zheng, X L; Arruda, V R; Camire, R M; Sabatino, D E

2017-01-01

Essentials Factor (F) VIII is an inefficiently expressed protein. Furin deletion FVIII variants were purified and characterized using in vitro and in vivo assays. These minimally modified novel FVIII variants have enhanced function. These variants provide a strategy for increasing FVIII expression in hemophilia A gene therapy. Background The major challenge for developing gene-based therapies for hemophilia A is that human factor VIII (hFVIII) has intrinsic properties that result in inefficient biosynthesis. During intracellular processing, hFVIII is predominantly cleaved at a paired basic amino acid cleaving enzyme (PACE) or furin cleavage site to yield a heterodimer that is the major form of secreted protein. Previous studies with B-domain-deleted (BDD) canine FVIII and hFVIII-R1645H, both differing from hFVIII by a single amino acid at this site, suggested that these proteins are secreted mainly in a single polypeptide chain (SC) form and exhibit enhanced function. Objective We hypothesized that deletion(s) of the furin site modulates FVIII biology and may enhance its function. Methods A series of recombinant hFVIII-furin deletion variants were introduced into hFVIII-BDD [Δ1645, 1645-46(Δ2), 1645-47(Δ3), 1645-48(Δ4), or Δ1648] and characterized. Results In vitro, recombinant purified Δ3 and Δ4 were primarily SC and, interestingly, had 2-fold higher procoagulant activity compared with FVIII-BDD. In vivo, the variants also have improved hemostatic function. After adeno-associated viral (AAV) vector delivery, the expression of these variants is 2-4-fold higher than hFVIII-BDD. Protein challenges of each variant in mice tolerant to hFVIII-BDD showed no anti-FVIII immune response. Conclusions These data suggest that the furin deletion hFVIII variants are superior to hFVIII-BDD without increased immunogenicity. In the setting of gene-based therapeutics, these novel variants provide a unique strategy to increase FVIII expression, thus lowering the vector dose, a critical factor for hemophilia A gene therapy. © 2016 The Authors. Journal of Thrombosis and Haemostasis published by Wiley Periodicals, Inc. on behalf of International Society on Thrombosis and Haemostasis.

Germ-Line Recombination Activity of the Widely Used hGFAP-Cre and Nestin-Cre Transgenes

PubMed Central

Zhang, Jiong; Dublin, Pavel; Griemsmann, Stephanie; Klein, Alexandra; Brehm, Ralph; Bedner, Peter; Fleischmann, Bernd K.; Steinhäuser, Christian; Theis, Martin

2013-01-01

Herein we demonstrate with PCR, immunodetection and reporter gene approaches that the widely used human Glial Fibrillary Acidic Protein (hGFAP)-Cre transgene exhibits spontaneous germ-line recombination activity in leading to deletion in brain, heart and tail tissue with high frequency. The ectopic activity of hGFAP-Cre requires a rigorous control. We likewise observed that a second widely used nestin-Cre transgene shows germ-line deletion. Here we describe procedures to identify mice with germ-line recombination mediated by the hGFAP-Cre and nestin-Cre transgenes. Such control is essential to avoid pleiotropic effects due to germ-line deletion of loxP-flanked target genes and to maintain the CNS-restricted deletion status in transgenic mouse colonies. PMID:24349371

Contributions of recombination and repair proteins to telomere maintenance in telomerase-positive and negative Ustilago maydis.

PubMed

Yu, Eun Young; Hsu, Min; Holloman, William K; Lue, Neal F

2018-01-01

Homologous recombination and repair factors are known to promote both telomere replication and recombination-based telomere extension. Herein, we address the diverse contributions of several recombination/repair proteins to telomere maintenance in Ustilago maydis, a fungus that bears strong resemblance to mammals with respect to telomere regulation and recombination mechanisms. In telomerase-positive U. maydis, deletion of rad51 and blm separately caused shortened but stably maintained telomeres, whereas deletion of both engendered similar telomere loss, suggesting that the repair proteins help to resolve similar problems in telomere replication. In telomerase-negative cells, the loss of Rad51 or Brh2 caused accelerated senescence and failure to generate survivors on semi-solid medium. However, slow growing survivors can be isolated through continuous liquid culturing, and these survivors exhibit type II-like as well as ALT-like telomere features. In contrast, the trt1Δ blmΔ double mutant gives rise to survivors as readily as the trt1Δ single mutant, and like the single mutant survivors, exhibit almost exclusively type I-like telomere features. In addition, we observed direct physical interactions between Blm and two telomere-binding proteins, which may thus recruit or regulate Blm at telomeres. Our findings provide the basis for further analyzing the interplays between telomerase, telomere replication, and telomere recombination. © 2017 John Wiley & Sons Ltd.

Mucopolysaccharidosis type IVA: Common double deletion in the N-Acetylgalactosamine-6-sulfatase gene (GALNS)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hori, Toshinori; Tomatsu, Shunji; Fukuda, Seiji

1995-04-10

Mucopolysaccharidosis IVA (MPS IVA) is an autosomal recessive disorder caused by a deficiency in N-acetylgalactosamine-6-sulfatase (GALNS). We found two separate deletions of nearly 8.0 and 6.0 kb in the GALNS gene, including some exons. There are Alu repetitive elements near the breakpoints of the 8.0-kb deletion, and this deletion resulted from an Alu-Alu recombination. The other 6.0-kb deletion involved illegitimate recombinational events between incomplete short direct repeats of 8 bp at deletion breakpoints. The same rearrangement has been observed in a heteroallelic state in four unrelated patients. This is the first documentation of a common double deletion a gene thatmore » is not a member of a gene cluster. 39 refs., 5 figs.« less

Rational development of an attenuated recombinant cyprinid herpesvirus 3 vaccine using prokaryotic mutagenesis and in vivo bioluminescent imaging.

PubMed

Boutier, Maxime; Ronsmans, Maygane; Ouyang, Ping; Fournier, Guillaume; Reschner, Anca; Rakus, Krzysztof; Wilkie, Gavin S; Farnir, Frédéric; Bayrou, Calixte; Lieffrig, François; Li, Hong; Desmecht, Daniel; Davison, Andrew J; Vanderplasschen, Alain

2015-02-01

Cyprinid herpesvirus 3 (CyHV 3) is causing severe economic losses worldwide in common and koi carp industries, and a safe and efficacious attenuated vaccine compatible with mass vaccination is needed. We produced single deleted recombinants using prokaryotic mutagenesis. When producing a recombinant lacking open reading frame 134 (ORF134), we unexpectedly obtained a clone with additional deletion of ORF56 and ORF57. This triple deleted recombinant replicated efficiently in vitro and expressed an in vivo safety/efficacy profile compatible with use as an attenuated vaccine. To determine the role of the double ORF56-57 deletion in the phenotype and to improve further the quality of the vaccine candidate, a series of deleted recombinants was produced and tested in vivo. These experiments led to the selection of a double deleted recombinant lacking ORF56 and ORF57 as a vaccine candidate. The safety and efficacy of this strain were studied using an in vivo bioluminescent imaging system (IVIS), qPCR, and histopathological examination, which demonstrated that it enters fish via skin infection similar to the wild type strain. However, compared to the parental wild type strain, the vaccine candidate replicated at lower levels and spread less efficiently to secondary sites of infection. Transmission experiments allowing water contamination with or without additional physical contact between fish demonstrated that the vaccine candidate has a reduced ability to spread from vaccinated fish to naïve sentinel cohabitants. Finally, IVIS analyses demonstrated that the vaccine candidate induces a protective mucosal immune response at the portal of entry. Thus, the present study is the first to report the rational development of a recombinant attenuated vaccine against CyHV 3 for mass vaccination of carp. We also demonstrated the relevance of the CyHV 3 carp model for studying alloherpesvirus transmission and mucosal immunity in teleost skin.

Recurrent reciprocal deletions and duplications of 16p13.11: the deletion is a risk factor for MR/MCA while the duplication may be a rare benign variant

PubMed Central

Hannes, F D; Sharp, A J; Mefford, H C; de Ravel, T; Ruivenkamp, C A; Breuning, M H; Fryns, J-P; Devriendt, K; Van Buggenhout, G; Vogels, A; Stewart, H; Hennekam, R C; Cooper, G M; Regan, R; Knight, S J L; Eichler, E E; Vermeesch, J R

2009-01-01

Background: Genomic disorders are often caused by non-allelic homologous recombination between segmental duplications. Chromosome 16 is especially rich in a chromosome-specific low copy repeat, termed LCR16. Methods and Results: A bacterial artificial chromosome (BAC) array comparative genome hybridisation (CGH) screen of 1027 patients with mental retardation and/or multiple congenital anomalies (MR/MCA) was performed. The BAC array CGH screen identified five patients with deletions and five with apparently reciprocal duplications of 16p13 covering 1.65 Mb, including 15 RefSeq genes. In addition, three atypical rearrangements overlapping or flanking this region were found. Fine mapping by high-resolution oligonucleotide arrays suggests that these deletions and duplications result from non-allelic homologous recombination (NAHR) between distinct LCR16 subunits with >99% sequence identity. Deletions and duplications were either de novo or inherited from unaffected parents. To determine whether these imbalances are associated with the MR/MCA phenotype or whether they might be benign variants, a population of 2014 normal controls was screened. The absence of deletions in the control population showed that 16p13.11 deletions are significantly associated with MR/MCA (p = 0.0048). Despite phenotypic variability, common features were identified: three patients with deletions presented with MR, microcephaly and epilepsy (two of these had also short stature), and two other deletion carriers ascertained prenatally presented with cleft lip and midline defects. In contrast to its previous association with autism, the duplication seems to be a common variant in the population (5/1682, 0.29%). Conclusion: These findings indicate that deletions inherited from clinically normal parents are likely to be causal for the patients’ phenotype whereas the role of duplications (de novo or inherited) in the phenotype remains uncertain. This difference in knowledge regarding the clinical relevance of the deletion and the duplication causes a paradigm shift in (cyto)genetic counselling. PMID:18550696

A VNTR element associated with steroid sulfatase gene deletions stimulates recombination in cultured cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gong, Y.; Li, X.M.; Shapiro, L.J.

1994-09-01

Steroid sulfatase deficiency is a common genetic disorder, with a prevalence of approximately one in every 3500 males world wide. About 90% of these patients have complete gene deletions, which appear to result from recombination between members of a low-copy repeat family (CRI-232 is the prototype) that flank the gene. RU1 and RU2 are two VNTR elements found within each of these family members. RU1 consists of 30 bp repeating units and its length shows minimal variation among individuals. The RU2 element consists of repeating sequences which are highly asymmetric, with about 90% purines and no C`s on one strand,more » and range from 0.6 kb to over 23 kb among different individuals. We conducted a study to determine if the RU1 or RU2 elements can promote recombination in an in vivo test system. We inserted these elements adjacent to the neo gene in each of two pSV2neo derivatives, one of which has a deletion in the 5{prime} portion of the neo gene and the other having a deletion in the 3{prime} portion. These plasmids were combined and used to transfect EJ cells. Survival of cells in G418 indicates restoration of a functional neo gene by recombination between two deletion constructs. Thus counting G418 resistant colonies gives a quantitative measure of the enhancement of recombination by the inserted VNTR elements. The results showed no effect on recombination by the inserted RU1 element (compared to the insertion of a nonspecific sequence), while the RU2 element stimulated recombination by 3.5-fold (P<0.01). A separate set of constructs placed RU1 or RU2 within the intron of an exon trapping vector. Following tranfection of cells, recombination events were monitored by a PCR assay that detected the approximation of primer binding sites (as a result of recombination). These studies showed that, as in the first set of experiments, the highly variable RU2 element is capable of stimulating somatic recombination in mammalian cells.« less

Genotype-phenotype analysis of recombinant chromosome 4 syndrome: an array-CGH study and literature review

PubMed Central

2013-01-01

Background Recombinant chromosome 4, a rare constitutional rearrangement arising from pericentric inversion, comprises a duplicated segment of 4p13~p15→4pter and a deleted segment of 4q35→4qter. To date, 10 cases of recombinant chromosome 4 have been reported. Result We describe the second case in which array-CGH was used to characterize recombinant chromosome 4 syndrome. The patient was a one-year old boy with consistent clinical features. Conventional cytogenetics and FISH documented a recombinant chromosome 4, derived from a paternal pericentric inversion, leading to partial trisomy 4p and partial monosomy of 4q. Array-CGH, performed to further characterize the rearranged chromosome 4 and delineate the breakpoints, documented a small (4.36 Mb) 4q35.1 terminal deletion and a large (23.81 Mb) 4p15.1 terminal duplication. Genotype-phenotype analysis of 10 previously reported cases and the present case indicated relatively consistent clinical features and breakpoints. This consistency was more evident in our case and another characterized by array-CGH, where both showed the common breakpoints of p15.1 and q35.1. A genotype-phenotype correlation study between rec(4), dup(4p), and del(4q) syndromes revealed that urogenital and cardiac defects are probably due to the deletion of 4q whereas the other clinical features are likely due to 4p duplication. Conclusion Our findings support that the clinical features of patients with rec(4) are relatively consistent and specific to the regions of duplication or deletion. Recombinant chromosome 4 syndrome thus appears to be a discrete entity that can be suspected on the basis of clinical features or specific deleted and duplicated chromosomal regions. PMID:23639048

Genotype-phenotype analysis of recombinant chromosome 4 syndrome: an array-CGH study and literature review.

PubMed

Hemmat, Morteza; Hemmat, Omid; Anguiano, Arturo; Boyar, Fatih Z; El Naggar, Mohammed; Wang, Jia-Chi; Wang, Borris T; Sahoo, Trilochan; Owen, Renius; Haddadin, Mary

2013-05-02

Recombinant chromosome 4, a rare constitutional rearrangement arising from pericentric inversion, comprises a duplicated segment of 4p13~p15→4pter and a deleted segment of 4q35→4qter. To date, 10 cases of recombinant chromosome 4 have been reported. We describe the second case in which array-CGH was used to characterize recombinant chromosome 4 syndrome. The patient was a one-year old boy with consistent clinical features. Conventional cytogenetics and FISH documented a recombinant chromosome 4, derived from a paternal pericentric inversion, leading to partial trisomy 4p and partial monosomy of 4q. Array-CGH, performed to further characterize the rearranged chromosome 4 and delineate the breakpoints, documented a small (4.36 Mb) 4q35.1 terminal deletion and a large (23.81 Mb) 4p15.1 terminal duplication. Genotype-phenotype analysis of 10 previously reported cases and the present case indicated relatively consistent clinical features and breakpoints. This consistency was more evident in our case and another characterized by array-CGH, where both showed the common breakpoints of p15.1 and q35.1. A genotype-phenotype correlation study between rec(4), dup(4p), and del(4q) syndromes revealed that urogenital and cardiac defects are probably due to the deletion of 4q whereas the other clinical features are likely due to 4p duplication. Our findings support that the clinical features of patients with rec(4) are relatively consistent and specific to the regions of duplication or deletion. Recombinant chromosome 4 syndrome thus appears to be a discrete entity that can be suspected on the basis of clinical features or specific deleted and duplicated chromosomal regions.

Fission yeast Tup1-like repressors repress chromatin remodeling at the fbp1+ promoter and the ade6-M26 recombination hotspot.

PubMed Central

Hirota, Kouji; Hoffman, Charles S; Shibata, Takehiko; Ohta, Kunihiro

2003-01-01

Chromatin remodeling plays crucial roles in the regulation of gene expression and recombination. Transcription of the fission yeast fbp1(+) gene and recombination at the meiotic recombination hotspot ade6-M26 (M26) are both regulated by cAMP responsive element (CRE)-like sequences and the CREB/ATF-type transcription factor Atf1*Pcr1. The Tup11 and Tup12 proteins, the fission yeast counterparts of the Saccharomyces cerevisiae Tup1 corepressor, are involved in glucose repression of the fbp1(+) transcription. We have analyzed roles of the Tup1-like corepressors in chromatin regulation around the fbp1(+) promoter and the M26 hotspot. We found that the chromatin structure around two regulatory elements for fbp1(+) was remodeled under derepressed conditions in concert with the robust activation of fbp1(+) transcription. Strains with tup11delta tup12delta double deletions grown in repressed conditions exhibited the chromatin state associated with wild-type cells grown in derepressed conditions. Interestingly, deletion of rst2(+), encoding a transcription factor controlled by the cAMP-dependent kinase, alleviated the tup11delta tup12delta defects in chromatin regulation but not in transcription repression. The chromatin at the M26 site in mitotic cultures of a tup11delta tup12delta mutant resembled that of wild-type meiotic cells. These observations suggest that these fission yeast Tup1-like corepressors repress chromatin remodeling at CRE-related sequences and that Rst2 antagonizes this function. PMID:14573465

Chromosomal instability in enterohaemorrhagic Escherichia coli O157:H7: impact on adherence, tellurite resistance and colony phenotype

PubMed Central

Bielaszewska, Martina; Middendorf, Barbara; Tarr, Phillip I; Zhang, Wenlan; Prager, Rita; Aldick, Thomas; Dobrindt, Ulrich; Karch, Helge; Mellmann, Alexander

2011-01-01

Tellurite (Tel) resistant enterohaemorrhagic Escherichia coli (EHEC) O157:H7 is a global pathogen. In strain EDL933 Tel resistance (TelR) is encoded by duplicate ter cluster in O islands (OI) 43 and 48, which also harbour iha, encoding the adhesin and siderophore receptor Iha. We identified five EHEC O157:H7 strains that differentiate into large (L) colonies and small (S) colonies with high and low Tel minimal inhibitory concentrations (MICs) respectively. S colonies (Tel-MICs ≤ 4 µg ml−1) sustained large internal deletions within the TelR OIs via homologous recombination between IS elements and lost ter and iha. Moreover, complete excision of the islands occurred by site-specific recombination between flanking direct repeats. Complete excision of OI 43 and OI 48 occurred in 1.81 × 10−3 and 1.97 × 10−4 cells in culture, respectively; internal deletion of OI 48 was more frequent (9.7 × 10−1 cells). Under iron limitation that promotes iha transcription, iha-negative derivatives adhered less well to human intestinal epithelial cells and grew slower than did their iha-positive counterparts. Experiments utilizing iha deletion and complementation mutants identified Iha as the major factor responsible for these phenotypic differences. Spontaneous deletions affecting TelR OIs contribute to EHEC O157 genome plasticity and might impair virulence and/or fitness. PMID:21299654

Molecular biology of Streptococcus pneumoniae: an everlasting challenge.

PubMed

Sicard, M; Gasc, A M; Giammarinaro, P; Lefrançois, J; Pasta, F; Samrakandi, M

2000-01-01

Streptococcus pneumoniae is a model for elucidating: 1) recombination steps of DNA, from its discovery to polarity of integration; 2) long-patch mismatch repair, short-patch repair triggered by A/G and exclusion of deletions; 3) resistance to beta-lactam antibiotics; and 4) factors of virulence. Several of these topics remain a challenge for future investigations.

Mechanism of homologous recombination and implications for aging-related deletions in mitochondrial DNA.

PubMed

Chen, Xin Jie

2013-09-01

Homologous recombination is a universal process, conserved from bacteriophage to human, which is important for the repair of double-strand DNA breaks. Recombination in mitochondrial DNA (mtDNA) was documented more than 4 decades ago, but the underlying molecular mechanism has remained elusive. Recent studies have revealed the presence of a Rad52-type recombination system of bacteriophage origin in mitochondria, which operates by a single-strand annealing mechanism independent of the canonical RecA/Rad51-type recombinases. Increasing evidence supports the notion that, like in bacteriophages, mtDNA inheritance is a coordinated interplay between recombination, repair, and replication. These findings could have profound implications for understanding the mechanism of mtDNA inheritance and the generation of mtDNA deletions in aging cells.

Mechanism of Homologous Recombination and Implications for Aging-Related Deletions in Mitochondrial DNA

PubMed Central

2013-01-01

SUMMARY Homologous recombination is a universal process, conserved from bacteriophage to human, which is important for the repair of double-strand DNA breaks. Recombination in mitochondrial DNA (mtDNA) was documented more than 4 decades ago, but the underlying molecular mechanism has remained elusive. Recent studies have revealed the presence of a Rad52-type recombination system of bacteriophage origin in mitochondria, which operates by a single-strand annealing mechanism independent of the canonical RecA/Rad51-type recombinases. Increasing evidence supports the notion that, like in bacteriophages, mtDNA inheritance is a coordinated interplay between recombination, repair, and replication. These findings could have profound implications for understanding the mechanism of mtDNA inheritance and the generation of mtDNA deletions in aging cells. PMID:24006472

Illegitimate V(D)J recombination-mediated deletions in Notch1 and Bcl11b are not sufficient for extensive clonal expansion and show minimal age or sex bias in frequency or junctional processing

PubMed Central

Champagne, Devin P.; Shockett, Penny E.

2014-01-01

Illegitimate V(D)J recombination at oncogenes and tumor suppressor genes is implicated in formation of several T cell malignancies. Notch1 and Bcl11b, genes involved in developing T cell specification, selection, proliferation, and survival, were previously shown to contain hotspots for deletional illegitimate V(D)J recombination associated with radiation-induced thymic lymphoma. Interestingly, these deletions were also observed in wild-type animals. In this study, we conducted frequency, clonality, and junctional processing analyses of Notch1 and Bcl11b deletions during mouse development and compared results to published analyses of authentic V(D)J rearrangements at the T cell receptor beta (TCRβ) locus and illegitimate V(D)J deletions observed at the human, nonimmune HPRT1 locus not involved in T cell malignancies. We detect deletions in Notch1 and Bcl11b in thymic and splenic T cell populations, consistent with cells bearing deletions in the circulating lymphocyte pool. Deletions in thymus can occur in utero, increase in frequency between fetal and postnatal stages, are detected at all ages examined between fetal and 7 months, exhibit only limited clonality (contrasting with previous results in radiation-sensitive mouse strains), and consistent with previous reports are more frequent in Bcl11b, partially explained by relatively high Recombination Signal Information Content (RIC) scores. Deletion junctions in Bcl11b exhibit greater germline nucleotide loss, while in Notch1 palindromic (P) nucleotides are more abundant, although average P nucleotide length is similar for both genes and consistent with results at the TCRβ locus. Non-templated (N) nucleotide insertions appear to increase between fetal and postnatal stages for Notch1, consistent with normal terminal deoxynucleotidyl transferase (TdT) activity; however, neonatal Bcl11b junctions contain elevated levels of N insertions. Finally, contrasting with results at the HPRT1 locus, we find no obvious age or gender bias in junctional processing, and inverted repeats at recessed coding ends (Pr nucleotides) correspond mostly to single-base additions consistent with normal TdT activity. PMID:24530429

Alu-mediated recombination defect in IGF1R: haploinsufficiency in a patient with short stature.

PubMed

Harmel, Eva-Maria; Binder, Gerhard; Barnikol-Oettler, Anja; Caliebe, Janina; Kiess, Wieland; Losekoot, Monique; Ranke, Michael B; Rappold, Gudrun A; Schlicke, Marina; Stobbe, Heike; Wit, Jan M; Pfäffle, Roland; Klammt, Jürgen

2013-01-01

The insulin-like growth factor (IGF) receptor (IGF1R) is essential for normal development and growth. IGF1R mutations cause IGF-1 resistance resulting in intrauterine and postnatal growth failure. The phenotypic spectrum related to IGF1R mutations remains to be fully understood. Auxological and endocrinological data of a patient identified previously were assessed. The patient's fibroblasts were studied to characterize the IGF1R deletion, mRNA fate, protein expression and signalling capabilities. The boy, who carries a heterozygous IGF1R exon 6 deletion caused by Alu element-mediated recombination and a heterozygous SHOX variant (p.Met240Ile), was born appropriate for gestational age but developed proportionate short stature postnatally. IGF-1 levels were low-normal. None of the stigmata associated with SHOX deficiency or sporadically observed in IGF1R mutation carriers were present. Nonsense-mediated mRNA decay led to a substantial decline of IGF1R dosage and IGF-1-dependent receptor autophosphorylation but not impaired downstream signalling. We present the first detailed report of an intragenic IGF1R deletion identified in a patient who, apart from short stature, deviates from all established markers that qualify a growth-retarded child for IGF1R analysis. Although such children will usually escape routine clinical mutation screenings, they can contribute to the understanding of factors and mechanisms that cooperate with the IGF1R. © 2013 S. Karger AG, Basel.

Identification of a DNA Segment Exhibiting Rearrangement Modifying Effects upon Transgenic δ-deleting Elements

PubMed Central

Janowski, Karen M.; Ledbetter, Stephanie; Mayo, Matthew S.; Hockett, Richard D.

1997-01-01

Control of the rearrangement and expression of the T cell receptor α and δ chains is critical for determining T cell type. The process of δ deletion is a candidate mechanism for maintaining separation of the α and δ loci. Mice harboring a transgenic reporter δ deletion construct show α/β T cell lineage–specific use of the transgenic elements. A 48-basepair segment of DNA, termed HPS1A, when deleted from this reporter construct, loses tight lineage-specific rearrangement control of transgenic elements, with abundant rearrangements of transgenic δ-deleting elements now in γ/δ T cells. Furthermore, HPS1A augments recombination frequency of extrachromosomal substrates in an in vitro recombination assay. DNA binding proteins recognizing HPS1A have been identified and are restricted to early B and T cells, during the time of active rearrangement of endogenous TCR and immunoglobulin loci. These data are consistent with δ deletion playing an important role in maintaining separate TCR α and δ loci. PMID:9207011

Targeting vector construction through recombineering.

PubMed

Malureanu, Liviu A

2011-01-01

Gene targeting in mouse embryonic stem cells is an essential, yet still very expensive and highly time-consuming, tool and method to study gene function at the organismal level or to create mouse models of human diseases. Conventional cloning-based methods have been largely used for generating targeting vectors, but are hampered by a number of limiting factors, including the variety and location of restriction enzymes in the gene locus of interest, the specific PCR amplification of repetitive DNA sequences, and cloning of large DNA fragments. Recombineering is a technique that exploits the highly efficient homologous recombination function encoded by λ phage in Escherichia coli. Bacteriophage-based recombination can recombine homologous sequences as short as 30-50 bases, allowing manipulations such as insertion, deletion, or mutation of virtually any genomic region. The large availability of mouse genomic bacterial artificial chromosome (BAC) libraries covering most of the genome facilitates the retrieval of genomic DNA sequences from the bacterial chromosomes through recombineering. This chapter describes a successfully applied protocol and aims to be a detailed guide through the steps of generation of targeting vectors through recombineering.

Recurrence and variability of germline EPCAM deletions in Lynch syndrome.

PubMed

Kuiper, Roland P; Vissers, Lisenka E L M; Venkatachalam, Ramprasath; Bodmer, Danielle; Hoenselaar, Eveline; Goossens, Monique; Haufe, Aline; Kamping, Eveline; Niessen, Renée C; Hogervorst, Frans B L; Gille, Johan J P; Redeker, Bert; Tops, Carli M J; van Gijn, Marielle E; van den Ouweland, Ans M W; Rahner, Nils; Steinke, Verena; Kahl, Philip; Holinski-Feder, Elke; Morak, Monika; Kloor, Matthias; Stemmler, Susanne; Betz, Beate; Hutter, Pierre; Bunyan, David J; Syngal, Sapna; Culver, Julie O; Graham, Tracy; Chan, Tsun L; Nagtegaal, Iris D; van Krieken, J Han J M; Schackert, Hans K; Hoogerbrugge, Nicoline; van Kessel, Ad Geurts; Ligtenberg, Marjolijn J L

2011-04-01

Recently, we identified 3' end deletions in the EPCAM gene as a novel cause of Lynch syndrome. These truncating EPCAM deletions cause allele-specific epigenetic silencing of the neighboring DNA mismatch repair gene MSH2 in tissues expressing EPCAM. Here we screened a cohort of unexplained Lynch-like families for the presence of EPCAM deletions. We identified 27 novel independent MSH2-deficient families from multiple geographical origins with varying deletions all encompassing the 3' end of EPCAM, but leaving the MSH2 gene intact. Within The Netherlands and Germany, EPCAM deletions appeared to represent at least 2.8% and 1.1% of the confirmed Lynch syndrome families, respectively. MSH2 promoter methylation was observed in epithelial tissues of all deletion carriers tested, thus confirming silencing of MSH2 as the causative defect. In a total of 45 families, 19 different deletions were found, all including the last two exons and the transcription termination signal of EPCAM. All deletions appeared to originate from Alu-repeat mediated recombination events. In 17 cases regions of microhomology around the breakpoints were found, suggesting nonallelic homologous recombination as the most likely mechanism. We conclude that 3' end EPCAM deletions are a recurrent cause of Lynch syndrome, which should be implemented in routine Lynch syndrome diagnostics. © 2011 Wiley-Liss, Inc.

Mechanisms of mutagenesis in human cells exposed to 55 MeV protons

NASA Technical Reports Server (NTRS)

Gauny, S.; Wiese, C.; Kronenberg, A.

2001-01-01

Protons represent the major type of charged particle radiation in spaceflight environments. The purpose of this study was to assess mutations arising in human lymphoid cells exposed to protons. Mutations were quantitated at the thymidine kinase (TK1) locus in cell lines derived from the same donor: TK6 cells (wt TP53) and WTK1 cells (mutant TP53). WTK1 cells were much more susceptible to mutagenesis following proton exposure than TK6 cells. Intragenic deletions were observed among early-arising TK1 mutants in TK6 cells, but not in WTK1 cells where all of the mutants arose by LOH. Deletion was the predominant mode of LOH in TK6 cells, while allelic recombination was the major mode of LOH in WTK1 cells. Deletions were of variable lengths, from <1 cM to 64 cM, while mutations that arose by allelic recombination often extended to the telomere. In summary, proton exposures elicited many types of mutations at an autosomal locus in human cells. Most involved large scale loss of genetic information, either through deletion or by recombination.

Normal growth and development in the absence of hepatic insulin-like growth factor I

PubMed Central

Yakar, Shoshana; Liu, Jun-Li; Stannard, Bethel; Butler, Andrew; Accili, Domenici; Sauer, Brian; LeRoith, Derek

1999-01-01

The somatomedin hypothesis proposed that insulin-like growth factor I (IGF-I) was a hepatically derived circulating mediator of growth hormone and is a crucial factor for postnatal growth and development. To reassess this hypothesis, we have used the Cre/loxP recombination system to delete the igf1 gene exclusively in the liver. igf1 gene deletion in the liver abrogated expression of igf1 mRNA and caused a dramatic reduction in circulating IGF-I levels. However, growth as determined by body weight, body length, and femoral length did not differ from wild-type littermates. Although our model proves that hepatic IGF-I is indeed the major contributor to circulating IGF-I levels in mice it challenges the concept that circulating IGF-I is crucial for normal postnatal growth. Rather, our model provides direct evidence for the importance of the autocrine/paracrine role of IGF-I. PMID:10377413

Identification of target genes to control acetate yield during aerobic fermentation with Saccharomyces cerevisiae.

PubMed

Curiel, José Antonio; Salvadó, Zoel; Tronchoni, Jordi; Morales, Pilar; Rodrigues, Alda Joao; Quirós, Manuel; Gonzalez, Ramón

2016-09-15

Aerobic fermentation of grape must, leading to respiro-fermentative metabolism of sugars, has been proposed as way of reducing alcohol content in wines. Two factors limit the usefulness of Saccharomyces cerevisiae for this application, the Crabtree effect, and excess volatile acidity under aerobic conditions. This work aimed to explore the impact on ethanol acetate production of different S. cerevisiae strains deleted for genes previously related with the Crabtree phenotype. Recombinant strains were constructed on a wine industrial genetic background, FX10. All yeast strains, including FX10, showed respiro-fermentative metabolism in natural grape must under aerobic conditions, as well as a concomitant reduction in ethanol yield. This indicates that the Crabtree effect is not a major constrain for reaching relevant respiration levels in grape must. Indeed, only minor differences in ethanol yield were observed between the original and some of the recombinant strains. In contrast, some yeast strains showed a relevant reduction of acetic acid production. This was identified as a positive feature for the feasibility of alcohol level reduction by respiration. Reduced acetic acid production was confirmed by a thorough analysis of these and some additional deletion strains (involving genes HXK2, PYK1, REG1, PDE2 and PDC1). Some recombinant yeasts showed altered production of glycerol and pyruvate derived metabolites. REG1 and PDC1 deletion strains showed a strong reduction of acetic acid yield in aerobic fermentations. Since REG1 defective strains may be obtained by non-GMO approaches, these gene modifications show good promise to help reducing ethanol content in wines.

Seven gene deletions in seven days: Fast generation of Escherichia coli strains tolerant to acetate and osmotic stress

PubMed Central

Jensen, Sheila I.; Lennen, Rebecca M.; Herrgård, Markus J.; Nielsen, Alex T.

2015-01-01

Generation of multiple genomic alterations is currently a time consuming process. Here, a method was established that enables highly efficient and simultaneous deletion of multiple genes in Escherichia coli. A temperature sensitive plasmid containing arabinose inducible lambda Red recombineering genes and a rhamnose inducible flippase recombinase was constructed to facilitate fast marker-free deletions. To further speed up the procedure, we integrated the arabinose inducible lambda Red recombineering genes and the rhamnose inducible FLP into the genome of E. coli K-12 MG1655. This system enables growth at 37 °C, thereby facilitating removal of integrated antibiotic cassettes and deletion of additional genes in the same day. Phosphorothioated primers were demonstrated to enable simultaneous deletions during one round of electroporation. Utilizing these methods, we constructed strains in which four to seven genes were deleted in E. coli W and E. coli K-12. The growth rate of an E. coli K-12 quintuple deletion strain was significantly improved in the presence of high concentrations of acetate and NaCl. In conclusion, we have generated a method that enables efficient and simultaneous deletion of multiple genes in several E. coli variants. The method enables deletion of up to seven genes in as little as seven days. PMID:26643270

Identification of a basic helix-loop-helix-type transcription regulator gene in Aspergillus oryzae by systematically deleting large chromosomal segments.

PubMed

Jin, Feng Jie; Takahashi, Tadashi; Machida, Masayuki; Koyama, Yasuji

2009-09-01

We previously developed two methods (loop-out and replacement-type recombination) for generating large-scale chromosomal deletions that can be applied to more effective chromosomal engineering in Aspergillus oryzae. In this study, the replacement-type method is used to systematically delete large chromosomal DNA segments to identify essential and nonessential regions in chromosome 7 (2.93 Mb), which is the smallest A. oryzae chromosome and contains a large number of nonsyntenic blocks. We constructed 12 mutants harboring deletions that spanned 16- to 150-kb segments of chromosome 7 and scored phenotypic changes in the resulting mutants. Among the deletion mutants, strains designated Delta5 and Delta7 displayed clear phenotypic changes involving growth and conidiation. In particular, the Delta5 mutant exhibited vigorous growth and conidiation, potentially beneficial characteristics for certain industrial applications. Further deletion analysis allowed identification of the AO090011000215 gene as the gene responsible for the Delta5 mutant phenotype. The AO090011000215 gene was predicted to encode a helix-loop-helix binding protein belonging to the bHLH family of transcription factors. These results illustrate the potential of the approach for identifying novel functional genes.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pfleger, Brian F.; Youngquist, Tyler J.

Recombinant cells and methods for improved yield of fatty alcohols. The recombinant cells harbor a recombinant thioesterase gene, a recombinant acyl-CoA synthetase gene, and a recombinant acyl-CoA reductase gene. In addition, a gene product from one or more of an acyl-CoA dehydrogenase gene, an enoyl-CoA hydratase gene, a 3-hydroxyacyl-CoA dehydrogenase gene, and a 3-ketoacyl-CoA thiolase gene in the recombinant cells is functionally deleted. Culturing the recombinant cells produces fatty alcohols at high yields.

The mouse lymphoma assay detects recombination, deletion, and aneuploidy.

PubMed

Wang, Jianyong; Sawyer, Jeffrey R; Chen, Ling; Chen, Tao; Honma, Masamitsu; Mei, Nan; Moore, Martha M

2009-05-01

The mouse lymphoma assay (MLA) uses the thymidine kinase (Tk) gene of the L5178Y/Tk(+/-)-3.7.2C mouse lymphoma cell line as a reporter gene to evaluate the mutagenicity of chemical and physical agents. The MLA is recommended by both the United States Food and Drug Administration and the United States Environmental Protection Agency as the preferred in vitro mammalian cell mutation assay for genetic toxicology screening because it detects a wide range of genetic alterations, including both point mutations and chromosomal mutations. However, the specific types of chromosomal mutations that can be detected by the MLA need further clarification. For this purpose, three chemicals, including two clastogens and an aneugen (3'-azido-3'-deoxythymidine, mitomycin C, and taxol), were used to induce Tk mutants. Loss of heterozygosity (LOH) analysis was used to select mutants that could be informative as to whether they resulted from deletion, mitotic recombination, or aneuploidy. A combination of additional methods, G-banding analysis, chromosome painting, and a real-time PCR method to detect the copy number (CN) of the Tk gene was then used to provide a detailed analysis. LOH involving at least 25% of chromosome 11, a normal karyotype, and a Tk CN of 2 would indicate that the mutant resulted from recombination, whereas LOH combined with a karyotypically visible deletion of chromosome 11 and a Tk CN of 1 would indicate a deletion. Aneuploidy was confirmed using G-banding combined with chromosome painting analysis for mutants showing LOH at every microsatellite marker on chromosome 11. From this analysis, it is clear that mouse lymphoma Tk mutants can result from recombination, deletion, and aneuploidy.

Overexpression of ppc or deletion of mdh for improving production of γ-aminobutyric acid in recombinant Corynebacterium glutamicum.

PubMed

Shi, Feng; Zhang, Ming; Li, Yongfu

2017-06-01

L-Glutamate decarboxylase (GAD) transforms L-glutamate into γ-aminobutyric acid (GABA). Corynebacterium glutamicum that expresses exogenous GAD gene(s) can synthesize GABA from its own produced L-glutamate. To enhance GABA production in recombinant C. glutamicum strain SH, metabolic engineering strategies were used to improve the supply of the GABA precursor, L-glutamate. Five new strains were constructed here. First, the ppc gene was coexpressed with two GAD genes (gadB1 and gadB2). Then, the mdh gene was deleted in C. glutamicum SH. Next, gadB1-gadB2 and gadB1-gadB2-ppc co-expression plasmids were transformed into C. glutamicum strains SH and Δmdh, resulting in four recombinant GAD strains SE1, SE2, SDE1, and SDE2, respectively. Finally, the mdh gene was overexpressed in mdh-deleted SDE1, generating the mdh-complemented GAD strain SDE3. After fermenting for 72 h, GABA production increased to 26.3 ± 3.4, 24.8 ± 0.7, and 25.5 ± 3.3 g/L in ppc-overexpressed SE2, mdh-deleted SDE1, and mdh-deleted ppc-overexpressed SDE2, respectively, which was higher than that in the control GAD strain SE1 (22.7 ± 0.5 g/L). While in the mdh-complemented SDE3, GABA production decreased to 20.0 ± 0.6 g/L. This study demonstrates that the recombinant strains SE2, SDE1, and SDE2 can be used as candidates for GABA production.

The Contribution of Genetic Recombination to CRISPR Array Evolution

PubMed Central

Kupczok, Anne; Landan, Giddy; Dagan, Tal

2015-01-01

CRISPR (clustered regularly interspaced short palindromic repeats) is a microbial immune system against foreign DNA. Recognition sequences (spacers) encoded within the CRISPR array mediate the immune reaction in a sequence-specific manner. The known mechanisms for the evolution of CRISPR arrays include spacer acquisition from foreign DNA elements at the time of invasion and array erosion through spacer deletion. Here, we consider the contribution of genetic recombination between homologous CRISPR arrays to the evolution of spacer repertoire. Acquisition of spacers from exogenic arrays via recombination may confer the recipient with immunity against unencountered antagonists. For this purpose, we develop a novel method for the detection of recombination in CRISPR arrays by modeling the spacer order in arrays from multiple strains from the same species. Because the evolutionary signal of spacer recombination may be similar to that of pervasive spacer deletions or independent spacer acquisition, our method entails a robustness analysis of the recombination inference by a statistical comparison to resampled and perturbed data sets. We analyze CRISPR data sets from four bacterial species: two Gammaproteobacteria species harboring CRISPR type I and two Streptococcus species harboring CRISPR type II loci. We find that CRISPR array evolution in Escherichia coli and Streptococcus agalactiae can be explained solely by vertical inheritance and differential spacer deletion. In Pseudomonas aeruginosa, we find an excess of single spacers potentially incorporated into the CRISPR locus during independent acquisition events. In Streptococcus thermophilus, evidence for spacer acquisition by recombination is present in 5 out of 70 strains. Genetic recombination has been proposed to accelerate adaptation by combining beneficial mutations that arose in independent lineages. However, for most species under study, we find that CRISPR evolution is shaped mainly by spacer acquisition and loss rather than recombination. Since the evolution of spacer content is characterized by a rapid turnover, it is likely that recombination is not beneficial for improving phage resistance in the strains under study, or that it cannot be detected in the resolution of intraspecies comparisons. PMID:26085541

Aberrant immunoglobulin class switch recombination and switch translocations in activated B cell-like diffuse large B cell lymphoma.

PubMed

Lenz, Georg; Nagel, Inga; Siebert, Reiner; Roschke, Anna V; Sanger, Warren; Wright, George W; Dave, Sandeep S; Tan, Bruce; Zhao, Hong; Rosenwald, Andreas; Muller-Hermelink, Hans Konrad; Gascoyne, Randy D; Campo, Elias; Jaffe, Elaine S; Smeland, Erlend B; Fisher, Richard I; Kuehl, W Michael; Chan, Wing C; Staudt, Louis M

2007-03-19

To elucidate the mechanisms underlying chromosomal translocations in diffuse large B cell lymphoma (DLBCL), we investigated the nature and extent of immunoglobulin class switch recombination (CSR) in these tumors. We used Southern blotting to detect legitimate and illegitimate CSR events in tumor samples of the activated B cell-like (ABC), germinal center B cell-like (GCB), and primary mediastinal B cell lymphoma (PMBL) subgroups of DLBCL. The frequency of legitimate CSR was lower in ABC DLBCL than in GCB DLBCL and PMBL. In contrast, ABC DLBCL had a higher frequency of internal deletions within the switch mu (Smu) region compared with GCB DLBCL and PMBL. ABC DLBCLs also had frequent deletions within Sgamma and other illegitimate switch recombinations. Sequence analysis revealed ongoing Smu deletions within ABC DLBCL tumor clones, which were accompanied by ongoing duplications and activation-induced cytidine deaminase-dependent somatic mutations. Unexpectedly, short fragments derived from multiple chromosomes were interspersed within Smu in one case. These findings suggest that ABC DLBCLs have abnormalities in the regulation of CSR that could predispose to chromosomal translocations. Accordingly, aberrant switch recombination was responsible for translocations in ABC DLBCLs involving BCL6, MYC, and a novel translocation partner, SPIB.

[Orthopoxvirus genes for kelch-like proteins. III. Construction of mousepox (ectromelia) virus variants with targeted gene deletions].

PubMed

Kochneva, G V; Kolosova, I V; Lupan, T A; Sivolobova, G F; Iudin, P V; Grazhdantseva, A A; Riabchikova, E I; Kandrina, N Iu; Shchelkunov, S N

2009-01-01

Mousepox (ectromelia) virus genome contains four genes encoding for kelch-like proteins EVM018, EVM027, EVM150 and EVM167. A complete set of insertion plasmids was constructed to allow the production of recombinant ectromelia viruses with targeted deletions of one to four genes of kelch family both individually (single mutants) and in different combinations (double, triple and quadruple mutants). It was shown that deletion of any of the three genes EVMO18, EVM027 or EVM167 resulted in reduction of 50% lethal dose (LD50) by five and more orders in outbred white mice infected intraperitoneally. Deletion of mousepox kelch-gene EVM150 did not influence the virus virulence. Two or more kelch-genes deletion also resulted in high level of attenuation, which could evidently be due to the lack of three genes EVM167, EVM018 and/or EVM027 identified as virulence factors. The local inflammatory process on the model of intradermal injection of mouse ear pinnae (vasodilatation level, hyperemia, cutaneous edema, arterial thrombosis) was significantly more intensive for wild type virus and virulent mutant deltaEVM150 in comparison with avirulent mutant AEVM167.

Rational development of an attenuated recombinant cyprinid herpesvirus 3 vaccine using prokaryotic mutagenesis and in vivo bioluminescent imaging

USDA-ARS?s Scientific Manuscript database

Cyprinid herpesvirus 3 (CyHV-3) is causing severe economic losses worldwide in the carp industry, and a safe and efficacious attenuated vaccine compatible with mass vaccination is needed. We produced single deleted recombinants using prokaryotic mutagenesis. When producing a recombinant lacking open...

[Construction and Function Verification of a Novel Shuttle Vector Containing a Marker Gene Self-deletion System].

PubMed

Li, Lili; Wang, Zhan; Zhou, Yubai; Zhang, Fang; Shen, Sisi; Li, Zelin; Zeng, Yi

2015-09-01

For rapid and accurate screening of recombinant modified vaccinia virus Ankara (rMVA) that satisfied the quality standards of clinical trials, a novel shuttle vector that can delete the marker gene automatically during virus propagation was construted: pZL-EGFP. To construct the pZL-EGFP, the original shuttle vector pSC11 was modified by replacing the LacZ marker gene with enhanced green fluorescent protein (EGFP) and then inserting homologous sequences of TKL into the flank regions of EGFP. Baby hamster kidney (BHK)-21 cells were cotransfected with pZL-EGFP and MVA, and underwent ten passages and one plaque screening to obtain the EGFP-free rMVA carrying the exogenous gene. Resulting rMVA was tested by polymerase chain reaction and western blotting to verify pZL-EGFP function. A novel shuttle vector pZL-EGFP containing an EGFP marker gene which could be deleted automatically was constructed. This gene deletion had no effect on the activities of rMVA, and the exogenous gene could be expressed stably. These results suggest that rMVA can be packaged efficiently by homologous recombination between pZL-EGFP and MVA in BHK-21 cells, and that the carried EGFP gene can be removed automatically by intramolecular homologous recombination during virus passage. Meanwhile, the gene deletion had no influence on the activities of rMVA and the expression of exogenous target gene. This study lays a solid foundation for the future research.

Characterization of the hyperrecombination phenotype of the pol3-t mutation of Saccharomyces cerevisiae.

PubMed

Galli, Alvaro; Cervelli, Tiziana; Schiestl, Robert H

2003-05-01

The DNA polymerase delta (Pol3p/Cdc2p) allele pol3-t of Saccharomyces cerevisiae has previously been shown to increase the frequency of deletions between short repeats (several base pairs), between homologous DNA sequences separated by long inverted repeats, and between distant short repeats, increasing the frequency of genomic deletions. We found that the pol3-t mutation increased intrachromosomal recombination events between direct DNA repeats up to 36-fold and interchromosomal recombination 14-fold. The hyperrecombination phenotype of pol3-t was partially dependent on the Rad52p function but much more so on Rad1p. However, in the double-mutant rad1 Delta rad52 Delta, the pol3-t mutation still increased spontaneous intrachromosomal recombination frequencies, suggesting that a Rad1p Rad52p-independent single-strand annealing pathway is involved. UV and gamma-rays were less potent inducers of recombination in the pol3-t mutant, indicating that Pol3p is partly involved in DNA-damage-induced recombination. In contrast, while UV- and gamma-ray-induced intrachromosomal recombination was almost completely abolished in the rad52 or the rad1 rad52 mutant, there was still good induction in those mutants in the pol3-t background, indicating channeling of lesions into the above-mentioned Rad1p Rad52p-independent pathway. Finally, a heterozygous pol3-t/POL3 mutant also showed an increased frequency of deletions and MMS sensitivity at the restrictive temperature, indicating that even a heterozygous polymerase delta mutation might increase the frequency of genetic instability.

Generation and characterization of functional mutants in the translation initiation factor IF1 of Escherichia coli.

PubMed

Croitoru, Victor; Bucheli-Witschel, Margarete; Hägg, Peter; Abdulkarim, Farhad; Isaksson, Leif A

2004-02-01

Three protein factors IF1, IF2 and IF3 are involved in the initiation of translation in prokaryotes. No clear function has been assigned to the smallest of these three factors, IF1. Therefore, to investigate the role of this protein in the initiation process in Escherichia coli we have mutated the corresponding gene infA. Because IF1 is essential for cell viability and no mutant selection has so far been described, the infA gene in a plasmid was mutated by site-directed mutagenesis in a strain with a chromosomal infA+ gene, followed by deletion of this infA+ gene. Using this approach, the six arginine residues of IF1 were altered to leucine or aspartate. Another set of plasmid-encoded IF1 mutants with a cold-sensitive phenotype was collected using localized random mutagenesis. All mutants with a mutated infA gene on a plasmid and a deletion of the chromosomal infA copy were viable, except for an R65D alteration. Differences in growth phenotypes of the mutants were observed in both minimal and rich media. Some of the mutated infA genes were successfully recombined into the chromosome thereby replacing the wild-type infA+ allele. Several of these recombinants showed reduced growth rate and a partial cold-sensitive phenotype. This paper presents a collection of IF1 mutants designed for in vivo and in vitro studies on the function of IF1.

Enhanced Maternal Origin of the 22q11.2 Deletion in Velocardiofacial and DiGeorge Syndromes

PubMed Central

Delio, Maria; Guo, Tingwei; McDonald-McGinn, Donna M.; Zackai, Elaine; Herman, Sean; Kaminetzky, Mark; Higgins, Anne Marie; Coleman, Karlene; Chow, Carolyn; Jarlbrzkowski, Maria; Bearden, Carrie E.; Bailey, Alice; Vangkilde, Anders; Olsen, Line; Olesen, Charlotte; Skovby, Flemming; Werge, Thomas M.; Templin, Ludivine; Busa, Tiffany; Philip, Nicole; Swillen, Ann; Vermeesch, Joris R.; Devriendt, Koen; Schneider, Maude; Dahoun, Sophie; Eliez, Stephan; Schoch, Kelly; Hooper, Stephen R.; Shashi, Vandana; Samanich, Joy; Marion, Robert; van Amelsvoort, Therese; Boot, Erik; Klaassen, Petra; Duijff, Sasja N.; Vorstman, Jacob; Yuen, Tracy; Silversides, Candice; Chow, Eva; Bassett, Anne; Frisch, Amos; Weizman, Abraham; Gothelf, Doron; Niarchou, Maria; van den Bree, Marianne; Owen, Michael J.; Suñer, Damian Heine; Andreo, Jordi Rosell; Armando, Marco; Vicari, Stefano; Digilio, Maria Cristina; Auton, Adam; Kates, Wendy R.; Wang, Tao; Shprintzen, Robert J.; Emanuel, Beverly S.; Morrow, Bernice E.

2013-01-01

Velocardiofacial and DiGeorge syndromes, also known as 22q11.2 deletion syndrome (22q11DS), are congenital-anomaly disorders caused by a de novo hemizygous 22q11.2 deletion mediated by meiotic nonallelic homologous recombination events between low-copy repeats, also known as segmental duplications. Although previous studies exist, each was of small size, and it remains to be determined whether there are parent-of-origin biases for the de novo 22q11.2 deletion. To address this question, we genotyped a total of 389 DNA samples from 22q11DS-affected families. A total of 219 (56%) individuals with 22q11DS had maternal origin and 170 (44%) had paternal origin of the de novo deletion, which represents a statistically significant bias for maternal origin (p = 0.0151). Combined with many smaller, previous studies, 465 (57%) individuals had maternal origin and 345 (43%) had paternal origin, amounting to a ratio of 1.35 or a 35% increase in maternal compared to paternal origin (p = 0.000028). Among 1,892 probands with the de novo 22q11.2 deletion, the average maternal age at time of conception was 29.5, and this is similar to data for the general population in individual countries. Of interest, the female recombination rate in the 22q11.2 region was about 1.6–1.7 times greater than that for males, suggesting that for this region in the genome, enhanced meiotic recombination rates, as well as other as-of-yet undefined 22q11.2-specific features, could be responsible for the observed excess in maternal origin. PMID:23453669

Simple Method for Markerless Gene Deletion in Multidrug-Resistant Acinetobacter baumannii

PubMed Central

Oh, Man Hwan; Lee, Je Chul; Kim, Jungmin

2015-01-01

The traditional markerless gene deletion technique based on overlap extension PCR has been used for generating gene deletions in multidrug-resistant Acinetobacter baumannii. However, the method is time-consuming because it requires restriction digestion of the PCR products in DNA cloning and the construction of new vectors containing a suitable antibiotic resistance cassette for the selection of A. baumannii merodiploids. Moreover, the availability of restriction sites and the selection of recombinant bacteria harboring the desired chimeric plasmid are limited, making the construction of a chimeric plasmid more difficult. We describe a rapid and easy cloning method for markerless gene deletion in A. baumannii, which has no limitation in the availability of restriction sites and allows for easy selection of the clones carrying the desired chimeric plasmid. Notably, it is not necessary to construct new vectors in our method. This method utilizes direct cloning of blunt-end DNA fragments, in which upstream and downstream regions of the target gene are fused with an antibiotic resistance cassette via overlap extension PCR and are inserted into a blunt-end suicide vector developed for blunt-end cloning. Importantly, the antibiotic resistance cassette is placed outside the downstream region in order to enable easy selection of the recombinants carrying the desired plasmid, to eliminate the antibiotic resistance cassette via homologous recombination, and to avoid the necessity of constructing new vectors. This strategy was successfully applied to functional analysis of the genes associated with iron acquisition by A. baumannii ATCC 19606 and to ompA gene deletion in other A. baumannii strains. Consequently, the proposed method is invaluable for markerless gene deletion in multidrug-resistant A. baumannii. PMID:25746991

Mitochondrial genetics in Bakers' yeast: a molecular mechanism for recombinational polarity and suppressiveness.

PubMed

Perlman, P S; Birky, C W

1974-11-01

Recombinational polarity and suppressiveness are two well-known but puzzling cytoplasmic genetic phenomena in bakers' yeast, Saccharomyces cerevisiae. Little progress has been made in characterizing the underlying molecular mechanisms of these phenomena. In this paper we describe a molecular model for recombinational polarity that is compatible with the available genetic evidence. The model stresses the role of small deletions and excision/repair processes in otherwise canonical recombinational events. According to the model, both phenomena require recombination and may share mechanistic elements.

CCAAT/enhancer-binding protein alpha (CEBPA) polymorphisms and mutations in healthy individuals and in patients with peripheral artery disease, ischaemic heart disease and hyperlipidaemia.

PubMed

Fuchs, O; Kostecka, A; Provazníková, D; Krásná, B; Kotlín, R; Stanková, M; Kobylka, P; Dostálová, G; Zeman, M; Chochola, M

2010-01-01

The CCAAT/enhancer-binding protein alpha, encoded by the intronless CEBPA gene, is a transcription factor that induces expression of genes involved in differentiation of granulocytes, monocytes, adipocytes and hepatocytes. Both mono- and bi-allelic CEBPA mutations were detected in acute myeloid leukaemia and myelodysplastic syndrome. In this study we also identified CEBPA mutations in healthy individuals and in patients with peripheral artery disease, ischaemic heart disease and hyperlipidaemia. We found 16 various deletions with the presence of two direct repeats in CEBPA by analysis of 431 individuals. Three most frequent repeats included in these deletions in CEBPA gene are CGCGAG (493- 498_865-870), GG (486-487_885-886), and GCCAAGCAGC (508-517_907-916), all according to GenBank Accession No. NM_004364.2. In one case we identified that a father with ischaemic heart disease and his healthy son had two identical deletions (493_864del and 508_906del, both according to GenBank Accession No. NM_004364.2) in CEBPA. The occurrence of deletions between two repetitive sequences may be caused by recombination events in the repair process. A double-stranded cut in DNA may initiate these recombination events in adjacent DNA sequences. Four types of polymorphisms in the CEBPA gene were also detected in the screened individuals. Polymorphism in CEBPA gene 690 G>T according to GenBank Accession No. NM_004364.2 is the most frequent type in our analysis. Statistical analysis did not find significant differences in the frequency of polymorphisms in CEBPA in patients and in healthy individuals with the exception of P4 polymorphism (580_585dup according to GenBank Accesion No. NM_004364.2). P4 polymorphism was significantly increased in ischaemic heart disease patients.

The Contribution of Genetic Recombination to CRISPR Array Evolution.

PubMed

Kupczok, Anne; Landan, Giddy; Dagan, Tal

2015-06-16

CRISPR (clustered regularly interspaced short palindromic repeats) is a microbial immune system against foreign DNA. Recognition sequences (spacers) encoded within the CRISPR array mediate the immune reaction in a sequence-specific manner. The known mechanisms for the evolution of CRISPR arrays include spacer acquisition from foreign DNA elements at the time of invasion and array erosion through spacer deletion. Here, we consider the contribution of genetic recombination between homologous CRISPR arrays to the evolution of spacer repertoire. Acquisition of spacers from exogenic arrays via recombination may confer the recipient with immunity against unencountered antagonists. For this purpose, we develop a novel method for the detection of recombination in CRISPR arrays by modeling the spacer order in arrays from multiple strains from the same species. Because the evolutionary signal of spacer recombination may be similar to that of pervasive spacer deletions or independent spacer acquisition, our method entails a robustness analysis of the recombination inference by a statistical comparison to resampled and perturbed data sets. We analyze CRISPR data sets from four bacterial species: two Gammaproteobacteria species harboring CRISPR type I and two Streptococcus species harboring CRISPR type II loci. We find that CRISPR array evolution in Escherichia coli and Streptococcus agalactiae can be explained solely by vertical inheritance and differential spacer deletion. In Pseudomonas aeruginosa, we find an excess of single spacers potentially incorporated into the CRISPR locus during independent acquisition events. In Streptococcus thermophilus, evidence for spacer acquisition by recombination is present in 5 out of 70 strains. Genetic recombination has been proposed to accelerate adaptation by combining beneficial mutations that arose in independent lineages. However, for most species under study, we find that CRISPR evolution is shaped mainly by spacer acquisition and loss rather than recombination. Since the evolution of spacer content is characterized by a rapid turnover, it is likely that recombination is not beneficial for improving phage resistance in the strains under study, or that it cannot be detected in the resolution of intraspecies comparisons. © The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Molecular analysis of mucopolysaccharidosis IVA: Common mutations and racial difference

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tomatsu, S.; Hori, T.; Nakashima, Y.

1994-09-01

Mucopolysaccharidosis IVA (MPS IVA) is an autosomal recessive disorder caused by a deficiency in N-acetylgalactosamine -6-sulfate sulfatase (GALNS). Studies on the molecular basis of MPS IVA have been facilitated following cloning of the full-length cDNA and genomic DNA. In this study we detected mutations from 20 Caucasian and 19 Japanese MPS IVA patients using SSCP system and compared mutations of Caucasian origin with those of Japanese origin. The results showed the presence of 16 various mutations (3 small, deletions, 2 nonsense and 11 missense mutations) for Caucasian patients and 15 (1 deletion, 1 large alteration and 13 missense mutations) formore » Japanese. Moreover, two common mutations existed; one is double gene deletion characteristic for Japanese (6 alleles; 15%) and the other is a point mutation (1113F A{yields}T transition) characteristic for Caucasian (9 alleles; 22.5%). And the clear genotype/phenotype relationship among 1342delCA, IVS1(-2), P151S, Q148X, R386C, I113F, Q473X, W220G, P151L, A291T, R90W, and P77R, for a severe type, G96B N204K and V138A for a milder type, was observed. Only R386 mutation was seen in both of the populations. Further, the precise DNA analysis for double gene deletion of a common double gene deletion has been performed by defining the breakpoints and the results showed that one deletion was caused by homologous recombination due to Alu repetitive sequences and the other was due to nonhomologous recombination of short direct repeat. Haplotype analysis for six alleles with double deletion were different, indicating the different origin of this mutation or the frequent recombination events before a mutational event. Thus the mutations in GALNS gene are very heterogeneous and the racial difference is characteristic.« less

A 590 kb deletion caused by non-allelic homologous recombination between two LINE-1 elements in a patient with mesomelia-synostosis syndrome.

PubMed

Kohmoto, Tomohiro; Naruto, Takuya; Watanabe, Miki; Fujita, Yuji; Ujiro, Sae; Okamoto, Nana; Horikawa, Hideaki; Masuda, Kiyoshi; Imoto, Issei

2017-04-01

Mesomelia-synostoses syndrome (MSS) is a rare, autosomal-dominant, syndromal osteochondrodysplasia characterized by mesomelic limb shortening, acral synostoses, and multiple congenital malformations due to a non-recurrent deletion at 8q13 that always encompasses two coding-genes, SULF1 and SLCO5A1. To date, five unrelated patients have been reported worldwide, and MMS was previously proposed to not be a genomic disorder associated with deletions recurring from non-allelic homologous recombination (NAHR) in at least two analyzed cases. We conducted targeted gene panel sequencing and subsequent array-based copy number analysis in an 11-year-old undiagnosed Japanese female patient with multiple congenital anomalies that included mesomelic limb shortening and detected a novel 590 Kb deletion at 8q13 encompassing the same gene set as reported previously, resulting in the diagnosis of MSS. Breakpoint sequences of the deleted region in our case demonstrated the first LINE-1s (L1s)-mediated unequal NAHR event utilizing two distant L1 elements as homology substrates in this disease, which may represent a novel causative mechanism of the 8q13 deletion, expanding the range of mechanisms involved in the chromosomal rearrangements responsible for MSS. © 2017 Wiley Periodicals, Inc.

Use of virion DNA as a cloning vector for the construction of mutant and recombinant herpesviruses.

PubMed

Duboise, S M; Guo, J; Desrosiers, R C; Jung, J U

1996-10-15

We have developed improved procedures for the isolation of deletion mutant, point mutant, and recombinant herpesvirus saimiri. These procedures take advantage of the absence of NotI and AscI restriction enzyme sites within the viral genome and use reporter genes for the identification of recombinant viruses. Genes for secreted engineered alkaline phosphatase and green fluorescent protein were placed under simian virus 40 early promoter control and flanked by NotI and AscI restriction sites. When permissive cells were cotransfected with herpesvirus saimiri virion DNA and one of the engineered reporter genes cloned within herpesvirus saimiri sequences, recombinant viruses were readily identified and purified on the basis of expression of the reporter gene. Digestion of recombinant virion DNA with NotI or AscI was used to delete the reporter gene from the recombinant herpesvirus saimiri. Replacement of the reporter gene can be achieved by NotI or AscI digestion of virion DNA and ligation with a terminally matched fragment or, alternatively, by homologous recombination in cotransfected cells. Any gene can, in theory, be cloned directly into the virion DNA when flanked by the appropriate NotI or AscI sites. These procedures should be widely applicable in their general form to most or all herpesviruses that replicate permissively in cultured cells.

The non-essential UL50 gene of avian infectious laryngotracheitis virus encodes a functional dUTPase which is not a virulence factor.

PubMed

Fuchs, W; Ziemann, K; Teifke, J P; Werner, O; Mettenleiter, T C

2000-03-01

The DNA sequence of the infectious laryngotracheitis virus (ILTV) UL50, UL51 and UL52 gene homologues was determined. Although the deduced UL50 protein lacks the first of five conserved domains of the corresponding proteins of mammalian alphaherpesviruses, the ILTV gene product was also shown to possess dUTPase activity. The generation of UL50-negative ILTV mutants was facilitated by recombination plasmids encoding green fluorescent protein (GFP), and expression constructs of predicted transactivator proteins of ILTV (alphaTIF, ICP4) were successfully used to increase the infectivity of viral genomic DNA. A GFP-expressing UL50-deletion mutant of ILTV showed reduced cell-to-cell spread in vitro, and was attenuated in vivo. A similar deletion mutant without the foreign gene, however, propagated like wild-type ILTV in cell culture and was pathogenic in chickens. We conclude that the viral dUTPase is not required for efficient replication of ILTV in the respiratory tract of infected animals. The replication defect of the GFP-expressing ILTV recombinant is most likely caused by toxic effects of the reporter gene product, since spontaneously occurring inactivation mutants exhibited wild-type-like growth.

N-terminal deletions in Rous sarcoma virus p60src: effects on tyrosine kinase and biological activities and on recombination in tissue culture with the cellular src gene.

PubMed Central

Cross, F R; Garber, E A; Hanafusa, H

1985-01-01

We have constructed deletions within the region of cloned Rous sarcoma virus DNA coding for the N-terminal 30 kilodaltons of p60src. Infectious virus was recovered after transfection. Deletions of amino acids 15 to 149, 15 to 169, or 149 to 169 attenuated but did not abolish transforming activity, as assayed by focus formation and anchorage-independent growth. These deletions also had only slight effects on the tyrosine kinase activity of the mutant src protein. Deletion of amino acids 169 to 264 or 15 to 264 completely abolished transforming activity, and src kinase activity was reduced at least 10-fold. However, these mutant viruses generated low levels of transforming virus by recombination with the cellular src gene. The results suggest that as well as previously identified functional domains for p60src myristylation and membrane binding (amino acids 1 to 14) and tyrosine kinase activity (amino acids 250 to 526), additional N-terminal sequences (particularly amino acids 82 to 169) can influence the transforming activity of the src protein. Images PMID:2426576

Cellular immunity to viral antigens limits E1-deleted adenoviruses for gene therapy.

PubMed Central

Yang, Y; Nunes, F A; Berencsi, K; Furth, E E; Gönczöl, E; Wilson, J M

1994-01-01

An important limitation that has emerged in the use of adenoviruses for gene therapy has been loss of recombinant gene expression that occurs concurrent with the development of pathology in the organ expressing the transgene. We have used liver-directed approaches to gene therapy in mice to study mechanisms that underlie the problems with transient expression and pathology that have characterized in vivo applications of first-generation recombinant adenoviruses (i.e., those deleted of E1a and E1b). Our data are consistent with the following hypothesis. Cells harboring the recombinant viral genome express the transgene as desired; however, low-level expression of viral genes also occurs. A virus-specific cellular immune response is stimulated that leads to destruction of the genetically modified hepatocytes, massive hepatitis, and repopulation of the liver with nontransgene-containing hepatocytes. These findings suggest approaches for improving recombinant adenoviruses that are based on further crippling the virus to limit expression of nondeleted viral genes. Images PMID:8183921

A Genetic System for Clostridium ljungdahlii: a Chassis for Autotrophic Production of Biocommodities and a Model Homoacetogen

DOE Office of Scientific and Technical Information (OSTI.GOV)

Leang, C; Ueki, T; Nevin, KP

Methods for genetic manipulation of Clostridium ljungdahlii are of interest because of the potential for production of fuels and other biocommodities from carbon dioxide via microbial electrosynthesis or more traditional modes of autotrophy with hydrogen or carbon monoxide as the electron donor. Furthermore, acetogenesis plays an important role in the global carbon cycle. Gene deletion strategies required for physiological studies of C. ljungdahlii have not previously been demonstrated. An electroporation procedure for introducing plasmids was optimized, and four different replicative origins for plasmid propagation in C. ljungdahlii were identified. Chromosomal gene deletion via double-crossover homologous recombination with a suicide vectormore » was demonstrated initially with deletion of the gene for FliA, a putative sigma factor involved in flagellar biogenesis and motility in C. ljungdahlii. Deletion of fliA yielded a strain that lacked flagella and was not motile. To evaluate the potential utility of gene deletions for functional genomic studies and to redirect carbon and electron flow, the genes for the putative bifunctional aldehyde/alcohol dehydrogenases, adhE1 and adhE2, were deleted individually or together. Deletion of adhE1, but not adhE2, diminished ethanol production with a corresponding carbon recovery in acetate. The double deletion mutant had a phenotype similar to that of the adhE1-deficient strain. Expression of adhE1 in trans partially restored the capacity for ethanol production. These results demonstrate the feasibility of genetic investigations of acetogen physiology and the potential for genetic manipulation of C. ljungdahlii to optimize autotrophic biocommodity production.« less

A family of selfish minicircular chromosomes with jumbled chloroplast gene fragments from a dinoflagellate.

PubMed

Zhang, Z; Cavalier-Smith, T; Green, B R

2001-08-01

Chloroplast genes of several dinoflagellate species are located on unigenic DNA minicircular chromosomes. We have now completely sequenced five aberrant minicircular chromosomes from the dinoflagellate Heterocapsa triquetra. These probably nonfunctional DNA circles lack complete genes, with each being composed of several short fragments of two or three different chloroplast genes and a common conserved region with a tripartite 9G-9A-9G core like the putative replicon origin of functional single-gene circular chloroplast chromosomes. Their sequences imply that all five circles evolved by differential deletions and duplications from common ancestral circles bearing fragments of four genes: psbA, psbC, 16S rRNA, and 23S rRNA. It appears that recombination between separate unigenic chromosomes initially gave intermediate heterodimers, which were subsequently stabilized by deletions that included part or all of one putative replicon origin. We suggest that homologous recombination at the 9G-9A-9G core regions produced a psbA/psbC heterodimer which generated two distinct chimeric circles by differential deletions and duplications. A 23S/16S rRNA heterodimer more likely formed by illegitimate recombination between 16S and 23S rRNA genes. Homologous recombination between the 9G-9A-9G core regions of both heterodimers and additional differential deletions and duplications could then have yielded the other three circles. Near identity of the gene fragments and 9G-9A-9G cores, despite diverging adjacent regions, may be maintained by gene conversion. The conserved organization of the 9G-9A-9G cores alone favors the idea that they are replicon origins and suggests that they may enable the aberrant minicircles to parasitize the chloroplast's replication machinery as selfish circles.

Attenuation and protection efficacy of ORF C gene-deleted recombinant of infectious laryngotracheitis virus.

PubMed

Garcia, Maricarmen; Spatz, S J; Cheng, Y; Riblet, S M; Volkening, J D; Schneiders, G H

2016-09-01

Infectious laryngotracheitis (ILT) is a highly contagious respiratory disease of chickens caused by infectious laryngotracheitis virus (ILTV). The disease is controlled by the use of live-attenuated vaccines. Previously we reported the complete nucleotide sequence of the ILTV vaccine strain (TCO) and identified a nonsense mutation in the gene encoding the ORF C protein. This suggested that the ORF C protein might be associated with viral virulence. To investigate this, an ILTV recombinant with a deletion in the gene encoding ORF C was constructed using the genome of the virulent United States Department of Agriculture (USDA) challenge strain (USDAch). Compared to the parental virus, the ΔORF C recombinant replicated in chicken kidney (CK) cells with similar kinetics and generated similar titres. This demonstrated that the ORF C deletion had no deleterious effects on replication efficacy in vitro. In chickens, the recombinant induced only minor microscopic tracheal lesions when inoculated via the intra-tracheal/ocular route, while the parental strain induced moderate to severe microscopic tracheal lesions, even though virus load in the tracheas were comparable. Groups of chickens vaccinated via eye-drop with the ∆ORFC-ILTV were protected to levels comparable to those elicited by TCO vaccination. To our knowledge, this is the first report that demonstrates the suitability of ∆ORFC as a live-attenuated vaccine to prevent the losses caused by ILTV.

Nonhomologous Pairing in Mice Heterozygous for a t Haplotype Can Produce Recombinant Chromosomes with Duplications and Deletions

PubMed Central

Sarvetnick, Nora; Fox, Howard S.; Mann, Elizabeth; Mains, Paul E.; Elliott, Rosemary W.; Silver, Lee M.

1986-01-01

We have investigated the structure and properties of a chromosomal product recovered from a rare recombination event between a t haplotype and a wild-type form of mouse chromosome 17. Our embryological and molecular studies indicate that this chromosome (twLub2 ) is characterized by both a deletion and duplication of adjacent genetic material. The deletion appears to be responsible for a dominant lethal maternal effect and a recessive embryonic lethality. The duplication provides an explanation for the twLub2 suppression of the dominant T locus phenotype. A reanalysis of previously described results with another chromosome 17 variant called TtOrl indicates a structure for this chromosome that is reciprocal to that observed for twLub2. We have postulated the existence of an inversion over the proximal portion of all complete t haplotypes in order to explain the generation of the partial t haplotypes t wLub2 and TtOrl. This proximal inversion and the previously described distal inversion are sufficient to account for all of the recombination properties that are characteristic of complete t haplotypes. The structures determined for twLub2 and TtOrl indicate that rare recombination can occur between nonequivalent genomic sequences within the inverted proximal t region when wild-type and t chromosomes are paired in a linear, nonhomologous configuration. PMID:3732789

Identification of 15 novel partial SHOX deletions and 13 partial duplications, and a review of the literature reveals intron 3 to be a hotspot region.

PubMed

Benito-Sanz, Sara; Belinchon-Martínez, Alberta; Aza-Carmona, Miriam; de la Torre, Carolina; Huber, Celine; González-Casado, Isabel; Ross, Judith L; Thomas, N Simon; Zinn, Andrew R; Cormier-Daire, Valerie; Heath, Karen E

2017-02-01

Short stature homeobox gene (SHOX) is located in the pseudoautosomal region 1 of the sex chromosomes. It encodes a transcription factor implicated in the skeletal growth. Point mutations, deletions or duplications of SHOX or its transcriptional regulatory elements are associated with two skeletal dysplasias, Léri-Weill dyschondrosteosis (LWD) and Langer mesomelic dysplasia (LMD), as well as in a small proportion of idiopathic short stature (ISS) individuals. We have identified a total of 15 partial SHOX deletions and 13 partial SHOX duplications in LWD, LMD and ISS patients referred for routine SHOX diagnostics during a 10 year period (2004-2014). Subsequently, we characterized these alterations using MLPA (multiplex ligation-dependent probe amplification assay), fine-tiling array CGH (comparative genomic hybridation) and breakpoint PCR. Nearly half of the alterations have a distal or proximal breakpoint in intron 3. Evaluation of our data and that in the literature reveals that although partial deletions and duplications only account for a small fraction of SHOX alterations, intron 3 appears to be a breakpoint hotspot, with alterations arising by non-allelic homologous recombination, non-homologous end joining or other complex mechanisms.

Problem-Solving Test: Conditional Gene Targeting Using the Cre/loxP Recombination System

ERIC Educational Resources Information Center

Szeberényi, József

2013-01-01

Terms to be familiar with before you start to solve the test: gene targeting, knock-out mutation, bacteriophage, complementary base-pairing, homologous recombination, deletion, transgenic organisms, promoter, polyadenylation element, transgene, DNA replication, RNA polymerase, Shine-Dalgarno sequence, restriction endonuclease, polymerase chain…

Enhanced production of recombinant proteins with Corynebacterium glutamicum by deletion of insertion sequences (IS elements).

PubMed

Choi, Jae Woong; Yim, Sung Sun; Kim, Min Jeong; Jeong, Ki Jun

2015-12-29

In most bacteria, various jumping genetic elements including insertion sequences elements (IS elements) cause a variety of genetic rearrangements resulting in harmful effects such as genome and recombinant plasmid instability. The genetic stability of a plasmid in a host is critical for high-level production of recombinant proteins, and in this regard, the development of an IS element-free strain could be a useful strategy for the enhanced production of recombinant proteins. Corynebacterium glutamicum, which is a workhorse in the industrial-scale production of various biomolecules including recombinant proteins, also has several IS elements, and it is necessary to identify the critical IS elements and to develop IS element deleted strain. From the cultivation of C. glutamicum harboring a plasmid for green fluorescent protein (GFP) gene expression, non-fluorescent clones were isolated by FACS (fluorescent activated cell sorting). All the isolated clones had insertions of IS elements in the GFP coding region, and two major IS elements (ISCg1 and ISCg2 families) were identified. By co-cultivating cells harboring either the isolated IS element-inserted plasmid or intact plasmid, it was clearly confirmed that cells harboring the IS element-inserted plasmids became dominant during the cultivation due to their growth advantage over cells containing intact plasmids, which can cause a significant reduction in recombinant protein production during cultivation. To minimize the harmful effects of IS elements on the expression of heterologous genes in C. glutamicum, two IS element free C. glutamicum strains were developed in which each major IS element was deleted, and enhanced productivity in the engineered C. glutamicum strain was successfully demonstrated with three models: GFP, poly(3-hydroxybutyrate) [P(3HB)] and γ-aminobutyrate (GABA). Our findings clearly indicate that the hopping of IS elements could be detrimental to the production of recombinant proteins in C. glutamicum, emphasizing the importance of developing IS element free host strains.

A new physical mapping approach refines the sex-determining gene positions on the Silene latifolia Y-chromosome

NASA Astrophysics Data System (ADS)

Kazama, Yusuke; Ishii, Kotaro; Aonuma, Wataru; Ikeda, Tokihiro; Kawamoto, Hiroki; Koizumi, Ayako; Filatov, Dmitry A.; Chibalina, Margarita; Bergero, Roberta; Charlesworth, Deborah; Abe, Tomoko; Kawano, Shigeyuki

2016-01-01

Sex chromosomes are particularly interesting regions of the genome for both molecular genetics and evolutionary studies; yet, for most species, we lack basic information, such as the gene order along the chromosome. Because they lack recombination, Y-linked genes cannot be mapped genetically, leaving physical mapping as the only option for establishing the extent of synteny and homology with the X chromosome. Here, we developed a novel and general method for deletion mapping of non-recombining regions by solving “the travelling salesman problem”, and evaluate its accuracy using simulated datasets. Unlike the existing radiation hybrid approach, this method allows us to combine deletion mutants from different experiments and sources. We applied our method to a set of newly generated deletion mutants in the dioecious plant Silene latifolia and refined the locations of the sex-determining loci on its Y chromosome map.

A Genome-Wide Map of Mitochondrial DNA Recombination in Yeast

PubMed Central

Fritsch, Emilie S.; Chabbert, Christophe D.; Klaus, Bernd; Steinmetz, Lars M.

2014-01-01

In eukaryotic cells, the production of cellular energy requires close interplay between nuclear and mitochondrial genomes. The mitochondrial genome is essential in that it encodes several genes involved in oxidative phosphorylation. Each cell contains several mitochondrial genome copies and mitochondrial DNA recombination is a widespread process occurring in plants, fungi, protists, and invertebrates. Saccharomyces cerevisiae has proved to be an excellent model to dissect mitochondrial biology. Several studies have focused on DNA recombination in this organelle, yet mostly relied on reporter genes or artificial systems. However, no complete mitochondrial recombination map has been released for any eukaryote so far. In the present work, we sequenced pools of diploids originating from a cross between two different S. cerevisiae strains to detect recombination events. This strategy allowed us to generate the first genome-wide map of recombination for yeast mitochondrial DNA. We demonstrated that recombination events are enriched in specific hotspots preferentially localized in non-protein-coding regions. Additionally, comparison of the recombination profiles of two different crosses showed that the genetic background affects hotspot localization and recombination rates. Finally, to gain insights into the mechanisms involved in mitochondrial recombination, we assessed the impact of individual depletion of four genes previously associated with this process. Deletion of NTG1 and MGT1 did not substantially influence the recombination landscape, alluding to the potential presence of additional regulatory factors. Our findings also revealed the loss of large mitochondrial DNA regions in the absence of MHR1, suggesting a pivotal role for Mhr1 in mitochondrial genome maintenance during mating. This study provides a comprehensive overview of mitochondrial DNA recombination in yeast and thus paves the way for future mechanistic studies of mitochondrial recombination and genome maintenance. PMID:25081569

A genome-wide map of mitochondrial DNA recombination in yeast.

PubMed

Fritsch, Emilie S; Chabbert, Christophe D; Klaus, Bernd; Steinmetz, Lars M

2014-10-01

In eukaryotic cells, the production of cellular energy requires close interplay between nuclear and mitochondrial genomes. The mitochondrial genome is essential in that it encodes several genes involved in oxidative phosphorylation. Each cell contains several mitochondrial genome copies and mitochondrial DNA recombination is a widespread process occurring in plants, fungi, protists, and invertebrates. Saccharomyces cerevisiae has proved to be an excellent model to dissect mitochondrial biology. Several studies have focused on DNA recombination in this organelle, yet mostly relied on reporter genes or artificial systems. However, no complete mitochondrial recombination map has been released for any eukaryote so far. In the present work, we sequenced pools of diploids originating from a cross between two different S. cerevisiae strains to detect recombination events. This strategy allowed us to generate the first genome-wide map of recombination for yeast mitochondrial DNA. We demonstrated that recombination events are enriched in specific hotspots preferentially localized in non-protein-coding regions. Additionally, comparison of the recombination profiles of two different crosses showed that the genetic background affects hotspot localization and recombination rates. Finally, to gain insights into the mechanisms involved in mitochondrial recombination, we assessed the impact of individual depletion of four genes previously associated with this process. Deletion of NTG1 and MGT1 did not substantially influence the recombination landscape, alluding to the potential presence of additional regulatory factors. Our findings also revealed the loss of large mitochondrial DNA regions in the absence of MHR1, suggesting a pivotal role for Mhr1 in mitochondrial genome maintenance during mating. This study provides a comprehensive overview of mitochondrial DNA recombination in yeast and thus paves the way for future mechanistic studies of mitochondrial recombination and genome maintenance. Copyright © 2014 by the Genetics Society of America.

Methods and materials for the production of L-lactic acid in yeast

DOEpatents

Hause, Ben [Jordan, MN; Rajgarhia, Vineet [Minnetonka, MN; Suominen, Pirkko [Maple Grove, MN

2009-05-19

Recombinant yeast are provided having, in one aspect, multiple exogenous LDH genes integrated into the genome, while leaving native PDC genes intact. In a second aspect, recombinant yeast are provided having an exogenous LDH gene integrated into its genome at the locus of a native PDC gene, with deletion of the native PDC gene. The recombinant yeast are useful in fermentation process for producing lactic acid.

21 CFR 514.8 - Supplements and other changes to an approved application.

Code of Federal Regulations, 2013 CFR

2013-04-01

... method(s) or an addition, deletion, or substitution of steps in an aseptic processing operation; (D... solely affecting a natural product, a recombinant DNA-derived protein/polypeptide, or a complex or...) or references to previously approved documentation; (H) For a natural product, a recombinant DNA...

21 CFR 514.8 - Supplements and other changes to an approved application.

Code of Federal Regulations, 2014 CFR

2014-04-01

... method(s) or an addition, deletion, or substitution of steps in an aseptic processing operation; (D... solely affecting a natural product, a recombinant DNA-derived protein/polypeptide, or a complex or...) or references to previously approved documentation; (H) For a natural product, a recombinant DNA...

21 CFR 514.8 - Supplements and other changes to an approved application.

Code of Federal Regulations, 2012 CFR

2012-04-01

... method(s) or an addition, deletion, or substitution of steps in an aseptic processing operation; (D... solely affecting a natural product, a recombinant DNA-derived protein/polypeptide, or a complex or...) or references to previously approved documentation; (H) For a natural product, a recombinant DNA...

Fibroblast Activation Protein Cleaves and Inactivates Fibroblast Growth Factor 21*

PubMed Central

Dunshee, Diana Ronai; Bainbridge, Travis W.; Kljavin, Noelyn M.; Zavala-Solorio, Jose; Schroeder, Amy C.; Chan, Ruby; Corpuz, Racquel; Wong, Manda; Zhou, Wei; Deshmukh, Gauri; Ly, Justin; Sutherlin, Daniel P.; Ernst, James A.; Sonoda, Junichiro

2016-01-01

FGF21 is a stress-induced hormone with potent anti-obesity, insulin-sensitizing, and hepatoprotective properties. Although proteolytic cleavage of recombinant human FGF21 in preclinical species has been observed previously, the regulation of endogenously produced FGF21 is not well understood. Here we identify fibroblast activation protein (FAP) as the enzyme that cleaves and inactivates human FGF21. A selective chemical inhibitor, immunodepletion, or genetic deletion of Fap stabilized recombinant human FGF21 in serum. In addition, administration of a selective FAP inhibitor acutely increased circulating intact FGF21 levels in cynomolgus monkeys. On the basis of our findings, we propose selective FAP inhibition as a potential therapeutic approach to increase endogenous FGF21 activity for the treatment of obesity, type 2 diabetes, non-alcoholic steatohepatitis, and related metabolic disorders. PMID:26797127

Genetic studies of Prader-Willi patients provide evidence for conservation of genomic architecture in proximal chromosome 15q.

PubMed

Hou, Aihua; Lin, Shuan-Pei; Ho, Shi Yun; Chen, Chi-Fung Jennifer; Lin, Hsiang-Yu; Chen, Yen-Juin; Huang, Chi-Yu; Chiu, Huei-Ching; Chuang, Chih-Kuang; Chen, Ken-Shiung

2011-03-01

Prader-Willi syndrome (PWS) is a neurogenetic disorder associated with recurrent genomic recombination involving low copy repeats (LCRs) located in the human chromosome 15q11-q13. Previous studies of PWS patients from Asia suggested that there is a higher incidence of deletion and lower incidence of maternal uniparental disomy (mUPD) compared to that of Western populations. In this report, we present genetic etiology of 28 PWS patients from Taiwan. Consistent with the genetic etiology findings from Western populations, the type II deletion appears to be the most common deletion subtype. Furthermore, the ratio of the two most common deletion subtypes and the ratio of the maternal heterodisomy to isodisomy cases observed from this study are in agreement with previous findings from Western populations. In addition, we identified and further mapped the deletion breakpoints in two patients with atypical deletions using array CGH (comparative genomic hybridization). Despite the relatively small numbers of patients in each subgroup, our findings suggest that the genomic architecture responsible for the recurrent recombination in PWS is conserved in Taiwanese of the Han Chinese heritage and Western populations, thereby predisposing chromosome 15q11-q13 to a similar risk of rearrangements. © 2010 The Authors Annals of Human Genetics © 2010 Blackwell Publishing Ltd/University College London.

A yeast-based genetic screening to identify human proteins that increase homologous recombination.

PubMed

Collavoli, Anita; Comelli, Laura; Rainaldi, Giuseppe; Galli, Alvaro

2008-05-01

To identify new human proteins implicated in homologous recombination (HR), we set up 'a papillae assay' to screen a human cDNA library using the RS112 strain of Saccharomyces cerevisiae containing an intrachromosomal recombination substrate. We isolated 23 cDNAs, 11 coding for complete proteins and 12 for partially deleted proteins that increased HR when overexpressed in yeast. We characterized the effect induced by the overexpression of the complete human proteasome subunit beta 2, the partially deleted proteasome subunits alpha 3 and beta 8, the ribosomal protein L12, the brain abundant membrane signal protein (BASP1) and the human homologue to v-Ha-RAS (HRAS), which elevated HR by 2-6.5-fold over the control. We found that deletion of the RAD52 gene, which has a key role in most HR events, abolished the increase of HR induced by the proteasome subunits and HRAS; by contrast, the RAD52 deletion did not affect the high level of HR due to BASP1 and RPL12. This suggests that the proteins stimulated yeast HR via different mechanisms. Overexpression of the complete beta 2 human proteasome subunit or the partially deleted alpha 3 and beta 8 subunits increased methyl methanesulphonate (MMS) resistance much more in the rad52 Delta mutant than in the wild-type. Overexpression of RPL12 and BASP1 did not affect MMS resistance in both the wild-type and the rad52 Delta mutant, whereas HRAS decreased MMS resistance in the rad52 Delta mutant. The results indicate that these proteins may interfere with the pathway(s) involved in the repair of MMS-induced DNA damage. Finally, we provide further evidence that yeast is a helpful tool to identify human proteins that may have a regulatory role in HR.

The Dual Regulatory Role of Amino Acids Leu480 and Gln481 of Prothrombin*

PubMed Central

Wiencek, Joesph R.; Hirbawi, Jamila; Yee, Vivien C.; Kalafatis, Michael

2016-01-01

Prothrombin (FII) is activated to α-thrombin (IIa) by prothrombinase. Prothrombinase is composed of a catalytic subunit, factor Xa (fXa), and a regulatory subunit, factor Va (fVa), assembled on a membrane surface in the presence of divalent metal ions. We constructed, expressed, and purified several mutated recombinant FII (rFII) molecules within the previously determined fVa-dependent binding site for fXa (amino acid region 473–487 of FII). rFII molecules bearing overlapping deletions within this significant region first established the minimal stretch of amino acids required for the fVa-dependent recognition exosite for fXa in prothrombinase within the amino acid sequence Ser478–Val479–Leu480–Gln481–Val482. Single, double, and triple point mutations within this stretch of rFII allowed for the identification of Leu480 and Gln481 as the two essential amino acids responsible for the enhanced activation of FII by prothrombinase. Unanticipated results demonstrated that although recombinant wild type α-thrombin and rIIaS478A were able to induce clotting and activate factor V and factor VIII with rates similar to the plasma-derived molecule, rIIaSLQ→AAA with mutations S478A/L480A/Q481A was deficient in clotting activity and unable to efficiently activate the pro-cofactors. This molecule was also impaired in protein C activation. Similar results were obtained with rIIaΔSLQ (where rIIaΔSLQ is recombinant human α-thrombin with amino acids Ser478/Leu480/Gln481 deleted). These data provide new evidence demonstrating that amino acid sequence Leu480–Gln481: 1) is crucial for proper recognition of the fVa-dependent site(s) for fXa within prothrombinase on FII, required for efficient initial cleavage of FII at Arg320; and 2) is compulsory for appropriate tethering of fV, fVIII, and protein C required for their timely activation by IIa. PMID:26601957

Gene Deletion in Barley Mediated by LTR-retrotransposon BARE

PubMed Central

Shang, Yi; Yang, Fei; Schulman, Alan H.; Zhu, Jinghuan; Jia, Yong; Wang, Junmei; Zhang, Xiao-Qi; Jia, Qiaojun; Hua, Wei; Yang, Jianming; Li, Chengdao

2017-01-01

A poly-row branched spike (prbs) barley mutant was obtained from soaking a two-rowed barley inflorescence in a solution of maize genomic DNA. Positional cloning and sequencing demonstrated that the prbs mutant resulted from a 28 kb deletion including the inflorescence architecture gene HvRA2. Sequence annotation revealed that the HvRA2 gene is flanked by two LTR (long terminal repeat) retrotransposons (BARE) sharing 89% sequence identity. A recombination between the integrase (IN) gene regions of the two BARE copies resulted in the formation of an intact BARE and loss of HvRA2. No maize DNA was detected in the recombination region although the flanking sequences of HvRA2 gene showed over 73% of sequence identity with repetitive sequences on 10 maize chromosomes. It is still unknown whether the interaction of retrotransposons between barley and maize has resulted in the recombination observed in the present study. PMID:28252053

Identification of an essential virulence gene of cyprinid herpesvirus 3.

PubMed

Boutier, Maxime; Gao, Yuan; Vancsok, Catherine; Suárez, Nicolás M; Davison, Andrew J; Vanderplasschen, Alain

2017-09-01

The genus Cyprinivirus consists of a growing list of phylogenetically related viruses, some of which cause severe economic losses to the aquaculture industry. The archetypal member, cyprinid herpesvirus 3 (CyHV-3) causes mass mortalities worldwide in koi and common carp. A CyHV-3 mutant was described previously that is attenuated in vivo by a deletion affecting two genes (ORF56 and ORF57). The relative contributions of ORF56 and ORF57 to the safety and efficacy profile of this vaccine candidate have now been assessed by analysing viruses individually deleted for ORF56 or ORF57. Inoculation of these viruses into carp demonstrated that the absence of ORF56 did not affect virulence, whereas the absence of ORF57 led to an attenuation comparable to, though slightly less than, that of the doubly deleted virus. To demonstrate further the role of ORF57 as a key virulence factor, a mutant retaining the ORF57 region but unable to express the ORF57 protein was produced by inserting multiple in-frame stop codons into the coding region. Analysis of this virus in vivo revealed a safety and efficacy profile comparable to that of the doubly deleted virus. These findings show that ORF57 encodes an essential CyHV-3 virulence factor. They also indicate that ORF57 orthologues in other cypriniviruses may offer promising targets for the rational design of attenuated recombinant vaccines. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Redundant function of DNA ligase 1 and 3 in alternative end-joining during immunoglobulin class switch recombination.

PubMed

Masani, Shahnaz; Han, Li; Meek, Katheryn; Yu, Kefei

2016-02-02

Nonhomologous end-joining (NHEJ) is the major DNA double-strand break (DSB) repair pathway in mammals and resolves the DSBs generated during both V(D)J recombination in developing lymphocytes and class switch recombination (CSR) in antigen-stimulated B cells. In contrast to the absolute requirement for NHEJ to resolve DSBs associated with V(D)J recombination, DSBs associated with CSR can be resolved in NHEJ-deficient cells (albeit at a reduced level) by a poorly defined alternative end-joining (A-EJ) pathway. Deletion of DNA ligase IV (Lig4), a core component of the NHEJ pathway, reduces CSR efficiency in a mouse B-cell line capable of robust cytokine-stimulated CSR in cell culture. Here, we report that CSR levels are not further reduced by deletion of either of the two remaining DNA ligases (Lig1 and nuclear Lig3) in Lig4(-/-) cells. We conclude that in the absence of Lig4, Lig1, and Lig3 function in a redundant manner in resolving switch region DSBs during CSR.

Intrachromosomal 3p insertion as a cause of reciprocal pure interstitial deletion and duplication in two siblings: further delineation of the emerging proximal 3p deletion syndrome.

PubMed

Lloveras, Elisabet; Vendrell, Teresa; Fernández, Asunción; Castells, Neus; Cueto, Ana; del Campo, Miguel; Hernando, Cristina; Villa, Olaya; Plaja, Alberto

2014-01-01

Very few cases of constitutional interstitial deletions of the proximal short arm of chromosome 3 have been reported; however, the proximal 3p deletion is emerging as a clinically recognizable syndrome. We present an intrachromosomal insertion of 3p12.3p14.1 in a phenotypic normal man (46,XY,ins(3)(p25p12.3p14.1)) which is responsible for the unbalanced karyotype in 2 affected offspring, one with a 3p12.3p14.1 interstitial deletion and the other with a reciprocal duplication. The exceptionality of these 2 reciprocal recombinants contributes to a better definition of the proximal 3p deletion syndrome and its duplication counterpart.

Generation and characterization of koi herpesvirus recombinants lacking viral enzymes of nucleotide metabolism.

PubMed

Fuchs, Walter; Fichtner, Dieter; Bergmann, Sven M; Mettenleiter, Thomas C

2011-06-01

Koi herpesvirus (KHV) causes a fatal disease in koi and common carp, but no reliable and genetically characterized vaccines are available up to now. Therefore, we generated KHV recombinants possessing deletions within the viral ribonucleotide reductase (RNR), thymidine kinase (TK), dUTPase, or TK and dUTPase genes, and their corresponding rescuants. All KHV mutants were replication competent in cultured cells. Whereas plaque sizes and titers of RNR-negative KHV were reduced, replication of the other mutants was not affected. Experimental infection of carp indicated attenuation of TK- or dUTPase-deleted KHV, and PCR analysis of tissue samples permitted differentiation of mutant from wild-type virus.

BRCT-domain protein BRIT1 influences class switch recombination

PubMed Central

Yen, Wei-Feng; Chaudhry, Ashutosh; Vaidyanathan, Bharat; Yewdell, William T.; Pucella, Joseph N.; Sharma, Rahul; Li, Kaiyi; Rudensky, Alexander Y.; Chaudhuri, Jayanta

2017-01-01

DNA double-strand breaks (DSBs) serve as obligatory intermediates for Ig heavy chain (Igh) class switch recombination (CSR). The mechanisms by which DSBs are resolved to promote long-range DNA end-joining while suppressing genomic instability inherently associated with DSBs are yet to be fully elucidated. Here, we use a targeted short-hairpin RNA screen in a B-cell lymphoma line to identify the BRCT-domain protein BRIT1 as an effector of CSR. We show that conditional genetic deletion of BRIT1 in mice leads to a marked increase in unrepaired Igh breaks and a significant reduction in CSR in ex vivo activated splenic B cells. We find that the C-terminal tandem BRCT domains of BRIT1 facilitate its interaction with phosphorylated H2AX and that BRIT1 is recruited to the Igh locus in an activation-induced cytidine deaminase (AID) and H2AX-dependent fashion. Finally, we demonstrate that depletion of another BRCT-domain protein, MDC1, in BRIT1-deleted B cells increases the severity of CSR defect over what is observed upon loss of either protein alone. Our results identify BRIT1 as a factor in CSR and demonstrate that multiple BRCT-domain proteins contribute to optimal resolution of AID-induced DSBs. PMID:28724724

Genetic correction of β-thalassemia patient-specific iPS cells and its use in improving hemoglobin production in irradiated SCID mice.

PubMed

Wang, Yixuan; Zheng, Chen-Guang; Jiang, Yonghua; Zhang, Jiqin; Chen, Jiayu; Yao, Chao; Zhao, Qingguo; Liu, Sheng; Chen, Ke; Du, Juan; Yang, Ze; Gao, Shaorong

2012-04-01

The generation of induced pluripotent stem cells (iPSCs) from differentiated somatic cells by over-expression of several transcription factors has the potential to cure many genetic and degenerative diseases currently recalcitrant to traditional clinical approaches. One such genetic disease is β-thalassemia major (Cooley's anemia). This disease is caused by either a point mutation or the deletion of several nucleotides in the β-globin gene, and it threatens the lives of millions of people in China. In the present study, we successfully generated iPSCs from fibroblasts collected from a 2-year-old patient who was diagnosed with a homozygous 41/42 deletion in his β-globin gene. More importantly, we successfully corrected this genetic mutation in the β-thalassemia iPSCs by homologous recombination. Furthermore, transplantation of the genetically corrected iPSCs-derived hematopoietic progenitors into sub-lethally irradiated immune deficient SCID mice showed improved hemoglobin production compared with the uncorrected iPSCs. Moreover, the generation of human β-globin could be detected in the mice transplanted with corrected iPSCs-derived hematopietic progenitors. Our study provides strong evidence that iPSCs generated from a patient with a genetic disease can be corrected by homologous recombination and that the corrected iPSCs have potential clinical uses.

Gene doctoring: a method for recombineering in laboratory and pathogenic Escherichia coli strains.

PubMed

Lee, David J; Bingle, Lewis E H; Heurlier, Karin; Pallen, Mark J; Penn, Charles W; Busby, Stephen J W; Hobman, Jon L

2009-12-09

Homologous recombination mediated by the lambda-Red genes is a common method for making chromosomal modifications in Escherichia coli. Several protocols have been developed that differ in the mechanisms by which DNA, carrying regions homologous to the chromosome, are delivered into the cell. A common technique is to electroporate linear DNA fragments into cells. Alternatively, DNA fragments are generated in vivo by digestion of a donor plasmid with a nuclease that does not cleave the host genome. In both cases the lambda-Red gene products recombine homologous regions carried on the linear DNA fragments with the chromosome. We have successfully used both techniques to generate chromosomal mutations in E. coli K-12 strains. However, we have had limited success with these lambda-Red based recombination techniques in pathogenic E. coli strains, which has led us to develop an enhanced protocol for recombineering in such strains. Our goal was to develop a high-throughput recombineering system, primarily for the coupling of genes to epitope tags, which could also be used for deletion of genes in both pathogenic and K-12 E. coli strains. To that end we have designed a series of donor plasmids for use with the lambda-Red recombination system, which when cleaved in vivo by the I-SceI meganuclease generate a discrete linear DNA fragment, allowing for C-terminal tagging of chromosomal genes with a 6xHis, 3xFLAG, 4xProteinA or GFP tag or for the deletion of chromosomal regions. We have enhanced existing protocols and technologies by inclusion of a cassette conferring kanamycin resistance and, crucially, by including the sacB gene on the donor plasmid, so that all but true recombinants are counter-selected on kanamycin and sucrose containing media, thus eliminating the need for extensive screening. This method has the added advantage of limiting the exposure of cells to the potential damaging effects of the lambda-Red system, which can lead to unwanted secondary alterations to the chromosome. We have developed a counter-selective recombineering technique for epitope tagging or for deleting genes in E. coli. We have demonstrated the versatility of the technique by modifying the chromosome of the enterohaemorrhagic O157:H7 (EHEC), uropathogenic CFT073 (UPEC), enteroaggregative O42 (EAEC) and enterotoxigenic H10407 (ETEC) E. coli strains as well as in K-12 laboratory strains.

Recombineering: A Homologous Recombination-Based Method of Genetic Engineering

PubMed Central

Sharan, Shyam K.; Thomason, Lynn C.; Kuznetsov, Sergey G.; Court, Donald L.

2009-01-01

Recombineering is an efficient method of in vivo genetic engineering applicable to chromosomal as well as episomal replicons in E. coli. This method circumvents the need for most standard in vitro cloning techniques. Recombineering allows construction of DNA molecules with precise junctions without constraints being imposed by restriction enzyme site location. Bacteriophage homologous recombination proteins catalyze these recombineering reactions using double- and single-strand linear DNA substrates, so-called targeting constructs, introduced by electroporation. Gene knockouts, deletions and point mutations are readily made, gene tags can be inserted, and regions of bacterial artificial chromosomes (BACs) or the E. coli genome can be subcloned by gene retrieval using recombineering. Most of these constructs can be made within about a week's time. PMID:19180090

In vitro V(D)J recombination: signal joint formation.

PubMed

Cortes, P; Weis-Garcia, F; Misulovin, Z; Nussenzweig, A; Lai, J S; Li, G; Nussenzweig, M C; Baltimore, D

1996-11-26

The first step of V(D)J recombination, specific cleavage at the recombination signal sequence (RSS), can be carried out by the recombination activating proteins RAG1 and RAG2. In vivo, the cleaved coding and signal ends must be rejoined to generate functional antigen receptors and maintain chromosomal integrity. We have investigated signal joint formation using deletion and inversion substrates in a cell free system. RAG1 and RAG2 alone or in combination were unable to generate signal joints. However, RAG1 and RAG2 complemented with nuclear extracts were able to recombine an extrachromosomal substrate and form precise signal joints. The in vitro reaction resembled authentic V(D)J recombination in being Ku-antigen-dependent.

Characterization of a genetically engineered mouse model of hemophilia A with complete deletion of the F8 gene.

PubMed

Chao, B N; Baldwin, W H; Healey, J F; Parker, E T; Shafer-Weaver, K; Cox, C; Jiang, P; Kanellopoulou, C; Lollar, P; Meeks, S L; Lenardo, M J

2016-02-01

ESSENTIALS: Anti-factor VIII (FVIII) inhibitory antibody formation is a severe complication in hemophilia A therapy. We genetically engineered and characterized a mouse model with complete deletion of the F8 coding region. F8(TKO) mice exhibit severe hemophilia, express no detectable F8 mRNA, and produce FVIII inhibitors. The defined background and lack of FVIII in F8(TKO) mice will aid in studying FVIII inhibitor formation. The most important complication in hemophilia A treatment is the development of inhibitory anti-Factor VIII (FVIII) antibodies in patients after FVIII therapy. Patients with severe hemophilia who express no endogenous FVIII (i.e. cross-reacting material, CRM) have the greatest incidence of inhibitor formation. However, current mouse models of severe hemophilia A produce low levels of truncated FVIII. The lack of a corresponding mouse model hampers the study of inhibitor formation in the complete absence of FVIII protein. We aimed to generate and characterize a novel mouse model of severe hemophilia A (designated the F8(TKO) strain) lacking the complete coding sequence of F8 and any FVIII CRM. Mice were created on a C57BL/6 background using Cre-Lox recombination and characterized using in vivo bleeding assays, measurement of FVIII activity by coagulation and chromogenic assays, and anti-FVIII antibody production using ELISA. All F8 exonic coding regions were deleted from the genome and no F8 mRNA was detected in F8(TKO) mice. The bleeding phenotype of F8(TKO) mice was comparable to E16 mice by measurements of factor activity and tail snip assay. Similar levels of anti-FVIII antibody titers after recombinant FVIII injections were observed between F8(TKO) and E16 mice. We describe a new C57BL/6 mouse model for severe hemophilia A patients lacking CRM. These mice can be directly bred to the many C57BL/6 strains of genetically engineered mice, which is valuable for studying the impact of a wide variety of genes on FVIII inhibitor formation on a defined genetic background. © 2015 International Society on Thrombosis and Haemostasis.

The substitution of cysteine 17 of recombinant human G-CSF with alanine greatly enhanced its stability.

PubMed

Ishikawa, M; Iijima, H; Satake-Ishikawa, R; Tsumura, H; Iwamatsu, A; Kadoya, T; Shimada, Y; Fukamachi, H; Kobayashi, K; Matsuki, S

1992-02-01

Human recombinant granulocyte-colony stimulating factor (rhG-CSF) has one free cysteine at position 17 and has two disulfide bridges (Cys36-Cys42 and Cys64-Cys74). The Cys17 of rhG-CSF was substituted with Gly, Ala, Ser, Ile, Tyr, Arg, and Pro, or deleted using site-directed mutagenesis in order to improve its thermostability. With the exception of Pro17-rhG-CSF, all mutant proteins retained biological activity which promotes the growth of mouse bone marrow cells in vitro. Among these mutant proteins, Ala17-rhG-CSF had more than 5 times higher stability than rhG-CSF. But Ser17-rhG-CSF had almost same stability as rhG-CSF and other mutant proteins had only lower stability.

Mdt1 Facilitates Efficient Repair of Blocked DNA Double-Strand Breaks and Recombinational Maintenance of Telomeres▿

PubMed Central

Pike, Brietta L.; Heierhorst, Jörg

2007-01-01

DNA recombination plays critical roles in DNA repair and alternative telomere maintenance. Here we show that absence of the SQ/TQ cluster domain-containing protein Mdt1 (Ybl051c) renders Saccharomyces cerevisiae particularly hypersensitive to bleomycin, a drug that causes 3′-phospho-glycolate-blocked DNA double-strand breaks (DSBs). mdt1Δ also hypersensitizes partially recombination-defective cells to camptothecin-induced 3′-phospho-tyrosyl protein-blocked DSBs. Remarkably, whereas mdt1Δ cells are unable to restore broken chromosomes after bleomycin treatment, they efficiently repair “clean” endonuclease-generated DSBs. Epistasis analyses indicate that MDT1 acts in the repair of bleomycin-induced DSBs by regulating the efficiency of the homologous recombination pathway as well as telomere-related functions of the KU complex. Moreover, mdt1Δ leads to severe synthetic growth defects with a deletion of the recombination facilitator and telomere-positioning factor gene CTF18 already in the absence of exogenous DNA damage. Importantly, mdt1Δ causes a dramatic shift from the usually prevalent type II to the less-efficient type I pathway of recombinational telomere maintenance in the absence of telomerase in liquid senescence assays. As telomeres resemble protein-blocked DSBs, the results indicate that Mdt1 acts in a novel blocked-end-specific recombination pathway that is required for the efficiency of both drug-induced DSB repair and telomerase-independent telomere maintenance. PMID:17636027

The first EGF domain of coagulation factor IX attenuates cell adhesion and induces apoptosis.

PubMed

Ishikawa, Tomomi; Kitano, Hisataka; Mamiya, Atsushi; Kokubun, Shinichiro; Hidai, Chiaki

2016-07-01

Coagulation factor IX (FIX) is an essential plasma protein for blood coagulation. The first epidermal growth factor (EGF) motif of FIX (EGF-F9) has been reported to attenuate cell adhesion to the extracellular matrix (ECM). The purpose of the present study was to determine the effects of this motif on cell adhesion and apoptosis. Treatment with a recombinant EGF-F9 attenuated cell adhesion to the ECM within 10 min. De-adhesion assays with native FIX recombinant FIX deletion mutant proteins suggested that the de-adhesion activity of EGF-F9 requires the same process of FIX activation as that which occurs for coagulation activity. The recombinant EGF-F9 increased lactate dehydrogenase (LDH) activity release into the medium and increased the number of cells stained with annexin V and activated caspase-3, by 8.8- and 2.7-fold respectively, indicating that EGF-F9 induced apoptosis. Activated caspase-3 increased very rapidly after only 5 min of administration of recombinant EGF-F9. Treatment with EGF-F9 increased the level of phosphorylated p38 mitogen-activated protein kinase (MAPK), but not that of phosphorylated MAPK 44/42 or c-Jun N-terminal kinase (JNK). Inhibitors of caspase-3 suppressed the release of LDH. Caspase-3 inhibitors also suppressed the attenuation of cell adhesion and phosphorylation of p38 MAPK by EGF-F9. Our data indicated that EGF-F9 activated signals for apoptosis and induced de-adhesion in a caspase-3 dependent manner. © 2016 The Author(s).

Experimental evidence that RNA recombination occurs in the Japanese encephalitis virus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chuang, C.-K.; Chen, W.-J., E-mail: wjchen@mail.cgu.edu.t; Department of Public Health and Parasitology, Chang Gung University, Kwei-San, Tao-Yuan 33332, Taiwan

2009-11-25

Due to the lack of a proofreading function and error-repairing ability of genomic RNA, accumulated mutations are known to be a force driving viral evolution in the genus Flavivirus, including the Japanese encephalitis (JE) virus. Based on sequencing data, RNA recombination was recently postulated to be another factor associated with genomic variations in these viruses. We herein provide experimental evidence to demonstrate the occurrence of RNA recombination in the JE virus using two local pure clones (T1P1-S1 and CJN-S1) respectively derived from the local strains, T1P1 and CJN. Based on results from a restriction fragment length polymorphism (RFLP) assay onmore » the C/preM junction comprising a fragment of 868 nucleotides (nt 10-877), the recombinant progeny virus was primarily formed in BHK-21 cells that had been co-infected with the two clones used in this study. Nine of 20 recombinant forms of the JE virus had a crossover in the nt 123-323 region. Sequencing data derived from these recombinants revealed that no nucleotide deletion or insertion occurred in this region favoring crossovers, indicating that precisely, not aberrantly, homologous recombination was involved. With site-directed mutagenesis, three stem-loop secondary structures were destabilized and re-stabilized in sequence, leading to changes in the frequency of recombination. This suggests that the conformation, not the free energy, of the secondary structure is important in modulating RNA recombination of the virus. It was concluded that because RNA recombination generates genetic diversity in the JE virus, this must be considered particularly in studies of viral evolution, epidemiology, and possible vaccine safety.« less

[Construction and characterization of enterohemorrhagic Escherichia coli O157:H7 ppk- deleted strain].

PubMed

Han, Peng; Sun, Qi; Zhao, Suhui; Zhang, Qiwei; Wan, Chengsong

2014-06-01

To construct enterohemorrhagic Escherichia coli (EHEC) O157: H7 ppk gene deletion strains and study its biological characteristics. The gene fragment of kanamycin resistance was amplified using a pair of homologous arm primers whose 5' and 3' ends were homologous with ppk gene and kanamycin resistance gene, respectively. EHEC O157: H7 EDL933w competent strains were prepared and transformed via electroporation with the amplification products. The ppk gene was replaced by kanamycin resistance gene using pKD46-mediated Red recombination system. The recombinant strain was confirmed by PCR and sequencing, and its morphology, growth ability and adhesion were assessed using Gram staining, OD600 value and Giemsa staining. We established a ppk-deleted EHEC O157:H7 EDL933w strain with kanamycin resistance and compared the biological characteristics of the wild-type and mutant strains, which may facilitate further study of the regulatory mechanism of ppk gene.

[Recombinant chromosome 4 with partial 4p deletion and 4q duplication inherited from paternal pericentric inversion].

PubMed

Mun, Se Jin; Cho, Eun Hae; Chey, Myoung-Jae; Shim, Gyu-Hong; Shin, Bo-Moon; Lee, Rae-Kyung; Ko, Ji-Kyung; Yoo, Soo Jin

2010-02-01

Pericentric inversion of chromosome 4 can give rise to 2 alternate recombinant (rec) chromosomesby duplication or deletion of 4p. The deletion of distal 4p manifests as Wolf-Hirschhorn syndrome (WHS). Here, we report the molecular cytogenetic findings and clinical manifestations observed in an infant with 46,XX,rec(4)dup(4q)inv(4)(p16q31.3)pat. The infant was delivered by Cesarean section at the 33rd week of gestation because pleural effusion and polyhydramnios were detected on ultrasonography. At birth, the infant showed no malformation or dysfunction, except for a preauricular skin tag. Array comparative genomic hybridization analysis of neonatal peripheral blood samples showed a gain of 38 Mb on 4q31.3-qter and a loss of 3 Mb on 4p16.3, and these results were consistent with WHS. At the last follow-up at 8 months of age (corrected age, 6 months), the infant had not achieved complete head control.

Identification and characterization of novel mutations of the major Fanconi anemia gene FANCA in the Japanese population.

PubMed

Yagasaki, Hiroshi; Hamanoue, Satoshi; Oda, Tsukasa; Nakahata, Tatsutoshi; Asano, Shigetaka; Yamashita, Takayuki

2004-12-01

Fanconi anemia (FA) is a rare autosomal recessive disorder of hematopoiesis, with at least 11 complementation groups. FANCA, a gene for group A, accounts for the majority of FA patients. Previous studies of FANCA mutations revealed high allelic heterogeneity, frequent occurrence of large deletions, and interpopulation differences. However, systematic mutational analysis, including gene dosage assay to detect large deletions, has not been documented for Asian populations. A newly developed TaqMan quantitative PCR-based gene dosage assay, combined with sequencing of exons and cDNA fragments, allowed for detection of 48 mutant alleles of FANCA in 27 (77%) of 35 unrelated Japanese FA families with no detectable mutations in FANCC or FANCG. We identified 29 different mutations (21 nucleotide substitutions or small deletions/insertions and eight large deletions), at least 20 of which were novel. The FANCA mutational spectrum of the Japanese was different from that of other ethnic groups so far studied. This is the largest scale of mutation analysis of FANCA in the Japanese population. Characterization of these mutations provided new information regarding the mutagenesis mechanisms and structure-function relationship of FANCA. Specifically, our data suggest that diverse mechanisms including nonhomologous recombination as well as Alu-mediated homologous recombination are involved in the generation of large deletions in FANCA. Copyright 2004 Wiley-Liss, Inc.

An Improved Single-Step Cloning Strategy Simplifies the Agrobacterium tumefaciens-Mediated Transformation (ATMT)-Based Gene-Disruption Method for Verticillium dahliae.

PubMed

Wang, Sheng; Xing, Haiying; Hua, Chenlei; Guo, Hui-Shan; Zhang, Jie

2016-06-01

The soilborne fungal pathogen Verticillium dahliae infects a broad range of plant species to cause severe diseases. The availability of Verticillium genome sequences has provided opportunities for large-scale investigations of individual gene function in Verticillium strains using Agrobacterium tumefaciens-mediated transformation (ATMT)-based gene-disruption strategies. Traditional ATMT vectors require multiple cloning steps and elaborate characterization procedures to achieve successful gene replacement; thus, these vectors are not suitable for high-throughput ATMT-based gene deletion. Several advancements have been made that either involve simplification of the steps required for gene-deletion vector construction or increase the efficiency of the technique for rapid recombinant characterization. However, an ATMT binary vector that is both simple and efficient is still lacking. Here, we generated a USER-ATMT dual-selection (DS) binary vector, which combines both the advantages of the USER single-step cloning technique and the efficiency of the herpes simplex virus thymidine kinase negative-selection marker. Highly efficient deletion of three different genes in V. dahliae using the USER-ATMT-DS vector enabled verification that this newly-generated vector not only facilitates the cloning process but also simplifies the subsequent identification of fungal homologous recombinants. The results suggest that the USER-ATMT-DS vector is applicable for efficient gene deletion and suitable for large-scale gene deletion in V. dahliae.

Identification of defective illegitimate recombinational repair of oxidatively-induced DNA double-strand breaks in ataxia-telangiectasia cells

NASA Technical Reports Server (NTRS)

Dar, M. E.; Winters, T. A.; Jorgensen, T. J.

1997-01-01

Ataxia-telangiectasia (A-T) is an autosomal-recessive lethal human disease. Homozygotes suffer from a number of neurological disorders, as well as very high cancer incidence. Heterozygotes may also have a higher than normal risk of cancer, particularly for the breast. The gene responsible for the disease (ATM) has been cloned, but its role in mechanisms of the disease remain unknown. Cellular A-T phenotypes, such as radiosensitivity and genomic instability, suggest that a deficiency in the repair of DNA double-strand breaks (DSBs) may be the primary defect; however, overall levels of DSB rejoining appear normal. We used the shuttle vector, pZ189, containing an oxidatively-induced DSB, to compare the integrity of DSB rejoining in one normal and two A-T fibroblast cells lines. Mutation frequencies were two-fold higher in A-T cells, and the mutational spectrum was different. The majority of the mutations found in all three cell lines were deletions (44-63%). The DNA sequence analysis indicated that 17 of the 17 plasmids with deletion mutations in normal cells occurred between short direct-repeat sequences (removing one of the repeats plus the intervening sequences), implicating illegitimate recombination in DSB rejoining. The combined data from both A-T cell lines showed that 21 of 24 deletions did not involve direct-repeats sequences, implicating a defect in the illegitimate recombination pathway. These findings suggest that the A-T gene product may either directly participate in illegitimate recombination or modulate the pathway. Regardless, this defect is likely to be important to a mechanistic understanding of this lethal disease.

Properties of a herpes simplex virus multiple immediate-early gene-deleted recombinant as a vaccine vector

DOE Office of Scientific and Technical Information (OSTI.GOV)

Watanabe, Daisuke; Brockman, Mark A.; Ndung'u, Thumbi

2007-01-20

Herpes simplex virus (HSV) recombinants induce durable immune responses in rhesus macaques and mice and have induced partial protection in rhesus macaques against mucosal challenge with virulent simian immunodeficiency virus (SIV). In this study, we evaluated the properties of a new generation HSV vaccine vector, an HSV-1 multiple immediate-early (IE) gene deletion mutant virus, d106, which contains deletions in the ICP4, ICP27, ICP22, and ICP47 genes. Because several of the HSV IE genes have been implicated in immune evasion, inactivation of the genes encoding these proteins was expected to result in enhanced immunogenicity. The d106 virus expresses few HSV genemore » products and shows minimal cytopathic effect in cultured cells. When d106 was inoculated into mice, viral DNA accumulated at high levels in draining lymph nodes, consistent with an ability to transduce dendritic cells and activate their maturation and movement to lymph nodes. A d106 recombinant expressing Escherichia coli {beta}-galactosidase induced durable {beta}-gal-specific IgG and CD8{sup +} T cell responses in naive and HSV-immune mice. Finally, d106-based recombinants have been constructed that express simian immunodeficiency virus (SIV) gag, env, or a rev-tat-nef fusion protein for several days in cultured cells. Thus, d106 shows many of the properties desirable in a vaccine vector: limited expression of HSV gene products and cytopathogenicity, high level expression of transgenes, ability to induce durable immune responses, and an ability to transduce dendritic cells and induce their maturation and migration to lymph nodes.« less

Establishment of a Cre recombinase based mutagenesis protocol for markerless gene deletion in Streptococcus suis.

PubMed

Koczula, A; Willenborg, J; Bertram, R; Takamatsu, D; Valentin-Weigand, P; Goethe, R

2014-12-01

The lack of knowledge about pathogenicity mechanisms of Streptococcus (S.) suis is, at least partially, attributed to limited methods for its genetic manipulation. Here, we established a Cre-lox based recombination system for markerless gene deletions in S. suis serotype 2 with high selective pressure and without undesired side effects. Copyright © 2014 Elsevier B.V. All rights reserved.

A critical developmental role for tgfbr2 in myogenic cell lineages is revealed in mice expressing SM22-Cre, not SMMHC-Cre.

PubMed

Frutkin, Andrew D; Shi, Haikun; Otsuka, Goro; Levéen, Per; Karlsson, Stefan; Dichek, David A

2006-10-01

Smooth muscle cell (SMC)-specific deletion of transforming growth factor beta (TGF-beta) signaling would help elucidate the mechanisms through which TGF-beta signaling contributes to vascular development and disease. We attempted to generate mice with SMC-specific deletion of TGF-beta signaling by mating mice with a conditional ("floxed") allele for the type II TGF-beta receptor (tgfbr2flox) to mice with SMC-targeted expression of Cre recombinase. We bred male mice transgenic for smooth muscle myosin heavy chain (SMMHC)-Cre with females carrying tgfbr2flox. Surprisingly, SMMHC-Cre mice recombined tgfbr2flox at low levels in SMC and at high levels in the testis. Recombination of tgfbr2flox in testis correlated with high-level expression of SMMHC-Cre in testis and germline transmission of tgfbr2null. In contrast, mice expressing Cre from a SM22alpha promoter (SM22-Cre) efficiently recombined tgfbr2flox in vascular and visceral SMC and the heart, but not in testis. Use of the R26R reporter allele confirmed that Cre-mediated recombination in vascular SMC was inefficient for SMMHC-Cre mice and highly efficient for SM22-Cre mice. Breedings that introduced the SM22-Cre allele into tgfbr2flox/flox zygotes in order to generate adult mice that are hemizygous for SM22-Cre and homozygous for tgfbr2flox- and would have conversion of tgfbr2flox/flox to tgfbr2null/null in SMC-produced no live SM22-Cre : tgfbr2flox/flox pups (P<0.001). We conclude: (1) "SMC-targeted" Cre lines vary significantly in specificity and efficiency of Cre expression; (2) TGF-beta signaling in the subset of cells that express SM22alpha is required for normal development; (3) generation of adult mice with absent TGF-beta signaling in SMC remains a challenge.

A recombination outside the BB deletion refines the location of the X-linked retinitis pigmentosa locus RP3

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fujita, R.; Bingham, E.; Forsythe, P.

1996-07-01

Genetic loci for X-linked retinitis pigmentosa (XLRP) have been mapped between Xp11.22 and Xp22.13 (RP2, RP3, RP6, and RP15). The RP3 gene, which is responsible for the predominant form of XLRP in most Caucasian populations, has been localized to Xp21.1 by linkage analysis and the map positions of chromosomal deletions associated with the disease. Previous linkage studies have suggested that RP3 is flanked by the markers DXS1110 (distal) and OTC (proximal). Patient BB was though to have RP because of a lesion at the RP3 locus, in addition to chronic granulomatous disease, Duchenne muscular dystrophy (DMD), mild mental retardation, andmore » the McLeod phenotype. This patient carried a deletion extending {approximately}3 Mb from DMD in Xp21.3 to Xp21.1, with the proximal breakpoint located {approximately}40 kb centromeric to DXS1110. The RP3 gene, therefore, is believed to reside between DXS1110 and the proximal breakpoint of the BB deletion. In order to refine the location of RP3 and to ascertain patients with RP3, we have been analyzing several XLRP families for linkage to Xp markers. Linkage analysis in an American family of 27 individuals demonstrates segregation of XLRP with markers in Xp21.1, consistent with the RP3 subtype. One affected male shows a recombination event proximal to DXS1110. Additional markers within the DXS1110-OTC interval show that the crossover is between two novel polymorphic markers, DXS8349 and M6, both of which are present in BB DNA and lie centromeric to the proximal breakpoint. This recombination places the XLRP mutation in this family outside the BB deletion and redefines the location of RP3. 22 refs., 3 figs., 2 tabs.« less

Mouse Hepatitis Virus Strain A59 and Blocking Antireceptor Monoclonal Antibody Bind to the N-Terminal Domain of Cellular Receptor

NASA Astrophysics Data System (ADS)

Dveksler, Gabriela S.; Pensiero, Michael N.; Dieffenbach, Carl W.; Cardellichio, Christine B.; Basile, Alexis A.; Elia, Patrick E.; Holmes, Kathryn V.

1993-03-01

Mouse hepatitis virus (MHV) strain A59 uses as cellular receptors members of the carcinoembryonic antigen family in the immunoglobulin superfamily. Recombinant receptor proteins with deletions of whole or partial immunoglobulin domains were used to identify the regions of receptor glycoprotein recognized by virus and by antireceptor monoclonal antibody CC1, which blocks infection of murine cells. Monoclonal antibody CC1 and MHV-A59 virions bound only to recombinant proteins containing the entire first domain of MHV receptor. To determine which of the proteins could serve as functional virus receptors, receptor-negative hamster cells were transfected with recombinant deletion clones and then challenged with MHV-A59 virions. Receptor activity required the entire N-terminal domain with either the second or the fourth domain and the transmembrane and cytoplasmic domains. Recombinant proteins lacking the first domain or its C-terminal portion did not serve as viral receptors. Thus, like other virus receptors in the immunoglobulin superfamily, including CD4, poliovirus receptor, and intercellular adhesion molecule 1, the N-terminal domain of MHV receptor is recognized by the virus and the blocking monoclonal antibody.

Nonhomologous recombination between defective poliovirus and coxsackievirus genomes suggests a new model of genetic plasticity for picornaviruses.

PubMed

Holmblat, Barbara; Jégouic, Sophie; Muslin, Claire; Blondel, Bruno; Joffret, Marie-Line; Delpeyroux, Francis

2014-08-05

Most of the circulating vaccine-derived polioviruses (cVDPVs) implicated in poliomyelitis outbreaks in Madagascar have been shown to be recombinants between the type 2 poliovirus (PV) strain of the oral polio vaccine (Sabin 2) and another species C human enterovirus (HEV-C), such as type 17 coxsackie A virus (CA17) in particular. We studied intertypic genetic exchanges between PV and non-PV HEV-C by developing a recombination model, making it possible to rescue defective type 2 PV RNA genomes with a short deletion at the 3' end by the cotransfection of cells with defective or infectious CA17 RNAs. We isolated over 200 different PV/CA17 recombinants, using murine cells expressing the human PV receptor (PVR) and selecting viruses with PV capsids. We found some homologous (H) recombinants and, mostly, nonhomologous (NH) recombinants presenting duplications of parental sequences preferentially located in the regions encoding proteins 2A, 2B, and 3A. Short duplications appeared to be stable, whereas longer duplications were excised during passaging in cultured cells or after multiplication in PVR-transgenic mice, generating H recombinants with diverse sites of recombination. This suggests that NH recombination events may be a transient, intermediate step in the generation and selection of the fittest H recombinants. In addition to the classical copy-choice mechanism of recombination thought to generate mostly H recombinants, there may also be a modular mechanism of recombination, involving NH recombinant precursors, shaping the genomes of recombinant enteroviruses and other picornaviruses. Importance: The multiplication of circulating vaccine-derived polioviruses (cVDPVs) in poorly immunized human populations can render these viruses pathogenic, causing poliomyelitis outbreaks. Most cVDPVs are intertypic recombinants between a poliovirus (PV) strain and another human enterovirus, such as type 17 coxsackie A viruses (CA17). For further studies of the genetic exchanges between PV and CA17, we have developed a model of recombination, making it possible to rescue defective PV RNA genomes with a short deletion by cotransfecting cells with the defective PV genome and CA17 genomic RNA. Numerous recombinants were found, including homologous PV/CA17 recombinants, but mostly nonhomologous recombinants presenting duplications of parental sequences preferentially located in particular regions. Long duplications were excised by passages in cultured cells or in mice, generating diverse homologous recombinants. Recombination leading to nonhomologous recombinants, which evolve into homologous recombinants, may therefore be seen as a model of genetic plasticity in enteroviruses and, possibly, in other RNA viruses. Copyright © 2014 Holmblat et al.

Recombinant chromosome 7 in a mosaic 45,X/47,XXX patient.

PubMed

Tirado, Carlos A; Gotway, Garrett; Torgbe, Emmanuel; Iyer, Santha; Dallaire, Stephanie; Appleberry, Taylor; Suterwala, Mohamed; Garcia, Rolando; Valdez, Federico; Patel, Sangeeta; Koduru, Prasad

2012-01-01

Individuals with pericentric inversions are at risk for producing offspring with chromosomal gains and losses, while those carrying paracentric inversions usually produce unviable gametes [Madan, 1995]. In this current study, we present a newborn with dysmorphic features and malformations, whose karyotype showed an abnormal copy of chromomosome 7 described at first as add(7)(q32) as well as mos 45,X/47,XXX. Array comparative genomic hybridization (CGH) revealed an interstitial deletion in the long arm of chromosome 7 involving bands q35 to q36.3 but retaining the 7q subtelomere. The patient's deletion is believed to be due to meiotic recombination in the inversion loop in the phenotypically normal father who seems to carry two paracentric inversions in the long arm of chromosome 7, which was described as rec(7)(7pter- > q35::q36.3- > 7qter)pat. The abnormal copy of chromosome 7 in the father has been described as: der(7)(7pter- > q22.1::q36.3- > q35::q22.1- > q35::q36.3- > 7qter). This is a unique karyotype that to our knowledge has not been previously reported in the literature and predisposes to meiotic recombination that can result in deletions or duplications of 7q35-36. Copyright © 2011 Wiley Periodicals, Inc.

The contribution of alu elements to mutagenic DNA double-strand break repair.

PubMed

Morales, Maria E; White, Travis B; Streva, Vincent A; DeFreece, Cecily B; Hedges, Dale J; Deininger, Prescott L

2015-03-01

Alu elements make up the largest family of human mobile elements, numbering 1.1 million copies and comprising 11% of the human genome. As a consequence of evolution and genetic drift, Alu elements of various sequence divergence exist throughout the human genome. Alu/Alu recombination has been shown to cause approximately 0.5% of new human genetic diseases and contribute to extensive genomic structural variation. To begin understanding the molecular mechanisms leading to these rearrangements in mammalian cells, we constructed Alu/Alu recombination reporter cell lines containing Alu elements ranging in sequence divergence from 0%-30% that allow detection of both Alu/Alu recombination and large non-homologous end joining (NHEJ) deletions that range from 1.0 to 1.9 kb in size. Introduction of as little as 0.7% sequence divergence between Alu elements resulted in a significant reduction in recombination, which indicates even small degrees of sequence divergence reduce the efficiency of homology-directed DNA double-strand break (DSB) repair. Further reduction in recombination was observed in a sequence divergence-dependent manner for diverged Alu/Alu recombination constructs with up to 10% sequence divergence. With greater levels of sequence divergence (15%-30%), we observed a significant increase in DSB repair due to a shift from Alu/Alu recombination to variable-length NHEJ which removes sequence between the two Alu elements. This increase in NHEJ deletions depends on the presence of Alu sequence homeology (similar but not identical sequences). Analysis of recombination products revealed that Alu/Alu recombination junctions occur more frequently in the first 100 bp of the Alu element within our reporter assay, just as they do in genomic Alu/Alu recombination events. This is the first extensive study characterizing the influence of Alu element sequence divergence on DNA repair, which will inform predictions regarding the effect of Alu element sequence divergence on both the rate and nature of DNA repair events.

N-linked oligosaccharides on chondroitin 6-sulfotransferase-1 are required for production of the active enzyme, Golgi localization, and sulfotransferase activity toward keratan sulfate.

PubMed

Yusa, Akiko; Kitajima, Ken; Habuchi, Osami

2006-07-21

We have shown previously that purified chondroitin 6-sulfotransferase-1 (C6ST-1) was a glycoprotein abundant in N-linked oligosaccharides and could sulfate both chondroitin (C6ST activity) and keratan sulfate (KSST activity); however, functional roles of the N-glycans have remained unclear. In the present study, we show essential roles of N-glycans attached to C6ST-1 in the generation of the active enzyme and in its KSST activity. Treatment with tunicamycin of COS-7 cells transfected with C6ST-1 cDNA totally abolished production of the active C6ST-1. A nearly complete removal of N-glycans of the recombinant C6ST-1 by peptide N-glycosidase F increased the C6ST activity but decreased the KSST activity. Among six potential N-glycosylation sites, deletion of the fourth or sixth site from the amino terminus inhibited production of the active C6ST-1, whereas deletion of the fifth site resulted in a marked loss of the KSST activity. Wild-type recombinant C6ST-1 showed a typical Golgi localization, whereas M-4 recombinant C6ST-1, in which the fourth N-glycosylation site was deleted, colocalized with calnexin, an endoplasmic reticulum-resident protein. Unlike wildtype recombinant C6ST-1, M-4 recombinant C6ST-1 showed a weak affinity toward wheat germ agglutinin and was converted completely to the nonglycosylated form by endoglycosidase H. These observations suggest that N-glycan attached to the fourth N-glycosylation site may function in the proper processing of N-glycans required for the Golgi localization, thereby causing the production of the active C6ST-1, and that N-glycan attached to the fifth N-glycosylation site may contribute to the KSST activity of C6ST-1.

The amyR-deletion strain of Aspergillus niger CICC2462 is a suitable host strain to express secreted protein with a low background.

PubMed

Zhang, Hui; Wang, Shuang; Zhang, Xiang Xiang; Ji, Wei; Song, Fuping; Zhao, Yue; Li, Jie

2016-04-28

The filamentous fungus Aspergillus niger is widely exploited as an important expression host for industrial production. The glucoamylase high-producing strain A. niger CICC2462 has been used as a host strain for the establishment of a secretion expression system. It expresses recombinant xylanase, mannase and asparaginase at a high level, but some high secretory background proteins in these recombinant strains still remain, such as alpha-amylase and alpha-glucosidase; lead to a low-purity of fermentation products. The aim was to construct an A. niger host strain with a low background of protein secretion. The transcription factor amyR was deleted in A. niger CICC2462, and the results from enzyme activity assays and SDS-PAGE analysis showed that the glucoamylase and amylase activities of the ∆amyR strains were significantly lower than those of the wild-type strain. High-throughput RNA-sequencing and shotgun LC-MS/MS proteomic technology analysis demonstrated that the expression of amylolytic enzymes was decreased at both the transcriptional and translational levels in the ∆amyR strain. Interestingly, the ∆amyR strain growth rate better than the wild-type strain. Our findings clearly indicated that the ∆amyR strain of A. niger CICC2462 can be used as a host strain with a low background of protein secretion.

Biochemical and functional characterization of a recombinant monomeric factor VIII-Fc fusion protein.

PubMed

Peters, R T; Toby, G; Lu, Q; Liu, T; Kulman, J D; Low, S C; Bitonti, A J; Pierce, G F

2013-01-01

Hemophilia A results from a deficiency in factor VIII activity. Current treatment regimens require frequent dosing, owing to the short half-life of FVIII. A recombinant FVIII-Fc fusion protein (rFVIIIFc) was molecularly engineered to increase the half-life of FVIII, by 1.5-2-fold, in several preclinical animal models and humans. To perform a biochemical and functional in vitro characterization of rFVIIIFc, with existing FVIII products as comparators.  rFVIIIFc was examined by utilizing a series of structural and analytic assays, including mass spectrometry following lysyl endopeptidase or thrombin digestion. rFVIIIFc activity was determined in both one-stage clotting (activated partial thromboplastin time) and chromogenic activity assays, in the context of the FXase complex with purified components, and in both in vitro and ex vivo rotational thromboelastometry (ROTEM) assays performed in whole blood.  rFVIIIFc contained the predicted primary structure and post-translational modifications, with an FVIII moiety that was similar to other recombinant FVIII products. The von Willebrand factor-binding and specific activity of rFVIIIFc were also found to be similar to those of other recombinant FVIII molecules. Both chromogenic and one-stage assays of rFVIIIFc gave similar results. Ex vivo ROTEM studies demonstrated that circulating rFVIIIFc activity was prolonged in mice with hemophilia A in comparison with B-domain-deleted or full-length FVIII. Clot parameters at early time points were similar to those for FVIII, whereas rFVIIIFc showed prolonged improvement of clot formation.  rFVIIIFc maintains normal FVIII interactions with other proteins necessary for its activity, with prolonged in vivo activity, owing to fusion with the Fc region of IgG(1) . © 2012 International Society on Thrombosis and Haemostasis.

The CCR4-NOT Complex Is Implicated in the Viability of Aneuploid Yeasts

PubMed Central

Tange, Yoshie; Kurabayashi, Atsushi; Goto, Bunshiro; Hoe, Kwang-Lae; Kim, Dong-Uk; Park, Han-Oh; Hayles, Jacqueline; Chikashige, Yuji; Tsutumi, Chihiro; Hiraoka, Yasushi; Yamao, Fumiaki; Nurse, Paul; Niwa, Osami

2012-01-01

To identify the genes required to sustain aneuploid viability, we screened a deletion library of non-essential genes in the fission yeast Schizosaccharomyces pombe, in which most types of aneuploidy are eventually lethal to the cell. Aneuploids remain viable for a period of time and can form colonies by reducing the extent of the aneuploidy. We hypothesized that a reduction in colony formation efficiency could be used to screen for gene deletions that compromise aneuploid viability. Deletion mutants were used to measure the effects on the viability of spores derived from triploid meiosis and from a chromosome instability mutant. We found that the CCR4-NOT complex, an evolutionarily conserved general regulator of mRNA turnover, and other related factors, including poly(A)-specific nuclease for mRNA decay, are involved in aneuploid viability. Defective mutations in CCR4-NOT complex components in the distantly related yeast Saccharomyces cerevisiae also affected the viability of spores produced from triploid cells, suggesting that this complex has a conserved role in aneuploids. In addition, our findings suggest that the genes required for homologous recombination repair are important for aneuploid viability. PMID:22737087

P53 Suppression of Homologous Recombination and Tumorigenesis

DTIC Science & Technology

2012-01-01

mutation acted on both rad51 dependent gene conversion events and deletion events (6). Willers et al. also showed an increase in recombination...suffer from sarcomas. MEFs from these mice show aneuploidy, allelic loss and gene amplification. Most of these germline mutations are missense...the absence of tumor suppressor gene activity, such as p53, results in increased genomic instability and increased cancer predisposition

Analysis of the functional consequences of targeted exon deletion in COL7A1 reveals prospects for dystrophic epidermolysis bullosa therapy

PubMed Central

Bornert, Olivier; Kühl, Tobias; Bremer, Jeroen; van den Akker, Peter C; Pasmooij, Anna MG; Nyström, Alexander

2016-01-01

Genetically evoked deficiency of collagen VII causes dystrophic epidermolysis bullosa (DEB)—a debilitating disease characterized by chronic skin fragility and progressive fibrosis. Removal of exons carrying frame-disrupting mutations can reinstate protein expression in genetic diseases. The therapeutic potential of this approach is critically dependent on gene, protein, and disease intrinsic factors. Naturally occurring exon skipping in COL7A1, translating collagen VII, suggests that skipping of exons containing disease-causing mutations may be feasible for the treatment of DEB. However, despite a primarily in-frame arrangement of exons in the COL7A1 gene, no general conclusion of the aptitude of exon skipping for DEB can be drawn, since regulation of collagen VII functionality is complex involving folding, intra- and intermolecular interactions. To directly address this, we deleted two conceptually important exons located at both ends of COL7A1, exon 13, containing recurrent mutations, and exon 105, predicted to impact folding. The resulting recombinantly expressed proteins showed conserved functionality in biochemical and in vitro assays. Injected into DEB mice, the proteins promoted skin stability. By demonstrating functionality of internally deleted collagen VII variants, our study provides support of targeted exon deletion or skipping as a potential therapy to treat a large number of individuals with DEB. PMID:27157667

A novel non prophage(-like) gene-intervening element within gerE that is reconstituted during sporulation in Bacillus cereus ATCC10987.

PubMed

Abe, Kimihiro; Shimizu, Shin-Ya; Tsuda, Shuhei; Sato, Tsutomu

2017-09-12

Gene rearrangement is a widely-shared phenomenon in spore forming bacteria, in which prophage(-like) elements interrupting sporulation-specific genes are excised from the host genome to reconstitute the intact gene. Here, we report a novel class of gene-intervening elements, named gin, inserted in the 225 bp gerE-coding region of the B. cereus ATCC10987 genome, which generates a sporulation-specific rearrangement. gin has no phage-related genes and possesses three site-specific recombinase genes; girA, girB, and girC. We demonstrated that the gerE rearrangement occurs at the middle stage of sporulation, in which site-specific DNA recombination took place within the 9 bp consensus sequence flanking the disrupted gerE segments. Deletion analysis of gin uncovered that GirC and an additional factor, GirX, are responsible for gerE reconstitution. Involvement of GirC and GirX in DNA recombination was confirmed by an in vitro recombination assay. These results broaden the definition of the sporulation-specific gene rearrangement phenomenon: gene-intervening elements are not limited to phage DNA but may include non-viral genetic elements that carry a developmentally-regulated site-specific recombination system.

Small rare recurrent deletions and reciprocal duplications in 2q21.1, including brain-specific ARHGEF4 and GPR148

PubMed Central

Dharmadhikari, Avinash V.; Kang, Sung-Hae L.; Szafranski, Przemyslaw; Person, Richard E.; Sampath, Srirangan; Prakash, Siddharth K.; Bader, Patricia I.; Phillips, John A.; Hannig, Vickie; Williams, Misti; Vinson, Sherry S.; Wilfong, Angus A.; Reimschisel, Tyler E.; Craigen, William J.; Patel, Ankita; Bi, Weimin; Lupski, James R.; Belmont, John; Cheung, Sau Wai; Stankiewicz, Pawel

2012-01-01

We have identified a rare small (∼450 kb unique sequence) recurrent deletion in a previously linked attention-deficit hyperactivity disorder (ADHD) locus at 2q21.1 in five unrelated families with developmental delay (DD)/intellectual disability (ID), ADHD, epilepsy and other neurobehavioral abnormalities from 17 035 samples referred for clinical chromosomal microarray analysis. Additionally, a DECIPHER (http://decipher.sanger.ac.uk) patient 2311 was found to have the same deletion and presented with aggressive behavior. The deletion was not found in either six control groups consisting of 13 999 healthy individuals or in the DGV database. We have also identified reciprocal duplications in five unrelated families with autism, developmental delay (DD), seizures and ADHD. This genomic region is flanked by large, complex low-copy repeats (LCRs) with directly oriented subunits of ∼109 kb in size that have 97.7% DNA sequence identity. We sequenced the deletion breakpoints within the directly oriented paralogous subunits of the flanking LCR clusters, demonstrating non-allelic homologous recombination as a mechanism of formation. The rearranged segment harbors five genes: GPR148, FAM123C, ARHGEF4, FAM168B and PLEKHB2. Expression of ARHGEF4 (Rho guanine nucleotide exchange factor 4) is restricted to the brain and may regulate the actin cytoskeletal network, cell morphology and migration, and neuronal function. GPR148 encodes a G-protein-coupled receptor protein expressed in the brain and testes. We suggest that small rare recurrent deletion of 2q21.1 is pathogenic for DD/ID, ADHD, epilepsy and other neurobehavioral abnormalities and, because of its small size, low frequency and more severe phenotype might have been missed in other previous genome-wide screening studies using single-nucleotide polymorphism analyses. PMID:22543972

Increased ethanol production by deletion of HAP4 in recombinant xylose-assimilating Saccharomyces cerevisiae.

PubMed

Matsushika, Akinori; Hoshino, Tamotsu

2015-12-01

The Saccharomyces cerevisiae HAP4 gene encodes a transcription activator that plays a key role in controlling the expression of genes involved in mitochondrial respiration and reductive pathways. This work examines the effect of knockout of the HAP4 gene on aerobic ethanol production in a xylose-utilizing S. cerevisiae strain. A hap4-deleted recombinant yeast strain (B42-DHAP4) showed increased maximum concentration, production rate, and yield of ethanol compared with the reference strain MA-B42, irrespective of cultivation medium (glucose, xylose, or glucose/xylose mixtures). Notably, B42-DHAP4 was capable of producing ethanol from xylose as the sole carbon source under aerobic conditions, whereas no ethanol was produced by MA-B42. Moreover, the rate of ethanol production and ethanol yield (0.44 g/g) from the detoxified hydrolysate of wood chips was markedly improved in B42-DHAP4 compared to MA-B42. Thus, the results of this study support the view that deleting HAP4 in xylose-utilizing S. cerevisiae strains represents a useful strategy in ethanol production processes.

Copy number variation at the 7q11.23 segmental duplications is a susceptibility factor for the Williams-Beuren syndrome deletion

PubMed Central

Cuscó, Ivon; Corominas, Roser; Bayés, Mònica; Flores, Raquel; Rivera-Brugués, Núria; Campuzano, Victoria; Pérez-Jurado, Luis A.

2008-01-01

Large copy number variants (CNVs) have been recently found as structural polymorphisms of the human genome of still unknown biological significance. CNVs are significantly enriched in regions with segmental duplications or low-copy repeats (LCRs). Williams-Beuren syndrome (WBS) is a neurodevelopmental disorder caused by a heterozygous deletion of contiguous genes at 7q11.23 mediated by nonallelic homologous recombination (NAHR) between large flanking LCRs and facilitated by a structural variant of the region, a ∼2-Mb paracentric inversion present in 20%–25% of WBS-transmitting progenitors. We now report that eight out of 180 (4.44%) WBS-transmitting progenitors are carriers of a CNV, displaying a chromosome with large deletion of LCRs. The prevalence of this CNV among control individuals and non-transmitting progenitors is much lower (1%, n = 600), thus indicating that it is a predisposing factor for the WBS deletion (odds ratio 4.6-fold, P = 0.002). LCR duplications were found in 2.22% of WBS-transmitting progenitors but also in 1.16% of controls, which implies a non–statistically significant increase in WBS-transmitting progenitors. We have characterized the organization and breakpoints of these CNVs, encompassing ∼100–300 kb of genomic DNA and containing several pseudogenes but no functional genes. Additional structural variants of the region have also been defined, all generated by NAHR between different blocks of segmental duplications. Our data further illustrate the highly dynamic structure of regions rich in segmental duplications, such as the WBS locus, and indicate that large CNVs can act as susceptibility alleles for disease-associated genomic rearrangements in the progeny. PMID:18292220

Simultaneous non-contiguous deletions using large synthetic DNA and site-specific recombinases

PubMed Central

Krishnakumar, Radha; Grose, Carissa; Haft, Daniel H.; Zaveri, Jayshree; Alperovich, Nina; Gibson, Daniel G.; Merryman, Chuck; Glass, John I.

2014-01-01

Toward achieving rapid and large scale genome modification directly in a target organism, we have developed a new genome engineering strategy that uses a combination of bioinformatics aided design, large synthetic DNA and site-specific recombinases. Using Cre recombinase we swapped a target 126-kb segment of the Escherichia coli genome with a 72-kb synthetic DNA cassette, thereby effectively eliminating over 54 kb of genomic DNA from three non-contiguous regions in a single recombination event. We observed complete replacement of the native sequence with the modified synthetic sequence through the action of the Cre recombinase and no competition from homologous recombination. Because of the versatility and high-efficiency of the Cre-lox system, this method can be used in any organism where this system is functional as well as adapted to use with other highly precise genome engineering systems. Compared to present-day iterative approaches in genome engineering, we anticipate this method will greatly speed up the creation of reduced, modularized and optimized genomes through the integration of deletion analyses data, transcriptomics, synthetic biology and site-specific recombination. PMID:24914053

Long-term auxological and pubertal outcome of patients with hereditary insulin-like growth factor-I deficiency (Laron and growth hormone-gene deletion syndrome) treated with recombinant human insulin-like growth factor-I.

PubMed

Messina, M F; Arrigo, T; Valenzise, M; Ghizzoni, L; Caruso-Nicoletti, M; Zucchini, S; Chiabotto, P; Crisafulli, G; Zirilli, G; De Luca, F

2011-04-01

GH-IGF-I axis is mainly involved in the complex process of somatic growth but emerging evidence suggests that it also influences hypothalamic-pituitary-gonadal (HPG) function. We report some data regarding long-term auxological and pubertal outcome of five female patients with hereditary forms of GH-IGF-I deficiency (Laron and GH-gene deletion syndrome) and a mean age of 23.4±5.3 yr (range 19-32). All the patients received recombinant human IGF-I (rhIGF-I, Pharmacia and Upjohn, Stockholm, Sweden, and rhIGF-I, Genentech, San Francisco, CA, USA) from a mean age of 8.6 yr (range 3.2-14.2) up to the final height. Final height was very disappointing (≤ -5.0 SD scores) and lower than target height in all the patients. Pubertal onset was delayed in most of them but menarche occurred spontaneously in all the patients. Median age at menarche was 15.1 yr. Menstrual cycles were regular for several years. Median duration of gynecological follow- up was 8.3 yr with the longest span of 17.2 yr. We can assert that GH-IGF-I axis has an essential role in promoting linear growth in humans and its physiological action cannot be replaced by pharmacological treatment in most patients with hereditary forms of IGF-I insufficiency as demonstrated by their subnormal final height. Our clinical observations can also support an essential role of IGF-I in genitalia growth but not in the function of HPG axis as demonstrated by the maintenance of regular menstrual cycles in the presence of subnormal levels of IGF-I after treatment discontinuation.

Prenatal Diagnosis and Molecular Analysis of a Large Novel Deletion (- -JS) Causing α0-Thalassemia.

PubMed

Cao, Jinru; He, Shuzhen; Pu, Yudong; Liu, Jingjing; Liu, Fuping; Feng, Jun

α-Thalassemia (α-thal) is a very common single gene hereditary disease caused by large deletions or point mutations of the α-globin gene cluster in tropical and subtropical regions of the world. Here, we report for the first time, a novel large α-thal deletion in a Chinese family from Jiangsu Province, People's Republic of China (PRC), which removes almost the entire α2 and α1 genes from the α-globin gene cluster. Thus, it was named the Jiangsu deletion (- - JS ) on the α-globin gene cluster causing α 0 -thal. Heterozygotes for this deletion showed an α-thal trait phenotype with reduced mean corpuscular volume (MCV) and mean corpuscular hemoglobin (Hb) (MCH) levels. The sequencing results showed that a 2538 bp deletion (NG_000006.1: g.35801_38338) existed in this novel genotype on the basis of -α 4.2 (leftward), indicating a deletion of about 6.8 kb from the α-globin cluster. In addition, a 29 bp sequence was inserted into the deletion during the recombination events that led to this deletion. Through pedigree analysis, we knew that the proband inherited the novel allele from his mother.

Illegitimate recombination mediated by calf thymus DNA topoisomerase II in vitro.

PubMed Central

Bae, Y S; Kawasaki, I; Ikeda, H; Liu, L F

1988-01-01

We have found that purified calf thymus DNA topoisomerase II mediates recombination between two phage lambda DNA molecules in an in vitro system. The enzyme mainly produced a linear monomer recombinant DNA that can be packaged in vitro. Novobiocin and anti-calf thymus DNA topoisomerase II antibody inhibit this ATP-dependent recombination. The recombinant molecules contain duplications or deletions, and most crossovers take place between nonhomologous sequences of lambda DNA, as judged by the sequences of recombination junctions. Therefore, the recombination mediated by the calf thymus DNA topoisomerase II is an illegitimate recombination that is similar to recombination mediated by Escherichia coli DNA gyrase or phage T4 DNA topoisomerase. The subunit exchange model, which has been suggested for the DNA gyrase-mediated recombination, is now generalized as follows: DNA topoisomerase II molecules bind to DNAs, associate with each other, and lead to the exchange of DNA strands through the exchange of topoisomerase II subunits. Illegitimate recombination might be carried out by a general mechanism in organisms ranging from prokaryotes to higher eukaryotes. Images PMID:2832845

Inhibition of colorectal cancer genomic copy number alterations and chromosomal fragile site tumor suppressor FHIT and WWOX deletions by DNA mismatch repair

PubMed Central

Gelincik, Ozkan; Blecua, Pedro; Edelmann, Winfried; Kucherlapati, Raju; Zhou, Kathy; Jasin, Maria; Gümüş, Zeynep H.; Lipkin, Steven M.

2017-01-01

Homologous recombination (HR) enables precise DNA repair after DNA double strand breaks (DSBs) using identical sequence templates, whereas homeologous recombination (HeR) uses only partially homologous sequences. Homeologous recombination introduces mutations through gene conversion and genomic deletions through single-strand annealing (SSA). DNA mismatch repair (MMR) inhibits HeR, but the roles of mammalian MMR MutL homologues (MLH1, PMS2 and MLH3) proteins in HeR suppression are poorly characterized. Here, we demonstrate that mouse embryonic fibroblasts (MEFs) carrying Mlh1, Pms2, and Mlh3 mutations have higher HeR rates, by using 7,863 uniquely mapping paired direct repeat sequences (DRs) in the mouse genome as endogenous gene conversion and SSA reporters. Additionally, when DSBs are induced by gamma-radiation, Mlh1, Pms2 and Mlh3 mutant MEFs have higher DR copy number alterations (CNAs), including DR CNA hotspots previously identified in mouse MMR-deficient colorectal cancer (dMMR CRC). Analysis of The Cancer Genome Atlas CRC data revealed that dMMR CRCs have higher genome-wide DR HeR rates than MMR proficient CRCs, and that dMMR CRCs have deletion hotspots in tumor suppressors FHIT/WWOX at chromosomal fragile sites FRA3B and FRA16D (which have elevated DSB rates) flanked by paired homologous DRs and inverted repeats (IR). Overall, these data provide novel insights into the MMR-dependent HeR inhibition mechanism and its role in tumor suppression. PMID:29069730

Efficient genome engineering of a virulent Klebsiella bacteriophage using CRISPR-Cas9.

PubMed

Shen, Juntao; Zhou, Jinjie; Chen, Guo-Qiang; Xiu, Zhi-Long

2018-06-13

Klebsiella pneumoniae is one of the most common nosocomial opportunistic pathogens usually with multiple drug-resistance. Phage therapy, a potential new therapeutics to replace or supplement antibiotics, has attracted much attention. However, very few Klebsiella phages have been well-characterized as the lack of efficient genome editing tools. Here, Cas9 from Streptococcus pyogenes and a single guide RNA (sgRNA) were used to modify a virulent Klebsiella bacteriophage phiKpS2. We firstly evaluated the distribution of sgRNA activity in phages and proved that it's largely inconsistent with the predicted activity from current models trained on eukaryotic cell datasets. A simple CRISPR-based phage genome editing procedure was developed based on the discovery that homologous arms as short as 30-60 bp was sufficient to introduce point mutation, gene deletion and swap. We also demonstrated that weak sgRNAs could be used for precise phage genome editing but failed to select random recombinants, possibly because inefficient cleavage can be tolerated through continuous repair by homologous recombination with the uncut genomes. Small frameshift deletion was proved to be an efficient way to evaluate the essentiality of phage genes. By using the above strategies, a putative promoter and nine genes of phiKpS2 were successfully deleted. Interestingly, the holin gene can be deleted with little effect on phiKpS2 infection, but the reason is not yet clear. This study established an efficient, time-saving, and cost-effective procedure for phage genome editing, which is expected to significantly promote the development of bacteriophage therapy. IMPORTANCE In the present study, we have addressed an efficient, time-saving and cost-effective CRISPR-based phage genome editing of Klebsiella phage, which has the potential to significantly expand our knowledge of phage-host interactions and to promote the applications of phage therapy. The distribution of sgRNA activity was first evaluated in phages. Short homologous arms were proved enough to introduce point mutation, small frameshift deletion, gene deletion and swap into phages, and weak sgRNAs were proved useful for precise phage genome editing but failed to select random recombinants, which all make the CRISPR-based phage genome editing easier to use. Copyright © 2018 American Society for Microbiology.

Trimeric autotransporter adhesins contribute to Actinobacillus pleuropneumoniae pathogenicity in mice and regulate bacterial gene expression during interactions between bacteria and porcine primary alveolar macrophages.

PubMed

Qin, Wanhai; Wang, Lei; Zhai, Ruidong; Ma, Qiuyue; Liu, Jianfang; Bao, Chuntong; Zhang, Hu; Sun, Changjiang; Feng, Xin; Gu, Jingmin; Du, Chongtao; Han, Wenyu; Langford, P R; Lei, Liancheng

2016-01-01

Actinobacillus pleuropneumoniae is an important pathogen that causes respiratory disease in pigs. Trimeric autotransporter adhesin (TAA) is a recently discovered bacterial virulence factor that mediates bacterial adhesion and colonization. Two TAA coding genes have been found in the genome of A. pleuropneumoniae strain 5b L20, but whether they contribute to bacterial pathogenicity is unclear. In this study, we used homologous recombination to construct a double-gene deletion mutant, ΔTAA, in which both TAA coding genes were deleted and used it in in vivo and in vitro studies to confirm that TAAs participate in bacterial auto-aggregation, biofilm formation, cell adhesion and virulence in mice. A microarray analysis was used to determine whether TAAs can regulate other A. pleuropneumoniae genes during interactions with porcine primary alveolar macrophages. The results showed that deletion of both TAA coding genes up-regulated 36 genes, including ene1514, hofB and tbpB2, and simultaneously down-regulated 36 genes, including lgt, murF and ftsY. These data illustrate that TAAs help to maintain full bacterial virulence both directly, through their bioactivity, and indirectly by regulating the bacterial type II and IV secretion systems and regulating the synthesis or secretion of virulence factors. This study not only enhances our understanding of the role of TAAs but also has significance for those studying A. pleuropneumoniae pathogenesis.

Insertion and deletion mutagenesis of the human cytomegalovirus genome

DOE Office of Scientific and Technical Information (OSTI.GOV)

Spaete, R.R.; Mocarski, E.S.

1987-10-01

Studies on human cytomegalovirus (CMV) have been limited by a paucity of molecular genetic techniques available for manipulating the viral genome. The authors have developed methods for site-specific insertion and deletion mutagenesis of CMV utilizing a modified Escherichia coli lacZ gene as a genetic marker. The lacZ gene was placed under the control of the major ..beta.. gene regulatory signals and inserted into the viral genome by homologous recombination, disrupting one of two copies of this ..beta.. gene within the L-component repeats of CMV DNA. They observed high-level expression of ..beta..-galactosidase by the recombinant in a temporally authentic manner, withmore » levels of this enzyme approaching 1% of total protein in infected cells. Thus, CMV is an efficient vector for high-level expression of foreign gene products in human cells. Using back selection of lacZ-deficient virus in the presence of the chromogenic substrate 5-bromo-4-chloro-3-indolyl ..beta..-D-galactoside, they generated random endpoint deletion mutants. Analysis of these mutant revealed that CMV DNA sequences flanking the insert had been removed, thereby establishing this approach as a means of determining whether sequences flanking a lacZ insertion are dispensable for viral growth. In an initial test of the methods, they have shown that 7800 base pairs of one copy of L-component repeat sequences can be deleted without affecting viral growth in human fibroblasts.« less

The fate of deleted DNA produced during programmed genomic deletion events in Tetrahymena thermophila.

PubMed Central

Saveliev, S V; Cox, M M

1994-01-01

Thousands of DNA deletion events occur during macronuclear development in the ciliate Tetrahymena thermophila. In two deleted genomic regions, designated M and R, the eliminated sequences form circles that can be detected by PCR. However, the circles are not normal products of the reaction pathway. The circular forms occur at very low levels in conjugating cells, but are stable. Sequencing analysis showed that many of the circles (as many as 50% of those examined) reflected a precise deletion in the M and R regions. The remaining circles were either smaller or larger and contained varying lengths of sequences derived from the chromosomal DNA surrounding the eliminated region. The chromosomal junctions left behind after deletion were more precise, although deletions in either the M or R regions can generate any of several alternative junctions (1). Some new chromosomal junctions were detected in the present study. The results suggest that the deleted segment is released as a linear DNA species that is degraded rapidly. The species is only rarely converted to the stable circles we detect. The deletion mechanism is different from those proposed for deletion events in hypotrichous ciliates (2-4), and does not reflect a conservative site-specific recombination process such as that promoted by the bacteriophage lambda integrase (5). Images PMID:7838724

Molecular Mechanisms Underlying Genomic Instability in Brca-Deficient Cells

DTIC Science & Technology

2014-03-01

in BRCA1, which is required for homologous recombination, or in any of the Fanconi anaemia complementation group ( FANC ) genes , which excise DNA...The Brca1 gene is required for DNA repair by homologous recombination and normal embryonic development. The protein 53BP1 promotes ligation and...in the right panel. Key Research Accomplishments • Deletion of the DNA damage response gene RIF1 mimics 53BP1 deficiency with respect to

RTEL1 contributes to DNA replication and repair and telomere maintenance.

PubMed

Uringa, Evert-Jan; Lisaingo, Kathleen; Pickett, Hilda A; Brind'Amour, Julie; Rohde, Jan-Hendrik; Zelensky, Alex; Essers, Jeroen; Lansdorp, Peter M

2012-07-01

Telomere maintenance and DNA repair are important processes that protect the genome against instability. mRtel1, an essential helicase, is a dominant factor setting telomere length in mice. In addition, mRtel1 is involved in DNA double-strand break repair. The role of mRtel1 in telomere maintenance and genome stability is poorly understood. Therefore we used mRtel1-deficient mouse embryonic stem cells to examine the function of mRtel1 in replication, DNA repair, recombination, and telomere maintenance. mRtel1-deficient mouse embryonic stem cells showed sensitivity to a range of DNA-damaging agents, highlighting its role in replication and genome maintenance. Deletion of mRtel1 increased the frequency of sister chromatid exchange events and suppressed gene replacement, demonstrating the involvement of the protein in homologous recombination. mRtel1 localized transiently at telomeres and is needed for efficient telomere replication. Of interest, in the absence of mRtel1, telomeres in embryonic stem cells appeared relatively stable in length, suggesting that mRtel1 is required to allow extension by telomerase. We propose that mRtel1 is a key protein for DNA replication, recombination, and repair and efficient elongation of telomeres by telomerase.

Isolation of a transcription factor expressed in neural crest from the region of 22q11 deleted in DiGeorge syndrome

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wadey, R.; Roberts, C.; Daw, S.

1994-09-01

Deletions within chromosome 22q11 cause a wide variety of birth defects including DiGeorge syndrome and Shprintzen syndrome. We have defined a commonly deleted region of over 2 Mb, and a critical region of 300 kb. A gene, TUPLE1, has been isolated from this critical region encoding a transcriptional regulator similar to the yeast HIR1 histone regulator gene. Since it has been suggested that DGS results from a defective neural crest, the expression of Tuple1 was examined in whole mouse and chick embryos, tissue sections and neural tube explants: Tuple1 is expressed in a dynamic pattern with high levels in regionsmore » containing migrating crest. Prior to crest migration Tuple1 is expressed in a rhombomere-specific expression pattern. Later Tuple1 is expressed in discrete domains within the developing neural tube. A remarkable feature of the experiments was the detection of a similar dynamic pattern with sense probe; i.e., there is an antisense Tuple1 transcript. This was confirmed using RPA. Tuple1 is being screened for mutations in non-deletion patients and constructs assembled for homologous recombination in ES cells. Tuple1 maps to MMU16 extending the homology of linkage with human chromosome 22. From these data we predict that the human homologue of the murine scid mutation maps to 22q11.« less

Molecular Characterization of Heterologous HIV-1gp120 Gene Expression Disruption in Mycobacterium bovis BCG Host Strain: A Critical Issue for Engineering Mycobacterial Based-Vaccine Vectors

PubMed Central

Joseph, Joan; Fernández-Lloris, Raquel; Pezzat, Elías; Saubi, Narcís; Cardona, Pere-Joan; Mothe, Beatriz; Gatell, Josep Maria

2010-01-01

Mycobacterium bovis Bacillus Calmette-Guérin (BCG) as a live vector of recombinant bacterial vaccine is a promising system to be used. In this study, we evaluate the disrupted expression of heterologous HIV-1gp120 gene in BCG Pasteur host strain using replicative vectors pMV261 and pJH222. pJH222 carries a lysine complementing gene in BCG lysine auxotrophs. The HIV-1 gp120 gene expression was regulated by BCG hsp60 promoter (in plasmid pMV261) and Mycobacteria spp. α-antigen promoter (in plasmid pJH222). Among 14 rBCG:HIV-1gp120 (pMV261) colonies screened, 12 showed a partial deletion and two showed a complete deletion. However, deletion was not observed in all 10 rBCG:HIV-1gp120 (pJH222) colonies screened. In this study, we demonstrated that E. coli/Mycobacterial expression vectors bearing a weak promoter and lysine complementing gene in a recombinant lysine auxotroph of BCG could prevent genetic rearrangements and disruption of HIV 1gp120 gene expression, a key issue for engineering Mycobacterial based vaccine vectors. PMID:20617151

aroA-Deficient Salmonella enterica Serovar Typhimurium Is More Than a Metabolically Attenuated Mutant

PubMed Central

Frahm, Michael; Kocijancic, Dino; Rohde, Manfred; Eckweiler, Denitsa; Bielecka, Agata; Bueno, Emilio; Cava, Felipe; Abraham, Wolf-Rainer; Curtiss, Roy; Häussler, Susanne; Erhardt, Marc; Weiss, Siegfried

2016-01-01

ABSTRACT Recombinant attenuated Salmonella enterica serovar Typhimurium strains are believed to act as powerful live vaccine carriers that are able to elicit protection against various pathogens. Auxotrophic mutations, such as a deletion of aroA, are commonly introduced into such bacteria for attenuation without incapacitating immunostimulation. In this study, we describe the surprising finding that deletion of aroA dramatically increased the virulence of attenuated Salmonella in mouse models. Mutant bacteria lacking aroA elicited increased levels of the proinflammatory cytokine tumor necrosis factor alpha (TNF-α) after systemic application. A detailed genetic and phenotypic characterization in combination with transcriptomic and metabolic profiling demonstrated that ΔaroA mutants display pleiotropic alterations in cellular physiology and lipid and amino acid metabolism, as well as increased sensitivity to penicillin, complement, and phagocytic uptake. In concert with other immunomodulating mutations, deletion of aroA affected flagellin phase variation and gene expression of the virulence-associated genes arnT and ansB. Finally, ΔaroA strains displayed significantly improved tumor therapeutic activity. These results highlight the importance of a functional shikimate pathway to control homeostatic bacterial physiology. They further highlight the great potential of ΔaroA-attenuated Salmonella for the development of vaccines and cancer therapies with important implications for host-pathogen interactions and translational medicine. PMID:27601574

The yeast Holliday junction resolvase, CCE1, can restore wild-type mitochondrial DNA to human cells carrying rearranged mitochondrial DNA.

PubMed

Sembongi, Hiroshi; Di Re, Miriam; Bokori-Brown, Monika; Holt, Ian J

2007-10-01

Rearrangements of mitochondrial DNA (mtDNA) are a well-recognized cause of human disease; deletions are more frequent, but duplications are more readily transmitted to offspring. In theory, partial duplications of mtDNA can be resolved to partially deleted and wild-type (WT) molecules, via homologous recombination. Therefore, the yeast CCE1 gene, encoding a Holliday junction resolvase, was introduced into cells carrying partially duplicated or partially triplicated mtDNA. Some cell lines carrying the CCE1 gene had substantial amounts of WT mtDNA suggesting that the enzyme can mediate intramolecular recombination in human mitochondria. However, high levels of expression of CCE1 frequently led to mtDNA loss, and so it is necessary to strictly regulate the expression of CCE1 in human cells to ensure the selection and maintenance of WT mtDNA.

Inter-Fork Strand Annealing causes genomic deletions during the termination of DNA replication.

PubMed

Morrow, Carl A; Nguyen, Michael O; Fower, Andrew; Wong, Io Nam; Osman, Fekret; Bryer, Claire; Whitby, Matthew C

2017-06-06

Problems that arise during DNA replication can drive genomic alterations that are instrumental in the development of cancers and many human genetic disorders. Replication fork barriers are a commonly encountered problem, which can cause fork collapse and act as hotspots for replication termination. Collapsed forks can be rescued by homologous recombination, which restarts replication. However, replication restart is relatively slow and, therefore, replication termination may frequently occur by an active fork converging on a collapsed fork. We find that this type of non-canonical fork convergence in fission yeast is prone to trigger deletions between repetitive DNA sequences via a mechanism we call Inter-Fork Strand Annealing (IFSA) that depends on the recombination proteins Rad52, Exo1 and Mus81, and is countered by the FANCM-related DNA helicase Fml1. Based on our findings, we propose that IFSA is a potential threat to genomic stability in eukaryotes.

Role of Fanconi Anemia FANCG in Preventing Double-Strand Breakage and Chromosomal Rearrangement during DNA Replication

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tebbs, R S; Hinz, J M; Yamada, N A

The Fanconi anemia (FA) proteins overlap with those of homologous recombination through FANCD1/BRCA2, but the biochemical functions of other FA proteins are unknown. By constructing and characterizing a null fancg mutant of hamster CHO cells, we present several new insights for FA. The fancg cells show a broad sensitivity to genotoxic agents, not supporting the conventional concept of sensitivity to only DNA crosslinking agents. The aprt mutation rate is normal, but hprt mutations are reduced, which we ascribe to the lethality of large deletions. CAD and dhfr gene amplification rates are increased, implying excess chromosomal breakage during DNA replication, andmore » suggesting amplification as a contributing factor to cancer-proneness in FA patients. In S-phase cells, both spontaneous and mutagen-induced Rad51 nuclear foci are elevated. These results support a model in which FancG protein helps to prevent collapse of replication forks by allowing translesion synthesis or lesion bypass through homologous recombination.« less

Rad52 phosphorylation by Ipl1 and Mps1 contributes to Mps1 kinetochore localization and spindle assembly checkpoint regulation

PubMed Central

Lim, Gyubum

2017-01-01

Rad52 is well known as a key factor in homologous recombination. Here, we report that Rad52 has functions unrelated to homologous recombination in Saccharomyces cerevisiae; it plays a role in the recruitment of Mps1 to the kinetochores and the maintenance of spindle assembly checkpoint (SAC) activity. Deletion of RAD52 causes various phenotypes related to the dysregulation of chromosome biorientation. Rad52 directly affects efficient operation of the SAC and accurate chromosome segregation. Remarkably, by using an in vitro kinase assay, we found that Rad52 is a substrate of Ipl1/Aurora and Mps1 in yeast and humans. Ipl1-dependent phosphorylation of Rad52 facilitates the kinetochore accumulation of Mps1, and Mps1-dependent phosphorylation of Rad52 is important for the accurate regulation of the SAC under spindle damage conditions. Taken together, our data provide detailed insights into the regulatory mechanism of chromosome biorientation by mitotic kinases. PMID:29078282

Rad52 phosphorylation by Ipl1 and Mps1 contributes to Mps1 kinetochore localization and spindle assembly checkpoint regulation.

PubMed

Lim, Gyubum; Huh, Won-Ki

2017-10-31

Rad52 is well known as a key factor in homologous recombination. Here, we report that Rad52 has functions unrelated to homologous recombination in Saccharomyces cerevisiae ; it plays a role in the recruitment of Mps1 to the kinetochores and the maintenance of spindle assembly checkpoint (SAC) activity. Deletion of RAD52 causes various phenotypes related to the dysregulation of chromosome biorientation. Rad52 directly affects efficient operation of the SAC and accurate chromosome segregation. Remarkably, by using an in vitro kinase assay, we found that Rad52 is a substrate of Ipl1/Aurora and Mps1 in yeast and humans. Ipl1-dependent phosphorylation of Rad52 facilitates the kinetochore accumulation of Mps1, and Mps1-dependent phosphorylation of Rad52 is important for the accurate regulation of the SAC under spindle damage conditions. Taken together, our data provide detailed insights into the regulatory mechanism of chromosome biorientation by mitotic kinases. Published under the PNAS license.

Recombination of mitochondrial DNA in skeletal muscle of individuals with multiple mitochondrial DNA heteroplasmy.

PubMed

Zsurka, Gábor; Kraytsberg, Yevgenia; Kudina, Tatiana; Kornblum, Cornelia; Elger, Christian E; Khrapko, Konstantin; Kunz, Wolfram S

2005-08-01

Experimental evidence for human mitochondrial DNA (mtDNA) recombination was recently obtained in an individual with paternal inheritance of mtDNA and in an in vitro cell culture system. Whether mtDNA recombination is a common event in humans remained to be determined. To detect mtDNA recombination in human skeletal muscle, we analyzed the distribution of alleles in individuals with multiple mtDNA heteroplasmy using single-cell PCR and allele-specific PCR. In all ten individuals who carried a heteroplasmic D-loop mutation and a distantly located tRNA point mutation or a large deletion, we observed a mixture of four allelic combinations (tetraplasmy), a hallmark of recombination. Twelve of 14 individuals with closely located heteroplasmic D-loop mutation pairs contained a mixture of only three types of mitochondrial genomes (triplasmy), consistent with the absence of recombination between adjacent markers. These findings indicate that mtDNA recombination is common in human skeletal muscle.

Complete Genome Sequence of a Recombinant NADC30-Like Strain, SCnj16, of Porcine Reproductive and Respiratory Syndrome Virus in Southwestern China

PubMed Central

Kang, Runmin; Xie, Bo; Tian, Yiming; Yang, Xin; Yu, Jifeng

2018-01-01

ABSTRACT The NADC30-like strains of porcine reproductive and respiratory syndrome virus (PRRSV) are characterized by a 131-amino-acid deletion in nonstructural protein 2 (NSP2). Here, we report the complete genome sequence of a recombinant NADC30-like PRRSV strain, SCnj16, that exhibits the molecular marker of the Chinese highly pathogenic PRRSV (HP-PRRSV) in NSP2. PMID:29439029

An efficient procedure for marker-free mutagenesis of S. coelicolor by site-specific recombination for secondary metabolite overproduction.

PubMed

Zhang, Bo; Zhang, Lin; Dai, Ruixue; Yu, Meiying; Zhao, Guoping; Ding, Xiaoming

2013-01-01

Streptomyces bacteria are known for producing important natural compounds by secondary metabolism, especially antibiotics with novel biological activities. Functional studies of antibiotic-biosynthesizing gene clusters are generally through homologous genomic recombination by gene-targeting vectors. Here, we present a rapid and efficient method for construction of gene-targeting vectors. This approach is based on Streptomyces phage φBT1 integrase-mediated multisite in vitro site-specific recombination. Four 'entry clones' were assembled into a circular plasmid to generate the destination gene-targeting vector by a one-step reaction. The four 'entry clones' contained two clones of the upstream and downstream flanks of the target gene, a selectable marker and an E. coli-Streptomyces shuttle vector. After targeted modification of the genome, the selectable markers were removed by φC31 integrase-mediated in vivo site-specific recombination between pre-placed attB and attP sites. Using this method, part of the calcium-dependent antibiotic (CDA) and actinorhodin (Act) biosynthetic gene clusters were deleted, and the rrdA encoding RrdA, a negative regulator of Red production, was also deleted. The final prodiginine production of the engineered strain was over five times that of the wild-type strain. This straightforward φBT1 and φC31 integrase-based strategy provides an alternative approach for rapid gene-targeting vector construction and marker removal in streptomycetes.

Exonal deletion of SLC24A4 causes hypomaturation amelogenesis imperfecta.

PubMed

Seymen, F; Lee, K-E; Tran Le, C G; Yildirim, M; Gencay, K; Lee, Z H; Kim, J-W

2014-04-01

Amelogenesis imperfecta is a heterogeneous group of genetic conditions affecting enamel formation. Recently, mutations in solute carrier family 24 member 4 (SLC24A4) have been identified to cause autosomal recessive hypomaturation amelogenesis imperfecta. We recruited a consanguineous family with hypomaturation amelogenesis imperfecta with generalized brown discoloration. Sequencing of the candidate genes identified a 10-kb deletion, including exons 15, 16, and most of the last exon of the SLC24A4 gene. Interestingly, this deletion was caused by homologous recombination between two 354-bp-long homologous sequences located in intron 14 and the 3' UTR. This is the first report of exonal deletion in SLC24A4 providing confirmatory evidence that the function of SLC24A4 in calcium transport has a crucial role in the maturation stage of amelogenesis.

[Biological characteristics of an enteroinvasive Escherichia coli strain with tatABC deletion].

PubMed

Gong, Zhaolong; Ye, Changyun; Liu, Xiaobing; Zhang, Min; Zhuo, Qin

2013-05-04

To study the relationship between twin-arginine translocation system (Tat) system with the biological characteristics of enteroinvasive Escherichia coli (EIEC). Through homologous recombination, we constructed EIEC's tatABC gene deletion strain and complementary strain, and explored their impact on bacterial form, substrate transport function as well as on HeLa cells and guinea pig's corneal invasion force. The tatABC gene deletion strain had apparent changes in bacterial form, loss of substrate transporter function, and significant weakened bacterial invasion force (the number of the deletion strain invading into HeLa cells was decreased significantly, and the ability of its corneal lesion capacity of the guinea pig was significantly weakened), while the complementary strain was similar to the wild strain in the above respects. EIEC's Tat protein transport system is closely related with the biological characteristics of EIEC.

The Norrie disease gene maps to a 150 kb region on chromosome Xp11.3.

PubMed

Sims, K B; Lebo, R V; Benson, G; Shalish, C; Schuback, D; Chen, Z Y; Bruns, G; Craig, I W; Golbus, M S; Breakefield, X O

1992-05-01

Norrie disease is a human X-linked recessive disorder of unknown etiology characterized by congenital blindness, sensory neural deafness and mental retardation. This disease gene was previously linked to the DXS7 (L1.28) locus and the MAO genes in band Xp11.3. We report here fine physical mapping of the obligate region containing the Norrie disease gene (NDP) defined by a recombination and by the smallest submicroscopic chromosomal deletion associated with Norrie disease identified to date. Analysis, using in addition two overlapping YAC clones from this region, allowed orientation of the MAOA and MAOB genes in a 5'-3'-3'-5' configuration. A recombination event between a (GT)n polymorphism in intron 2 of the MAOB gene and the NDP locus, in a family previously reported to have a recombination between DXS7 and NDP, delineates a flanking marker telomeric to this disease gene. An anonymous DNA probe, dc12, present in one of the YACs and in a patient with a submicroscopic deletion which includes MAOA and MAOB but not L1.28, serves as a flanking marker centromeric to the disease gene. An Alu-PCR fragment from the right arm of the MAO YAC (YMAO.AluR) is not deleted in this patient and also delineates the centromeric extent of the obligate disease region. The apparent order of these loci is telomere ... DXS7-MAOA-MAOB-NDP-dc12-YMAO.AluR ... centromere. Together these data define the obligate region containing the NDP gene to a chromosomal segment less than 150 kb.

Reverse genetics of Newcastle disease virus

USDA-ARS?s Scientific Manuscript database

Reverse genetics allows the generation of recombinant viruses or vectors used in functional studies, vaccine development, and gene therapy. This technique allows genetic manipulation and cloning of viral genomes, mutation through site-directed mutagenesis, and gene insertion or deletion, among othe...

Translocation and gross deletion breakpoints in human inherited disease and cancer II: Potential involvement of repetitive sequence elements in secondary structure formation between DNA ends.

PubMed

Chuzhanova, Nadia; Abeysinghe, Shaun S; Krawczak, Michael; Cooper, David N

2003-09-01

Translocations and gross deletions are responsible for a significant proportion of both cancer and inherited disease. Although such gene rearrangements are nonuniformly distributed in the human genome, the underlying mutational mechanisms remain unclear. We have studied the potential involvement of various types of repetitive sequence elements in the formation of secondary structure intermediates between the single-stranded DNA ends that recombine during rearrangements. Complexity analysis was used to assess the potential of these ends to form secondary structures, the maximum decrease in complexity consequent to a gross rearrangement being used as an indicator of the type of repeat and the specific DNA ends involved. A total of 175 pairs of deletion/translocation breakpoint junction sequences available from the Gross Rearrangement Breakpoint Database [GRaBD; www.uwcm.ac.uk/uwcm/mg/grabd/grabd.html] were analyzed. Potential secondary structure was noted between the 5' flanking sequence of the first breakpoint and the 3' flanking sequence of the second breakpoint in 49% of rearrangements and between the 5' flanking sequence of the second breakpoint and the 3' flanking sequence of the first breakpoint in 36% of rearrangements. Inverted repeats, inversions of inverted repeats, and symmetric elements were found in association with gross rearrangements at approximately the same frequency. However, inverted repeats and inversions of inverted repeats accounted for the vast majority (83%) of deletions plus small insertions, symmetric elements for one-half of all antigen receptor-mediated translocations, while direct repeats appear only to be involved in mediating simple deletions. These findings extend our understanding of illegitimate recombination by highlighting the importance of secondary structure formation between single-stranded DNA ends at breakpoint junctions. Copyright 2003 Wiley-Liss, Inc.

Mechanisms of formation and accumulation of mitochondrial DNA deletions in aging neurons.

PubMed

Fukui, Hirokazu; Moraes, Carlos T

2009-03-15

Age-dependent accumulation of partially deleted mitochondrial DNA (DeltamtDNA) has been suggested to contribute to aging and the development of age-associated diseases including Parkinson's disease. However, the molecular mechanisms underlying the generation and accumulation of DeltamtDNA have not been addressed in vivo. In this study, we have developed a mouse model expressing an inducible mitochondria-targeted restriction endonuclease (PstI). Using this system, we could trigger mtDNA double-strand breaks (DSBs) in adult neurons. We found that this transient event leads to the generation of a family of DeltamtDNA with features that closely resemble naturally-occurring mtDNA deletions. The formation of these deleted species is likely to be mediated by yet uncharacterized DNA repairing machineries that participate in homologous recombination and non-homologous end-joining. Furthermore, we obtained in vivo evidence that DeltamtDNAs with larger deletions accumulate faster than those with smaller deletions, implying a replicative advantage of smaller mtDNAs. These findings identify DSB, DNA repair systems and replicative advantage as likely mechanisms underlying the generation and age-associated accumulation of DeltamtDNA in mammalian neurons.

Functional Genomics Using the Saccharomyces cerevisiae Yeast Deletion Collections.

PubMed

Nislow, Corey; Wong, Lai Hong; Lee, Amy Huei-Yi; Giaever, Guri

2016-09-01

Constructed by a consortium of 16 laboratories, the Saccharomyces genome-wide deletion collections have, for the past decade, provided a powerful, rapid, and inexpensive approach for functional profiling of the yeast genome. Loss-of-function deletion mutants were systematically created using a polymerase chain reaction (PCR)-based gene deletion strategy to generate a start-to-stop codon replacement of each open reading frame by homologous recombination. Each strain carries two molecular barcodes that serve as unique strain identifiers, enabling their growth to be analyzed in parallel and the fitness contribution of each gene to be quantitatively assessed by hybridization to high-density oligonucleotide arrays or through the use of next-generation sequencing technologies. Functional profiling of the deletion collections, using either strain-by-strain or parallel assays, provides an unbiased approach to systematically survey the yeast genome. The Saccharomyces yeast deletion collections have proved immensely powerful in contributing to the understanding of gene function, including functional relationships between genes and genetic pathways in response to diverse genetic and environmental perturbations. © 2016 Cold Spring Harbor Laboratory Press.

Members of the RAD52 Epistasis Group Contribute to Mitochondrial Homologous Recombination and Double-Strand Break Repair in Saccharomyces cerevisiae.

PubMed

Stein, Alexis; Kalifa, Lidza; Sia, Elaine A

2015-11-01

Mitochondria contain an independently maintained genome that encodes several proteins required for cellular respiration. Deletions in the mitochondrial genome have been identified that cause several maternally inherited diseases and are associated with certain cancers and neurological disorders. The majority of these deletions in human cells are flanked by short, repetitive sequences, suggesting that these deletions may result from recombination events. Our current understanding of the maintenance and repair of mtDNA is quite limited compared to our understanding of similar events in the nucleus. Many nuclear DNA repair proteins are now known to also localize to mitochondria, but their function and the mechanism of their action remain largely unknown. This study investigated the contribution of the nuclear double-strand break repair (DSBR) proteins Rad51p, Rad52p and Rad59p in mtDNA repair. We have determined that both Rad51p and Rad59p are localized to the matrix of the mitochondria and that Rad51p binds directly to mitochondrial DNA. In addition, a mitochondrially-targeted restriction endonuclease (mtLS-KpnI) was used to produce a unique double-strand break (DSB) in the mitochondrial genome, which allowed direct analysis of DSB repair in vivo in Saccharomyces cerevisiae. We find that loss of these three proteins significantly decreases the rate of spontaneous deletion events and the loss of Rad51p and Rad59p impairs the repair of induced mtDNA DSBs.

Members of the RAD52 Epistasis Group Contribute to Mitochondrial Homologous Recombination and Double-Strand Break Repair in Saccharomyces cerevisiae

PubMed Central

Stein, Alexis; Kalifa, Lidza; Sia, Elaine A.

2015-01-01

Mitochondria contain an independently maintained genome that encodes several proteins required for cellular respiration. Deletions in the mitochondrial genome have been identified that cause several maternally inherited diseases and are associated with certain cancers and neurological disorders. The majority of these deletions in human cells are flanked by short, repetitive sequences, suggesting that these deletions may result from recombination events. Our current understanding of the maintenance and repair of mtDNA is quite limited compared to our understanding of similar events in the nucleus. Many nuclear DNA repair proteins are now known to also localize to mitochondria, but their function and the mechanism of their action remain largely unknown. This study investigated the contribution of the nuclear double-strand break repair (DSBR) proteins Rad51p, Rad52p and Rad59p in mtDNA repair. We have determined that both Rad51p and Rad59p are localized to the matrix of the mitochondria and that Rad51p binds directly to mitochondrial DNA. In addition, a mitochondrially-targeted restriction endonuclease (mtLS-KpnI) was used to produce a unique double-strand break (DSB) in the mitochondrial genome, which allowed direct analysis of DSB repair in vivo in Saccharomyces cerevisiae. We find that loss of these three proteins significantly decreases the rate of spontaneous deletion events and the loss of Rad51p and Rad59p impairs the repair of induced mtDNA DSBs. PMID:26540255

Epstein-Barr virus (EBV) recombinants: use of positive selection markers to rescue mutants in EBV-negative B-lymphoma cells.

PubMed Central

Wang, F; Marchini, A; Kieff, E

1991-01-01

The objective of these experiments was to develop strategies for creation and identification of recombinant mutant Epstein-Barr viruses (EBV). EBV recombinant molecular genetics has been limited to mutations within a short DNA segment deleted from a nontransforming EBV and an underlying strategy which relies on growth transformation of primary B lymphocytes for identification of recombinants. Thus, mutations outside the deletion or mutations which affect transformation cannot be easily recovered. In these experiments we investigated whether a toxic drug resistance gene, guanine phosphoribosyltransferase or hygromycin phosphotransferase, driven by the simian virus 40 promoter can be recombined into the EBV genome and can function to identify B-lymphoma cells infected with recombinant virus. Two different strategies were used to recombine the drug resistance marker into the EBV genome. Both utilized transfection of partially permissive, EBV-infected B95-8 cells and positive selection for cells which had incorporated a functional drug resistance gene. In the first series of experiments, B95-8 clones were screened for transfected DNA that had recombined into the EBV genome. In the second series of experiments, the transfected drug resistance marker was linked to the plasmid and lytic EBV origins so that it was maintained as an episome and could recombine with the B95-8 EBV genome during virus replication. The recombinant EBV from either experiment could be recovered by infection and toxic drug selection of EBV-negative B-lymphoma cells. The EBV genome in these B-lymphoma cells is frequently an episome. Virus genes associated with latent infection of primary B lymphocytes are expressed. Expression of Epstein-Barr virus nuclear antigen 2 (EBNA-2) and the EBNA-3 genes is variable relative to that of EBNA-1, as is characteristic of some naturally infected Burkitt tumor cells. Moreover, the EBV-infected B-lymphoma cells are often partially permissive for early replicative cycle gene expression and virus replication can be induced, in contrast to previously reported in vitro infected B-lymphoma cells. These studies demonstrate that dominant selectable markers can be inserted into the EBV genome, are active in the context of the EBV genome, and can be used to recover recombinant EBV in B-lymphoma cells. This system should be particularly useful for recovering EBV genomes with mutations in essential transforming genes. Images PMID:1848303

Epstein-Barr virus (EBV) recombinants: use of positive selection markers to rescue mutants in EBV-negative B-lymphoma cells.

PubMed

Wang, F; Marchini, A; Kieff, E

1991-04-01

The objective of these experiments was to develop strategies for creation and identification of recombinant mutant Epstein-Barr viruses (EBV). EBV recombinant molecular genetics has been limited to mutations within a short DNA segment deleted from a nontransforming EBV and an underlying strategy which relies on growth transformation of primary B lymphocytes for identification of recombinants. Thus, mutations outside the deletion or mutations which affect transformation cannot be easily recovered. In these experiments we investigated whether a toxic drug resistance gene, guanine phosphoribosyltransferase or hygromycin phosphotransferase, driven by the simian virus 40 promoter can be recombined into the EBV genome and can function to identify B-lymphoma cells infected with recombinant virus. Two different strategies were used to recombine the drug resistance marker into the EBV genome. Both utilized transfection of partially permissive, EBV-infected B95-8 cells and positive selection for cells which had incorporated a functional drug resistance gene. In the first series of experiments, B95-8 clones were screened for transfected DNA that had recombined into the EBV genome. In the second series of experiments, the transfected drug resistance marker was linked to the plasmid and lytic EBV origins so that it was maintained as an episome and could recombine with the B95-8 EBV genome during virus replication. The recombinant EBV from either experiment could be recovered by infection and toxic drug selection of EBV-negative B-lymphoma cells. The EBV genome in these B-lymphoma cells is frequently an episome. Virus genes associated with latent infection of primary B lymphocytes are expressed. Expression of Epstein-Barr virus nuclear antigen 2 (EBNA-2) and the EBNA-3 genes is variable relative to that of EBNA-1, as is characteristic of some naturally infected Burkitt tumor cells. Moreover, the EBV-infected B-lymphoma cells are often partially permissive for early replicative cycle gene expression and virus replication can be induced, in contrast to previously reported in vitro infected B-lymphoma cells. These studies demonstrate that dominant selectable markers can be inserted into the EBV genome, are active in the context of the EBV genome, and can be used to recover recombinant EBV in B-lymphoma cells. This system should be particularly useful for recovering EBV genomes with mutations in essential transforming genes.

Construction of a psb C deletion strain in Synechocystis 6803.

PubMed

Goldfarb, N; Knoepfle, N; Putnam-Evans, C

1997-01-01

Synechocystis 6803 is a cyanobacterium that carries out-oxygenic photosynthesis. We are interested in the introduction of mutations in the large extrinsic loop region of the CP43 protein of Photosystem II (PSII). CP43 appears to be required for the stable assembly of the PSII complex and also appears to play a role in photosynthetic oxygen evolution. Deletion of short segments of the large extrinsic loop results in mutants incapable of evolving oxygen. Alterations in psbC, the gene encoding CP43, are introduced into Synechocystis 6803 by transformation and homologous recombination. Specifically, plasmid constructs bearing the site-directed mutations are introduced into a deletion strain where the portion of the gene encoding the area of mutation has been deleted and replaced by a gene conferring antibiotic resistance. We have constructed a deletion strain of Synechocystis appropriate for the introduction of mutations in the large extrinsic loop of CP43 and have used it successfully to produce site-directed mutants.

Alu-mediated large deletion of the CDSN gene as a cause of peeling skin disease.

PubMed

Wada, T; Matsuda, Y; Muraoka, M; Toma, T; Takehara, K; Fujimoto, M; Yachie, A

2014-10-01

Peeling skin disease (PSD) is an autosomal recessive skin disorder caused by mutations in CDSN and is characterized by superficial peeling of the upper epidermis. Corneodesmosin (CDSN) is a major component of corneodesmosomes that plays an important role in maintaining epidermis integrity. Herein, we report a patient with PSD caused by a novel homozygous large deletion in the 6p21.3 region encompassing the CDSN gene, which abrogates CDSN expression. Several genes including C6orf15, PSORS1C1, PSORS1C2, CCHCR1, and TCF19 were also deleted, however, the patient showed only clinical features typical of PSD. The deletion size was 59.1 kb. Analysis of the sequence surrounding the breakpoint showed that both telomeric and centromeric breakpoints existed within Alu-S sequences that were oriented in opposite directions. These results suggest an Alu-mediated recombination event as the mechanism underlying the deletion in our patient. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Normal development of mice lacking PAXX, the paralogue of XRCC4 and XLF.

PubMed

Gago-Fuentes, Raquel; Xing, Mengtan; Sæterstad, Siri; Sarno, Antonio; Dewan, Alisa; Beck, Carole; Bradamante, Stefano; Bjørås, Magnar; Oksenych, Valentyn

2018-03-01

DNA repair consists of several cellular pathways which recognize and repair damaged DNA. The classical nonhomologous DNA end-joining (NHEJ) pathway repairs double-strand breaks in DNA. It is required for maturation of both B and T lymphocytes by supporting V(D)J recombination as well as B-cell differentiation during class switch recombination (CSR). Inactivation of NHEJ factors Ku70, Ku80, XRCC4, DNA ligase 4, DNA-PKcs, and Artemis impairs V(D)J recombination and blocks lymphocyte development. Paralogue of XRCC4 and XLF (PAXX) is an accessory NHEJ factor that has a significant impact on the repair of DNA lesions induced by ionizing radiation in human, murine, and chicken cells. However, the role of PAXX during development is poorly understood. To determine the physiological role of PAXX, we deleted part of the Paxx promoter and the first two exons in mice. Further, we compared Paxx -knockout mice with wild-type (WT) and NHEJ-deficient controls including Ku80- and Dna-pkcs -null and severe combined immunodeficiency mice. Surprisingly, Paxx -deficient mice were not distinguishable from the WT littermates; they were the same weight and size, fertility status, had normal spleen, thymus, and bone marrow. Paxx -deficient mice had the same number of chromosomal and chromatid breaks as WT mice. Moreover, Paxx -deficient primary B lymphocytes had the same level of CSR as lymphocytes isolated from WT mice. We concluded that PAXX is dispensable for normal mouse development.

Testicular expressed genes are missing in familial X-Linked Kallmann syndrome due to two large different deletions in daughter's X chromosomes.

PubMed

Hershkovitz, Eli; Loewenthal, Neta; Peretz, Asaf; Parvari, Ruti

2008-01-01

X-linked Kallmann syndrome (KS) is caused mainly by point mutations, in the KAL1 gene. Large deletions >1 Mb are rare events in the human population and commonly result in contiguous gene syndromes. A search for the mutation causing KS carried out on two pairs of first-degree cousins of 2 sisters. Two different apparently independent deletions were found. The deleted sequences encompass the KAL1 gene and four known additional genes exclusively expressed in testis. Two of these genes belong to the FAM9 gene family, which shares some homology with the SCYP3 gene, previously implicated in azoospermia. One of the events causing the deletion may have been mediated by an L1 transposition, the other by a non-homologous end joining. Such non-homologous recombinations have not yet been reported in the KAL genomic region and thus this area may be more prone to deletions than previously expected. This is the first report on genetic characterization of KS with a deletion of solely testis-expressed genes. The absence of these genes may have unfavorable implications for the patients regarding future fertility. (c) 2008 S. Karger AG, Basel

Identification of sites of STAT3 action in the female reproductive tract through conditional gene deletion.

PubMed

Robker, Rebecca L; Watson, Laura N; Robertson, Sarah A; Dunning, Kylie R; McLaughlin, Eileen A; Russell, Darryl L

2014-01-01

The STAT3 transcription factor is a pleiotropic transducer of signalling by hormones, growth factors and cytokines that has been identified in the female reproductive tract from oocytes and granulosa cells of the ovary to uterine epithelial and stromal cells. In the present study we used transgenic models to investigate the importance of STAT3 for reproductive performance in these different tissues. The Cre-LoxP system was used to delete STAT3 in oocytes by crossing Stat3fl/fl with Zp3-cre+ mice, or in ovarian granulosa cells and uterine stroma by crossing with Amhr2-Cre+ mice. Surprisingly, deletion of STAT3 in oocytes had no effect on fertility indicating that the abundance of STAT3 protein in maturing oocytes and fertilized zygotes is not essential to these developmental stages. In Stat3fl/fl;Amhr2-cre+ females impaired fertility was observed through significantly fewer litters and smaller litter size. Ovulation rate, oocyte fertilization and development to blastocyst were unaffected in this line; however, poor recombination efficiency in granulosa cells had yielded no net change in STAT3 protein abundance. In contrast, uteri from these mice showed STAT3 protein depletion selectively from the stomal compartment. A significant reduction in number of viable fetuses on gestational day 18, increased fetal resorptions and disrupted placental morphology were evident causes of the reduced fertility. In conclusion, this study defines an important role for STAT3 in uterine stromal cells during embryo implantation and the development of a functional placenta.

A method for the generation of ectromelia virus (ECTV) recombinants: in vivo analysis of ECTV vCD30 deletion mutants.

PubMed

Alejo, Ali; Saraiva, Margarida; Ruiz-Argüello, Maria Begoña; Viejo-Borbolla, Abel; de Marco, Mar Fernández; Salguero, Francisco Javier; Alcami, Antonio

2009-01-01

Ectromelia virus (ECTV) is the causative agent of mousepox, a lethal disease of mice with similarities to human smallpox. Mousepox progression involves replication at the initial site of infection, usually the skin, followed by a rapid spread to the secondary replicative organs, spleen and liver, and finally a dissemination to the skin, where the typical rash associated with this and other orthopoxviral induced diseases appears. Case fatality rate is genetically determined and reaches up to 100% in susceptible mice strains. Like other poxviruses, ECTV encodes a number of proteins with immunomodulatory potential, whose role in mousepox progression remains largely undescribed. Amongst these is a secreted homologue of the cellular tumour necrosis factor receptor superfamily member CD30 which has been proposed to modulate a Th1 immune response in vivo. To evaluate the contribution of viral CD30 (vCD30) to virus pathogenesis in the infected host, we have adapted a novel transient dominant method for the selection of recombinant ECTVs. Using this method, we have generated an ECTV vCD30 deletion mutant, its corresponding revertant control virus as well as a virus encoding the extracellular domain of murine CD30. These viruses contain no exogenous marker DNA sequences in their genomes, as opposed to other ECTVs reported up to date. We show that the vCD30 is expressed as a secreted disulfide linked trimer and that the absence of vCD30 does not impair mousepox induced fatality in vivo. Replacement of vCD30 by a secreted version of mouse CD30 caused limited attenuation of ECTV. The recombinant viruses generated may be of use in the study of the role of the cellular CD30-CD30L interaction in the development of the immune response. The method developed might be useful for the construction of ECTV mutants for the study of additional genes.

Delimitation of the Earliness per se D1 (Eps-D1) flowering gene to a subtelomeric chromosomal deletion in bread wheat (Triticum aestivum).

PubMed

Zikhali, Meluleki; Wingen, Luzie U; Griffiths, Simon

2016-01-01

Earliness per se (Eps) genes account for the variation in flowering time when vernalization and photoperiod requirements are satisfied. Genomics and bioinformatics approaches were used to describe allelic variation for 40 Triticum aestivum genes predicted, by synteny with Brachypodium distachyon, to be in the 1DL Eps region. Re-sequencing 1DL genes revealed that varieties carrying early heading alleles at this locus, Spark and Cadenza, carry a subtelomeric deletion including several genes. The equivalent region in Rialto and Avalon is intact. A bimodal distribution in the segregating Spark X Rialto single seed descent (SSD) populations enabled the 1DL QTL to be defined as a discrete Mendelian factor, which we named Eps-D1. Near isogenic lines (NILs) and NIL derived key recombinants between markers flanking Eps-D1 suggest that the 1DL deletion contains the gene(s) underlying Eps-D1. The deletion spans the equivalent of the Triticum monoccocum Eps-A (m) 1 locus, and hence includes MODIFIER OF TRANSCRIPTION 1 (MOT1) and FTSH PROTEASE 4 (FTSH4), the candidates for Eps-A (m) 1. The deletion also contains T. aestivum EARLY FLOWERING 3-D1 (TaELF3-D1) a homologue of the Arabidopsis thaliana circadian clock gene EARLY FLOWERING 3. Eps-D1 is possibly a homologue of Eps-B1 on chromosome 1BL. NILs carrying the Eps-D1 deletion have significantly reduced total TaELF3 expression and altered TaGIGANTEA (TaGI) expression compared with wild type. Altered TaGI expression is consistent with an ELF3 mutant, hence we propose TaELF3-D1 as the more likely candidate for Eps-D1. This is the first direct fine mapping of Eps effect in bread wheat. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Characterization of recombinant human HBP/CAP37/azurocidin, a pleiotropic mediator of inflammation-enhancing LPS-induced cytokine release from monocytes.

PubMed

Rasmussen, P B; Bjørn, S; Hastrup, S; Nielsen, P F; Norris, K; Thim, L; Wiberg, F C; Flodgaard, H

1996-07-15

Neutrophil-derived heparin-binding protein (HBP) is a strong chemoattractant for monocytes. We report here for the first time the expression of recombinant HBP. A baculovirus containing the human HBP cDNA mediated in insect cells the secretion of a 7-residue N-terminally extended HBP form (pro-HBP). Deletion of the pro-peptide-encoding cDNA sequence resulted in correctly processed HBP at the N-terminus. Electrospray mass spectrum analysis of recombinant HBP yielded a molecular weight of 27.237 +/- 3 amu. Consistent with this mass is a HBP form of 225 amino acids (mature part +3 amino acid C-terminal extension). The biological activity of recombinant HBP was confirmed by its chemotactic action towards monocytes. Furthermore, we have shown that recombinant HBP stimulates in a dose-dependent manner the lipopolysaccharide (LPS)-induced cytokine release from human monocytes.

Identification of a novel 15.5 kb SHOX deletion associated with marked intrafamilial phenotypic variability and analysis of its molecular origin.

PubMed

Alexandrou, Angelos; Papaevripidou, Ioannis; Tsangaras, Kyriakos; Alexandrou, Ioanna; Tryfonidis, Marios; Christophidou-Anastasiadou, Violetta; Zamba-Papanicolaou, Eleni; Koumbaris, George; Neocleous, Vassos; Phylactou, Leonidas A; Skordis, Nicos; Tanteles, George A; Sismani, Carolina

2016-12-01

Haploinsufficiency of the short stature homeobox contaning SHOX gene has been shown to result in a spectrum of phenotypes ranging from Leri-Weill dyschondrosteosis (LWD) at the more severe end to SHOX-related short stature at the milder end of the spectrum. Most alterations are whole gene deletions, point mutations within the coding region, or microdeletions in its flanking sequences. Here, we present the clinical and molecular data as well as the potential molecular mechanism underlying a novel microdeletion, causing a variable SHOX-related haploinsufficiency disorder in a three-generation family. The phenotype resembles that of LWD in females, in males, however, the phenotypic expression is milder. The 15523-bp SHOX intragenic deletion, encompassing exons 3-6, was initially detected by array-CGH, followed by MLPA analysis. Sequencing of the breakpoints indicated an Alu recombination-mediated deletion (ARMD) as the potential causative mechanism.

CRISPR-Cas9, a tool to efficiently increase the development of recombinant African swine fever viruses.

PubMed

Borca, Manuel V; Holinka, Lauren G; Berggren, Keith A; Gladue, Douglas P

2018-02-16

African swine fever virus (ASFV) causes a highly contagious disease called African swine fever. This disease is often lethal for domestic pigs, causing extensive losses for the swine industry. ASFV is a large and complex double stranded DNA virus. Currently there is no commercially available treatment or vaccine to prevent this devastating disease. Development of recombinant ASFV for producing live-attenuated vaccines or studying the involvement of specific genes in virus virulence has relied on the relatively rare event of homologous recombination in primary swine macrophages, causing difficulty to purify the recombinant virus from the wild-type parental ASFV. Here we present the use of the CRISPR-Cas9 gene editing system as a more robust and efficient system to produce recombinant ASFVs. Using CRISPR-Cas9 a recombinant virus was efficiently developed by deleting the non-essential gene 8-DR from the genome of the highly virulent field strain Georgia07 using swine macrophages as cell substrate.

A recombinant pseudorabies virus co-expressing capsid proteins precursor P1-2A of FMDV and VP2 protein of porcine parvovirus: a trivalent vaccine candidate.

PubMed

Hong, Qi; Qian, Ping; Li, Xiang-Min; Yu, Xiao-Lan; Chen, Huan-Chun

2007-11-01

Pseudorabies (PR), foot-and-mouth disease (FMD), and porcine parvovirus disease are three important infectious diseases in swine worldwide. The gene-deleted pseudorabies virus (PRV) has been used as a live-viral vector to develop multivalent genetic engineering vaccine. In this study, a recombinant PRV, which could co-express protein precursor P1-2A of FMDV and VP2 protein of PPV, was constructed using PRV TK(-)/gE(-)/LacZ(+) mutant as the vector. After homologous recombination and plaque purification, recombinant virus PRV TK(-)/gE(-)/P1-2A-VP2 was acquired and identified. Immunogenicity, safety of the recombinant PRV and its protection against PRV were confirmed in a mouse model by indirect ELISA and serum neutralization test. The results show that the recombinant PRV is a candidate vaccine strain to develop a novel trivalent vaccine against PRV, FMDV and PPV in swine.

Suppressor of fusion, a Fusarium oxysporum homolog of Ndt80, is required for nutrient-dependent regulation of anastomosis.

PubMed

Shahi, Shermineh; Fokkens, Like; Houterman, Petra M; Rep, Martijn

2016-10-01

Heterokaryon formation is an essential step in asexual recombination in Fusarium oxysporum. Filamentous fungi have an elaborate nonself recognition machinery to prevent formation and proliferation of heterokaryotic cells, called heterokaryon incompatibility (HI). In F. oxysporum the regulation of this machinery is not well understood. In Neurospora crassa, Vib-1, a putative transcription factor of the p53-like Ndt80 family of transcription factors, has been identified as global regulator of HI. In this study we investigated the role of the F. oxysporum homolog of Vib-1, called Suf, in vegetative hyphal and conidial anastomosis tube (CAT) fusion and HI. We identified a novel function for an Ndt80 homolog as a nutrient-dependent regulator of anastomosis. Strains carrying the SUF deletion mutation display a hyper-fusion phenotype during vegetative growth as well as germling development. In addition, conidial paring of incompatible SUF deletion strains led to more heterokaryon formation, which is independent of suppression of HI. Our data provides further proof for the divergence in the functions of different members Ndt80 family. We propose that Ndt80 homologs mediate responses to nutrient quality and quantity, with specific responses varying between species. Copyright © 2016 Elsevier Inc. All rights reserved.

Recurrent Deletions and Reciprocal Duplications of 10q11.21q11.23 Including CHAT and SLC18A3 are Likely Mediated by Complex Low-Copy Repeats

PubMed Central

Stankiewicz, Paweł; Kulkarni, Shashikant; Dharmadhikari, Avinash V.; Sampath, Srirangan; Bhatt, Samarth S.; Shaikh, Tamim H.; Xia, Zhilian; Pursley, Amber N.; Cooper, M. Lance; Shinawi, Marwan; Paciorkowski, Alex R.; Grange, Dorothy K.; Noetzel, Michael J.; Saunders, Scott; Simons, Paul; Summar, Marshall; Lee, Brendan; Scaglia, Fernando; Fellmann, Florence; Martinet, Danielle; Beckmann, Jacques S.; Asamoah, Alexander; Platky, Kathryn; Sparks, Susan; Martin, Ann S.; Madan-Khetarpal, Suneeta; Hoover, Jacqueline; Medne, Livija; Bonnemann, Carsten G.; Moeschler, John B.; Vallee, Stephanie E.; Parikh, Sumit; Irwin, Polly; Dalzell, Victoria P.; Smith, Wendy E.; Banks, Valerie C.; Flannery, David B.; Lovell, Carolyn M.; Bellus, Gary A.; Golden-Grant, Kathryn; Gorski, Jerome L.; Kussmann, Jennifer L.; McGregor, Tracy L.; Hamid, Rizwan; Pfotenhauer, Jean; Ballif, Blake C.; Shaw, Chad A.; Kang, Sung-Hae L.; Bacino, Carlos A.; Patel, Ankita; Rosenfeld, Jill A.; Cheung, Sau Wai; Shaffer, Lisa G.

2013-01-01

We report 24 unrelated individuals with deletions and 17 additional cases with duplications at 10q11.21q21.1 identified by chromosomal microarray analysis. The rearrangements range in size from 0.3 to 12 Mb. Nineteen of the deletions and eight duplications are flanked by large, directly oriented segmental duplications of >98% sequence identity, suggesting that nonallelic homologous recombination (NAHR) caused these genomic rearrangements. Nine individuals with deletions and five with duplications have additional copy number changes. Detailed clinical evaluation of 20 patients with deletions revealed variable clinical features, with developmental delay (DD) and/or intellectual disability (ID) as the only features common to a majority of individuals. We suggest that some of the other features present in more than one patient with deletion, including hypotonia, sleep apnea, chronic constipation, gastroesophageal and vesicoureteral refluxes, epilepsy, ataxia, dysphagia, nystagmus, and ptosis may result from deletion of the CHAT gene, encoding choline acetyltransferase, and the SLC18A3 gene, mapping in the first intron of CHAT and encoding vesicular acetylcholine transporter. The phenotypic diversity and presence of the deletion in apparently normal carrier parents suggest that subjects carrying 10q11.21q11.23 deletions may exhibit variable phenotypic expressivity and incomplete penetrance influenced by additional genetic and nongenetic modifiers. PMID:21948486

Homozygous hereditary C3 deficiency due to a partial gene deletion.

PubMed Central

Botto, M; Fong, K Y; So, A K; Barlow, R; Routier, R; Morley, B J; Walport, M J

1992-01-01

The molecular mechanism of C3 deficiency in an Afrikaans patient with recurrent pyogenic infections was studied. Restriction enzyme analysis showed a gene deletion of 800 base pairs (bp) mapping to the alpha chain of C3. Amplification of genomic DNA, using the PCR, demonstrated that the deletion included exons 22 and 23 of the C3 gene. Truncated mRNA was shown in an Epstein-Barr virus-transformed B-cell line by PCR amplification of first-strand cDNA. A consequence of this deletion was that the RNA transcribed 3' to the deletion was out of frame, resulting in formation of a stop codon 19 bp downstream from the deletion. The molecular basis of the deletion was compatible with homologous recombination between two Alu sequences located in introns 21 and 23. An unrelated nonconsanguineous relative and two of a sample of 174 Afrikaans-speaking individuals were heterozygous carriers of the same gene deletion. The wide prevalence of this null allele in this population is probably due to the effects of a small founder population. The presence of this deletion in the C3 gene is not compatible with production of any functional C3, supporting the idea that study of such patients offers a valid model for understanding physiological activities of C3 in vivo in humans. Images PMID:1350678

Insulin-like growth factor-I treatment of children with Laron syndrome (primary growth hormone insensitivity).

PubMed

Laron, Zvi

2008-03-01

Laron syndrome (LS, congenital primary GH insensitivity) is caused by deletions or mutations in the GH receptor gene, resulting in an inability to generate insulin-like growth factor-I (IGF-I). If untreated, the deficiency of IGF-I results in severe dwarfism, as well as skeletal and muscular underdevelopment. The only treatment is the daily administration of recombinant IGF-I. This review summarizes the present experience by several groups worldwide. The main conclusions are: A. The one or two injections regimen result in the same growth velocity; B. The growth velocity obtained with IGF-I administration is smaller than that observed with hGH in children with congenital isolated GH deficiency; C. Overdosage of IGF-I causes a series of adverse effects which can be avoided by carefully monitoring the serum IGF-I and GH levels.

A highly efficient targeted recombination system for engineering linear chromosomes of industrial bacteria Streptomyces.

PubMed

Pan, Hung-Yin; Chen, Carton W; Huang, Chih-Hung

2018-04-17

Soil bacteria Streptomyces are the most important producers of secondary metabolites, including most known antibiotics. These bacteria and their close relatives are unique in possessing linear chromosomes, which typically harbor 20 to 30 biosynthetic gene clusters of tens to hundreds of kb in length. Many Streptomyces chromosomes are accompanied by linear plasmids with sizes ranging from several to several hundred kb. The large linear plasmids also often contain biosynthetic gene clusters. We have developed a targeted recombination procedure for arm exchanges between a linear plasmid and a linear chromosome. A chromosomal segment inserted in an artificially constructed plasmid allows homologous recombination between the two replicons at the homology. Depending on the design, the recombination may result in two recombinant replicons or a single recombinant chromosome with the loss of the recombinant plasmid that lacks a replication origin. The efficiency of such targeted recombination ranges from 9 to 83% depending on the locations of the homology (and thus the size of the chromosomal arm exchanged), essentially eliminating the necessity of selection. The targeted recombination is useful for the efficient engineering of the Streptomyces genome for large-scale deletion, addition, and shuffling.

Gfi1-Cre knock-in mouse line: A tool for inner ear hair cell-specific gene deletion

PubMed Central

Yang, Hua; Gan, Jean; Xie, Xiaoling; Deng, Min; Feng, Liang; Chen, Xiaowei; Gao, Zhiqiang; Gan, Lin

2010-01-01

Summary Gfi1encodes a zinc-finger transcription factor essential for the development and maintenance of haematopoiesis and the inner ear. In mouse inner ear, Gfi1 expression is confined to hair cells during development and in adulthood. To construct a genetic tool for inner ear hair cell-specific gene deletion, we generated a Gfi1-Cre mouse line by knocking-in Cre coding sequences into the Gfi1 locus and inactivating the endogenous Gfi1. The specificity and efficiency of Gfi1-Cre recombinase-mediated recombination in the developing inner ear was revealed through the expression of the conditional R26R-lacZ reporter gene. The onset of lacZ expression in the Gfi1Cre/+ inner ear was first detected at E13.5 in the vestibule and at E15.5 in the cochlea, coinciding with the generation of hair cells. Throughout inner ear development, lacZ expression was detected only in hair cells. Thus, Gfi1-Cre knock-in mouse line provides a useful tool for gene manipulations specifically in inner ear hair cells. PMID:20533399

A non-canonical transferred DNA insertion at the BRI1 locus in Arabidopsis thaliana.

PubMed

Zhao, Zhong; Zhu, Yan; Erhardt, Mathieu; Ruan, Ying; Shen, Wen-Hui

2009-04-01

Agrobacterium-mediated transformation is widely used in transgenic plant engineering and has been proven to be a powerful tool for insertional mutagenesis of the plant genome. The transferred DNA (T-DNA) from Agrobacterium is integrated into the plant genome through illegitimate recombination between the T-DNA and the plant DNA. Contrasting to the canonical insertion, here we report on a locus showing a complex mutation associated with T-DNA insertion at the BRI1 gene in Arabidopsis thaliana. We obtained a mutant line, named salade for its phenotype of dwarf stature and proliferating rosette. Molecular characterization of this mutant revealed that in addition to T-DNA a non-T-DNA-localized transposon from bacteria was inserted in the Arabidopsis genome and that a region of more than 11.5 kb of the Arabidopsis genome was deleted at the insertion site. The deleted region contains the brassinosteroid receptor gene BRI1 and the transcription factor gene WRKY13. Our finding reveals non-canonical T-DNA insertion, implicating horizontal gene transfer and cautioning the use of T-DNA as mutagen in transgenic research.

Going, going, gone: predicting the fate of genomic insertions in plant RNA viruses.

PubMed

Willemsen, Anouk; Carrasco, José L; Elena, Santiago F; Zwart, Mark P

2018-05-10

Horizontal gene transfer is common among viruses, while they also have highly compact genomes and tend to lose artificial genomic insertions rapidly. Understanding the stability of genomic insertions in viral genomes is therefore relevant for explaining and predicting their evolutionary patterns. Here, we revisit a large body of experimental research on a plant RNA virus, tobacco etch potyvirus (TEV), to identify the patterns underlying the stability of a range of homologous and heterologous insertions in the viral genome. We obtained a wide range of estimates for the recombination rate-the rate at which deletions removing the insertion occur-and these appeared to be independent of the type of insertion and its location. Of the factors we considered, recombination rate was the best predictor of insertion stability, although we could not identify the specific sequence characteristics that would help predict insertion instability. We also considered experimentally the possibility that functional insertions lead to higher mutational robustness through increased redundancy. However, our observations suggest that both functional and non-functional increases in genome size decreased the mutational robustness. Our results therefore demonstrate the importance of recombination rates for predicting the long-term stability and evolution of viral RNA genomes and suggest that there are unexpected drawbacks to increases in genome size for mutational robustness.

Sperm-FISH analysis in a pericentric chromosome 1 inversion, 46,XY,inv(1)(p22q42), associated with infertility.

PubMed

Chantot-Bastaraud, S; Ravel, C; Berthaut, I; McElreavey, K; Bouchard, P; Mandelbaum, J; Siffroi, J P

2007-01-01

No phenotypic effect is observed in most inversion heterozygotes. However, reproductive risks may occur in the form of infertility, spontaneous abortions or chromosomally unbalanced children as a consequence of meiotic recombination between inverted and non-inverted chromosomes. An odd number of crossovers within the inverted segment results in gametes bearing recombinant chromosomes with a duplication of the region outside of the inversion segment of one arm and a deletion of the terminal segment of the other arm [dup(p)/del(q) and del(p)/dup(q)]. Using fluorescence in-situ hybridization (FISH), the chromosome segregation of a pericentric inversion of chromosome 1 was studied in spermatozoa of a inv(1)(p22q42) heterozygous carrier. Three-colour FISH was performed on sperm samples using a probe mixture consisting of chromosome 1p telomere-specific probe, chromosome 1q telomere-specific probe and chromosome 18 centromere-specific alpha satellite DNA probe. The frequency of the non-recombinant product was 80.1%. The frequencies of the two types of recombinants carrying a duplication of the short arm and a deletion of the long arm, and vice versa, were respectively 7.6 and 7.2%, and these frequencies were not statistically significant from the expected ratio of 1:1. Sperm-FISH allows the further understanding of segregation patterns and their effect on reproductive failure and allows an accurate genetic counselling.

The molecular biology of environmental aromatic hydrocarbons

DOE Office of Scientific and Technical Information (OSTI.GOV)

Weiss, S.B.

The induction of mutations in living cells by polycyclic aromatic hydrocarbons (PAH) has been recognized for many years. Although the mechanism for this occurrence has been examined by numerous investigators, the precise nature and type of mutations induced is still unclear. Earlier investigations of DNA damage and repair were primarily examined by the random alkylation of bacterial and mammalian DNAs, in vivo, using a variety of different PAH agents. This procedure is still used today. Though informative, such studies have not offered any explanation of the mechanism by which PAH agents induce carcinogenesis. We have attempted to examine the repairmore » of PAH-damaged DNA using small DNA oligomer constructs as targets for site-specific alkylation. DNA constructs containing a single BPDE alkylated site in each duplex strand were ligated into M13 RF DNA and used to transfect E. coli. Progeny M13 DNA was isolated from E. coli colonies grown on agar plates containing IPTG and Xgal. DNA sequence analysis of the isolated progeny M13 DNA, at the site of construct insertion, was found to contain large deletions and illegitimate recombinants. These sequence rearrangements occurred in either recA{sup +} or recA{sup -} host cells suggesting that SOS processing was not involved in the deletions and the recombinants observed. The mechanism by which BPDE induces illegitimate recombinants has not been resolved, however, it is possible that the closely spaced adducts activate the recombinant machinery in our DNA-damaged cells. 1 ref., 6 figs., 1 tab.« less

Recombinant levels of Escherichia coli K-12 mutants deficient in various replication, recombination, or repair genes.

PubMed Central

Zieg, J; Maples, V F; Kushner, S R

1978-01-01

Escherichia coli strains containing mutations in lexA, rep, uvrA, uvrD, uvrE, lig, polA, dam, or xthA were constructed and tested for conjugation and transduction proficiencies and ability to form Lac+ recombinants in an assay system utilizing a nontandem duplication of two partially deleted lactose operons (lacMS286phi80dIIlacBK1). lexA and rep mutants were as deficient (20% of wild type) as recB and recC strains in their ability to produce Lac+ progeny. All the other strains exhibited increased frequencies of Lac+ recombinant formation, compared with wild type, ranging from 2- to 13-fold. Some strains showed markedly increased conjugation proficiency (dam uvrD) compared to wild type, while others appeared deficient (polA107). Some differences in transduction proficiency were also observed. Analysis of the Lac+ recombinants formed by the various mutants indicated that they were identical to the recombinants formed by a wild-type strain. The results indicate that genetic recombination in E. coli is a highly regulated process involving multiple gene products. PMID:350859

Correlation of Meiotic DSB Formation and Transcription Initiation Around Fission Yeast Recombination Hotspots.

PubMed

Yamada, Shintaro; Okamura, Mika; Oda, Arisa; Murakami, Hiroshi; Ohta, Kunihiro; Yamada, Takatomi

2017-06-01

Meiotic homologous recombination, a critical event for ensuring faithful chromosome segregation and creating genetic diversity, is initiated by programmed DNA double-strand breaks (DSBs) formed at recombination hotspots. Meiotic DSB formation is likely to be influenced by other DNA-templated processes including transcription, but how DSB formation and transcription interact with each other has not been understood well. In this study, we used fission yeast to investigate a possible interplay of these two events. A group of hotspots in fission yeast are associated with sequences similar to the cyclic AMP response element and activated by the ATF/CREB family transcription factor dimer Atf1-Pcr1. We first focused on one of those hotspots, ade6-3049 , and Atf1. Our results showed that multiple transcripts, shorter than the ade6 full-length messenger RNA, emanate from a region surrounding the ade6-3049 hotspot. Interestingly, we found that the previously known recombination-activation region of Atf1 is also a transactivation domain, whose deletion affected DSB formation and short transcript production at ade6-3049 These results point to a possibility that the two events may be related to each other at ade6-3049 In fact, comparison of published maps of meiotic transcripts and hotspots suggested that hotspots are very often located close to meiotically transcribed regions. These observations therefore propose that meiotic DSB formation in fission yeast may be connected to transcription of surrounding regions. Copyright © 2017 by the Genetics Society of America.

Mapping of herpes simplex virus-1 neurovirulence to. gamma. sub 1 34. 5, a gene nonessential for growth in culture

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chou, J.; Roizman, B.; Kern, E.R.

1990-11-30

The gene designated {gamma}{sub 1}34.5 maps in the inverted repeats flanking the long unique sequence of herpes simplex virus-1 (HSV-1) DNA, and therefore it is present in two copies per genome. This gene is not essential for viral growth in cell culture. Four recombinant viruses were genetically engineered to test the function of this gene. These were (i) a virus from which both copies of the gene were deleted, (ii) a virus containing a stop codon in both copies of the gene, (iii) a virus containing after the first codon an insert encoding a 16-amino acid epitope known to reactmore » with a specific monoclonal antibody, and (iv) a virus in which the deleted sequences were restored. The viruses from which the gene was deleted or which carried stop codons were avirulent on intracerebral inoculation of mice. The virus with the gene tagged by the sequence encoding the epitope was moderately virulent, whereas the restored virus reacquired the phenotype of the parent virus. Significant amounts of virus were recovered only from brains of animals inoculated with virulent viruses. Inasmuch as the product of the {gamma}{sub 1}34.5 gene extended the host range of the virus by enabling it to replicate and destroy brain cells, it is a viral neurovirulence factor.« less

Switch junction sequences in PMS2-deficient mice reveal a microhomology-mediated mechanism of Ig class switch recombination

PubMed Central

Ehrenstein, Michael R.; Rada, Cristina; Jones, Anne-Marie; Milstein, César; Neuberger, Michael S.

2001-01-01

Isotype switching involves a region-specific, nonhomologous recombinational deletion that has been suggested to occur by nonhomologous joining of broken DNA ends. Here, we find increased donor/acceptor homology at switch junctions from PMS2-deficient mice and propose that class switching can occur by microhomology-mediated end-joining. Interestingly, although isotype switching and somatic hypermutation show many parallels, we confirm that PMS2 deficiency has no major effect on the pattern of nucleotide substitutions generated during somatic hypermutation. This finding is in contrast to MSH2 deficiency. With MSH2, the altered pattern of switch recombination and hypermutation suggests parallels in the mechanics of the two processes, whereas the fact that PMS2 deficiency affects only switch recombination may reflect differences in the pathways of break resolution. PMID:11717399

Repeat-associated plasticity in the Helicobacter pylori RD Gene Family

USDA-ARS?s Scientific Manuscript database

epetitive DNA facilitates genomic flexibility via increased recombination, deletion, and insertion. The bacterium Helicobacter pylori is remarkable for its ability to persist in the human stomach for decades without provoking sterilizing immunity. Examining the genomes of two H. pylori strains, we d...

Imbalanced PTEN and Phosphoinositide 3-kinase signaling impairs class switch recombination1

PubMed Central

Chen, Xiaomi; Dollin, Yonatan; Cambier, John C.; Wang, Jing H.

2015-01-01

Class switch recombination (CSR) generates isotype-switched antibodies with distinct effector functions. B cells express phosphatase and tensin homolog (PTEN) and multiple isoforms of class IA phosphoinositide 3-kinase (PI3K) catalytic subunits, including p110α and p110δ, whose roles in CSR remain unknown or controversial. Here, we demonstrate a direct effect of PTEN on CSR signaling by acute deletion of Pten specifically in mature B cells, thereby excluding the developmental impact of Pten deletion. We show that mature B cell-specific PTEN overexpression enhances CSR. More importantly, we establish a critical role of p110α in CSR. Furthermore, we identify a cooperative role of p110α and p110δ in suppressing CSR. Mechanistically, dysregulation of p110α or PTEN reversely affects activation-induced deaminase expression via modulating AKT activity. Thus, our study reveals that a signaling balance between PTEN and PI3K isoforms is essential to maintain normal CSR. PMID:26500350

Clinical polyomavirus BK variants with agnogene deletion are non-functional but rescued by trans-complementation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Myhre, Marit Renee; Olsen, Gunn-Hege; Gosert, Rainer

High-level replication of polyomavirus BK (BKV) in kidney transplant recipients is associated with the emergence of BKV variants with rearranged (rr) non-coding control region (NCCR) increasing viral early gene expression and cytopathology. Cloning and sequencing revealed the presence of a BKV quasispecies which included non-functional variants when assayed in a recombinant virus assay. Here we report that the rr-NCCR of BKV variants RH-3 and RH-12, both bearing a NCCR deletion including the 5' end of the agnoprotein coding sequence, mediated early and late viral reporter gene expression in kidney cells. However, in a recombinant virus they failed to produce infectiousmore » progeny despite large T-antigen and VP1 expression and the formation of nuclear virus-like particles. Infectious progeny was generated when the agnogene was reconstructed in cis or agnoprotein provided in trans from a co-existing BKV rr-NCCR variant. We conclude that complementation can rescue non-functional BKV variants in vitro and possibly in vivo.« less

Construction of upp deletion mutant strains of Lactobacillus casei and Lactococcus lactis based on counterselective system using temperature-sensitive plasmid.

PubMed

Song, Li; Cui, Hongyu; Tang, Lijie; Qiao, Xinyuan; Liu, Min; Jiang, Yanping; Cui, Wen; Li, Yijing

2014-07-01

Integration plasmids are often used in constructing chromosomal mutations, as it enables the alternation of genes at any location by integration or replacement. Food-grade integration vectors can integrate into the host genome without introducing any selectable markers or residual bases, and the recombination often happens in non-coding region. In this study we used the temperature-sensitive pWV01 replicon to construct 2 chloramphenicol-resistant integration plasmids (pGBHC32-upp) containing the uracil phosphoribosyl transferase (upp) gene as a counterselective marker for Lactobacillus casei (L. casei) ATCC393 and Lactococcus lactis (L. lactis) MG1363. We then ligated the designed homologous arms to the pGBHC32-upp plasmids to allow their integration to the bacterial chromosome, and selected upp deletion mutants of L. casei ATCC393 and L. lactis MG1363 in the presence of 5-fluorouracil (5-FU). Analysis of genetic stability, growth curve, carbon utilization and scanning electronic microscopy showed that, except for 5-FU resistance, there were no significant differences between the wild type and mutant lactic acid bacteria. The integration system and the upp deletion strains could be used in the insertion or deletion of genes at any location of the chromosome of both L. casei ATCC 393 and L. lactis MG1363, and the homologous recombination would not introduce any selectable markers or residual bases. These mutant strains can be further investigated for heterologous protein expression and construction of a live mucosal vaccine carrier. Copyright © 2014 Elsevier B.V. All rights reserved.

Involvement of Escherichia coli K1 ibeT in bacterial adhesion that is associated with the entry into human brain microvascular endothelial cells.

PubMed

Zou, Yanming; He, Lina; Chi, Feng; Jong, Ambrose; Huang, Sheng-He

2008-12-01

IbeT is a downstream gene of the invasion determinant ibeA in the chromosome of a clinical isolate of Escherichia coli K1 strain RS218 (serotype 018:K1:H7). Both ibeT and ibeA are in the same operon. Our previous mutagenesis and complementation studies suggested that ibeT may coordinately contribute to E. coli K1 invasion with ibeA. An isogenic in-frame deletion mutant of ibeT has been made by chromosomal gene replacement with a recombinant suicide vector carrying a fragment with an ibeT internal deletion. The characteristics of the mutant in meningitic E. coli infection were examined in vitro [cell culture of human brain microvascular endothelial cells (HBMEC)] and in vivo (infant rat model of E. coli meningitis) in comparison with the parent strain. The ibeT deletion mutant was significantly less adhesive and invasive than its parent strain E. coli E44 in vitro, and the adhesion- and invasion-deficient phenotypes of the mutant can be complemented by the ibeT gene. Recombinant IbeT protein is able to block E. coli E44 invasion of HBMEC. Furthermore, the ibeT deletion mutant is less capable of colonizing intestine and less virulent in bacterial translocation across the blood-brain barrier (BBB) than its parent E. coli E44 in vivo. These data suggest that ibeT-mediated E. coli K1 adhesion is associated with the bacterial invasion process.

Immune Response to Recombinant Adenovirus in Humans: Capsid Components from Viral Input Are Targets for Vector-Specific Cytotoxic T Lymphocytes

PubMed Central

Molinier-Frenkel, Valérie; Gahery-Segard, Hanne; Mehtali, Majid; Le Boulaire, Christophe; Ribault, Sébastien; Boulanger, Pierre; Tursz, Thomas; Guillet, Jean-Gérard; Farace, Françoise

2000-01-01

We previously demonstrated that a single injection of 109 PFU of recombinant adenovirus into patients induces strong vector-specific immune responses (H. Gahéry-Ségard, V. Molinier-Frenkel, C. Le Boulaire, P. Saulnier, P. Opolon, R. Lengagne, E. Gautier, A. Le Cesne, L. Zitvogel, A. Venet, C. Schatz, M. Courtney, T. Le Chevalier, T. Tursz, J.-G. Guillet, and F. Farace, J. Clin. Investig. 100:2218–2226, 1997). In the present study we analyzed the mechanism of vector recognition by cytotoxic T lymphocytes (CTL). CD8+ CTL lines were derived from two patients and maintained in long-term cultures. Target cell infections with E1-deleted and E1-plus E2-deleted adenoviruses, as well as transcription-blocking experiments with actinomycin D, revealed that host T-cell recognition did not require viral gene transcription. Target cells treated with brefeldin A were not lysed, indicating that viral input protein-derived peptides are associated with HLA class I molecules. Using recombinant capsid component-loaded targets, we observed that the three major proteins could be recognized. These results raise the question of the use of multideleted adenoviruses for gene therapy in the quest to diminish antivector CTL responses. PMID:10906225

Characterization of contiguous gene deletions in COL4A6 and COL4A5 in Alport syndrome-diffuse leiomyomatosis.

PubMed

Nozu, Kandai; Minamikawa, Shogo; Yamada, Shiro; Oka, Masafumi; Yanagita, Motoko; Morisada, Naoya; Fujinaga, Shuichiro; Nagano, China; Gotoh, Yoshimitsu; Takahashi, Eihiko; Morishita, Takahiro; Yamamura, Tomohiko; Ninchoji, Takeshi; Kaito, Hiroshi; Morioka, Ichiro; Nakanishi, Koichi; Vorechovsky, Igor; Iijima, Kazumoto

2017-07-01

Alport syndrome-diffuse leiomyomatosis (AS-DL, OMIM: 308940) is a rare variant of the X-linked Alport syndrome that shows overgrowth of visceral smooth muscles in the gastrointestinal, respiratory and female reproductive tracts in addition to renal symptoms. AS-DL results from deletions that encompass the 5' ends of the COL4A5 and COL4A6 genes, but deletion breakpoints between COL4A5 and COL4A6 have been determined in only four cases. Here, we characterize deletion breakpoints in five AS-DL patients and show a contiguous COL4A6/COL4A5 deletion in each case. We also demonstrate that eight out of nine deletion alleles involved sequences homologous between COL4A5 and COL4A6. Most breakpoints took place in recognizable transposed elements, including long and short interspersed repeats, DNA transposons and long-terminal repeat retrotransposons. Because deletions involved the bidirectional promoter region in each case, we suggest that the occurrence of leiomyomatosis in AS-DL requires inactivation of both genes. Altogether, our study highlights the importance of homologous recombination involving multiple transposed elements for the development of this continuous gene syndrome and other atypical loss-of-function phenotypes.

Phase I study of BAY 94-9027, a PEGylated B-domain-deleted recombinant factor VIII with an extended half-life, in subjects with hemophilia A.

PubMed

Coyle, T E; Reding, M T; Lin, J C; Michaels, L A; Shah, A; Powell, J

2014-04-01

BAY 94-9027 is a B-domain-deleted recombinant factor VIII (rFVIII) with site-specific attachment of poly(ethylene glycol) that has shown an extended half-life in animal models of hemophilia. To assess the pharmacokinetics and safety of BAY 94-9027 after single and repeated administration in subjects with severe hemophilia A. This 8-week, prospective, multicenter, open-label, phase I trial was conducted in 14 subjects aged 21–58 years with FVIII of < 1%, ≥ 150 days of exposure to FVIII, and no history of FVIII inhibitors. After a ≥ 3-day washout, subjects received a single dose of sucrose-formulated rFVIII (rFVIII-FS) (cohort 1 [n = 7], 25 IU kg−1; cohort 2 [n = 7], 50 IU kg−1) for a 48-h pharmacokinetic (PK) study. After another ≥ 3-day washout, cohort 1 received twice-weekly BAY 94-9027 at 25 IU kg−1 (16 doses), and cohort 2 received once-weekly BAY 94-9027 at 60 IU kg−1 (nine doses). A 168-h PK study was performed after the first and last BAY 94-9027 doses. BAY 94-9027 showed equivalent recovery and an improved PK profile vs. rFVIII-FS, with a half-life of ~ 19 h (vs. ~ 13.0 h for rFVIII-FS). BAY 94-9027 was well tolerated, and no immunogenicity was observed. This phase I study demonstrates that BAY 94-9027 has an extended half-life in subjects with hemophilia A and, after multiple dosing, was well tolerated with no immunogenicity during the 8-week trial. A phase III study in a larger number of subjects is underway to fully characterize how this prolonged half-life will permit less frequent prophylaxis dosing for patients with hemophilia.

Recombination in Enteroviruses Is a Biphasic Replicative Process Involving the Generation of Greater-than Genome Length ‘Imprecise’ Intermediates

PubMed Central

Lowry, Kym; Woodman, Andrew; Cook, Jonathan; Evans, David J.

2014-01-01

Recombination in enteroviruses provides an evolutionary mechanism for acquiring extensive regions of novel sequence, is suggested to have a role in genotype diversity and is known to have been key to the emergence of novel neuropathogenic variants of poliovirus. Despite the importance of this evolutionary mechanism, the recombination process remains relatively poorly understood. We investigated heterologous recombination using a novel reverse genetic approach that resulted in the isolation of intermediate chimeric intertypic polioviruses bearing genomes with extensive duplicated sequences at the recombination junction. Serial passage of viruses exhibiting such imprecise junctions yielded progeny with increased fitness which had lost the duplicated sequences. Mutations or inhibitors that changed polymerase fidelity or the coalescence of replication complexes markedly altered the yield of recombinants (but did not influence non-replicative recombination) indicating both that the process is replicative and that it may be possible to enhance or reduce recombination-mediated viral evolution if required. We propose that extant recombinants result from a biphasic process in which an initial recombination event is followed by a process of resolution, deleting extraneous sequences and optimizing viral fitness. This process has implications for our wider understanding of ‘evolution by duplication’ in the positive-strand RNA viruses. PMID:24945141

The Unstructured Paramyxovirus Nucleocapsid Protein Tail Domain Modulates Viral Pathogenesis through Regulation of Transcriptase Activity.

PubMed

Thakkar, Vidhi D; Cox, Robert M; Sawatsky, Bevan; da Fontoura Budaszewski, Renata; Sourimant, Julien; Wabbel, Katrin; Makhsous, Negar; Greninger, Alexander L; von Messling, Veronika; Plemper, Richard K

2018-04-15

The paramyxovirus replication machinery comprises the viral large (L) protein and phosphoprotein (P-protein) in addition to the nucleocapsid (N) protein, which encapsidates the single-stranded RNA genome. Common to paramyxovirus N proteins is a C-terminal tail (Ntail). The mechanistic role and relevance for virus replication of the structurally disordered central Ntail section are unknown. Focusing initially on members of the Morbillivirus genus, a series of measles virus (MeV) and canine distemper virus (CDV) N proteins were generated with internal deletions in the unstructured tail section. N proteins with large tail truncations remained bioactive in mono- and polycistronic minireplicon assays and supported efficient replication of recombinant viruses. Bioactivity of Ntail mutants extended to N proteins derived from highly pathogenic Nipah virus. To probe an effect of Ntail truncations on viral pathogenesis, recombinant CDVs were analyzed in a lethal CDV/ferret model of morbillivirus disease. The recombinant viruses displayed different stages of attenuation ranging from ameliorated clinical symptoms to complete survival of infected animals, depending on the molecular nature of the Ntail truncation. Reinfection of surviving animals with pathogenic CDV revealed robust protection against a lethal challenge. The highly attenuated virus was genetically stable after ex vivo passaging and recovery from infected animals. Mechanistically, gradual viral attenuation coincided with stepwise altered viral transcriptase activity in infected cells. These results identify the central Ntail section as a determinant for viral pathogenesis and establish a novel platform to engineer gradual virus attenuation for next-generation paramyxovirus vaccine design. IMPORTANCE Investigating the role of the paramyxovirus N protein tail domain (Ntail) in virus replication, we demonstrated in this study that the structurally disordered central Ntail region is a determinant for viral pathogenesis. We show that internal deletions in this Ntail region of up to 55 amino acids in length are compatible with efficient replication of recombinant viruses in cell culture but result in gradual viral attenuation in a lethal canine distemper virus (CDV)/ferret model. Mechanistically, we demonstrate a role of the intact Ntail region in the regulation of viral transcriptase activity. Recombinant viruses with Ntail truncations induce protective immunity against lethal challenge of ferrets with pathogenic CDV. This identification of the unstructured central Ntail domain as a nonessential paramyxovirus pathogenesis factor establishes a foundation for harnessing Ntail truncations for vaccine engineering against emerging and reemerging members of the paramyxovirus family. Copyright © 2018 American Society for Microbiology.

Expression of deleted, atoxic atypical recombinant beta2 toxin in a baculovirus system and production of polyclonal and monoclonal antibodies.

PubMed

Serroni, Anna; Magistrali, Chiara Francesca; Pezzotti, Giovanni; Bano, Luca; Pellegrini, Martina; Severi, Giulio; Di Pancrazio, Chiara; Luciani, Mirella; Tittarelli, Manuela; Tofani, Silvia; De Giuseppe, Antonio

2017-05-25

Clostridium perfringens is an important animal and human pathogen that can produce more than 16 different major and minor toxins. The beta-2 minor toxin (CPB2), comprising atypical and consensus variants, appears to be involved in both human and animal enterotoxaemia syndrome. The exact role of CPB2 in pathogenesis is poorly investigated, and its mechanism of action at the molecular level is still unknown because of the lack of specific reagents such as monoclonal antibodies against the CPB2 protein and/or the availability of a highly purified antigen. Previous studies have reported that purified wild-type or recombinant CPB2 toxin, expressed in a heterologous system, presented cytotoxic effects on human intestinal cell lines. Undoubtedly, for this reason, to date, these purified proteins have not yet been used for the production of monoclonal antibodies (MAbs). Recently, monoclonal antibodies against CPB2 were generated using peptides designed on predicted antigenic epitopes of this toxin. In this paper we report, for the first time, the expression in a baculovirus system of a deleted recombinant C-terminal 6xHis-tagged atypical CPB2 toxin (rCPB2 Δ1-25 -His 6 ) lacking the 25 amino acids (aa) of the N-terminal putative signal sequence. A high level of purified recombinant rCPB2 Δ1-25 -His 6 was obtained after purification by Ni 2+ affinity chromatography. The purified product showed no in vitro and in vivo toxicity. Polyclonal antibodies and twenty hybridoma-secreting Mabs were generated using purified rCPB2 Δ1-25 -His 6 . Finally, the reactivity and specificity of the new antibodies were tested against both recombinant and wild-type CPB2 toxins. The high-throughput of purified atoxic recombinant CPB2 produced in insect cells, allowed to obtain monoclonal and polyclonal antibodies. The availability of these molecules could contribute to develop immunoenzymatic methods and/or to perform studies about the biological activity of CPB2 toxin.

Genomic characterization of two large Alu-mediated rearrangements of the BRCA1 gene.

PubMed

Peixoto, Ana; Pinheiro, Manuela; Massena, Lígia; Santos, Catarina; Pinto, Pedro; Rocha, Patrícia; Pinto, Carla; Teixeira, Manuel R

2013-02-01

To determine whether a large genomic rearrangement is actually novel and to gain insight about the mutational mechanism responsible for its occurrence, molecular characterization with breakpoint identification is mandatory. We here report the characterization of two large deletions involving the BRCA1 gene. The first rearrangement harbored a 89,664-bp deletion comprising exon 7 of the BRCA1 gene to exon 11 of the NBR1 gene (c.441+1724_oNBR1:c.1073+480del). Two highly homologous Alu elements were found in the genomic sequences flanking the deletion breakpoints. Furthermore, a 20-bp overlapping sequence at the breakpoint junction was observed, suggesting that the most likely mechanism for the occurrence of this rearrangement was nonallelic homologous recombination. The second rearrangement fully characterized at the nucleotide level was a BRCA1 exons 11-15 deletion (c.671-319_4677-578delinsAlu). The case harbored a 23,363-bp deletion with an Alu element inserted at the breakpoints of the deleted region. As the Alu element inserted belongs to a still active AluY family, the observed rearrangement could be due to an insertion-mediated deletion mechanism caused by Alu retrotransposition. To conclude, we describe the breakpoints of two novel large deletions involving the BRCA1 gene and analysis of their genomic context allowed us to gain insight about the respective mutational mechanism.

Identification and characterization of cryptic SHOX intragenic deletions in three Japanese patients with Léri-Weill dyschondrosteosis.

PubMed

Fukami, Maki; Dateki, Sumito; Kato, Fumiko; Hasegawa, Yukihiro; Mochizuki, Hiroshi; Horikawa, Reiko; Ogata, Tsutomu

2008-01-01

Although short-stature homeobox-containing gene (SHOX ) haploinsufficiency is responsible for Léri-Weill dyschondrosteosis (LWD), the molecular defect has not been identified in approximately 20% of Japanese LWD patients. Furthermore, although high prevalence of microdeletions affecting SHOX is primarily ascribed to the presence of repeat sequences such as Alu elements around SHOX, it remains to be determined whether microdeletions are actually mediated by repeat sequences. We performed multiple ligation probe amplification (MLPA) assay in six Japanese LWD patients with apparently normal SHOX, followed by fluorescent in situ hybridization (FISH) analysis and sequencing for polymerase chain reaction (PCR) products encompassing the deletion junctions in patients with abnormal MLPA patterns. Consequently, heterozygous intragenic deletions were identified in three cases, i.e., a 5,906-bp deletion involving exons 4-5 in case 1, a 5,594-bp deletion involving exons 4-6a in case 2, and a 50,199-bp deletion involving exons 4-6b in case 3. The deletion breakpoints of cases 1 and 2 were present in nonrepeat sequences, whereas those of case 3 resided within Alu elements. The results suggest that cryptic SHOX intragenic deletions account for a small fraction of LWD and that microdeletions affecting SHOX can be generated by repeat-sequence-mediated aberrant recombinations and by nonhomologous end joining.

Structural characterization of the α-mating factor prepro-peptide for secretion of recombinant proteins in Pichia pastoris.

PubMed

Chahal, Sabreen; Wei, Peter; Moua, Pachai; Park, Sung Pil James; Kwon, Janet; Patel, Arth; Vu, Anthony T; Catolico, Jason A; Tsai, Yu Fang Tina; Shaheen, Nadia; Chu, Tiffany T; Tam, Vivian; Khan, Zill-E-Huma; Joo, Hyun Henry; Xue, Liang; Lin-Cereghino, Joan; Tsai, Jerry W; Lin-Cereghino, Geoff P

2017-01-20

The methylotrophic yeast Pichia pastoris has been used extensively for expressing recombinant proteins because it combines the ease of genetic manipulation, the ability to provide complex posttranslational modifications and the capacity for efficient protein secretion. The most successful and commonly used secretion signal leader in Pichia pastoris has been the alpha mating factor (MATα) prepro secretion signal. However, limitations exist as some proteins cannot be secreted efficiently, leading to strategies to enhance secretion efficiency by modifying the secretion signal leader. Based on a Jpred secondary structure prediction and knob-socket modeling of tertiary structure, numerous deletions and duplications of the MATα prepro leader were engineered to evaluate the correlation between predicted secondary structure and the secretion level of the reporters horseradish peroxidase (HRP) and Candida antarctica lipase B. In addition, circular dichroism analyses were completed for the wild type and several mutant pro-peptides to evaluate actual differences in secondary structure. The results lead to a new model of MATα pro-peptide signal leader, which suggests that the N and C-termini of MATα pro-peptide need to be presented in a specific orientation for proper interaction with the cellular secretion machinery and for efficient protein secretion. Copyright © 2016 Elsevier B.V. All rights reserved.

Performance of an ultra-sensitive Plasmodium falciparum HRP2-based rapid diagnostic test with recombinant HRP2, culture parasites, and archived whole blood samples.

PubMed

Das, Smita; Peck, Roger B; Barney, Rebecca; Jang, Ihn Kyung; Kahn, Maria; Zhu, Meilin; Domingo, Gonzalo J

2018-03-17

As malaria endemic countries shift from control to elimination, the proportion of low density Plasmodium falciparum infections increases. Current field diagnostic tools, such as microscopy and rapid diagnostic tests (RDT), with detection limits of approximately 100-200 parasites/µL (p/µL) and 800-1000 pg/mL histidine-rich protein 2 (HRP2), respectively, are unable to detect these infections. A novel ultra-sensitive HRP2-based Alere™ Malaria Ag P.f RDT (uRDT) was evaluated in laboratory conditions to define the test's performance against recombinant HRP2 and native cultured parasites. The uRDT detected dilutions of P. falciparum recombinant GST-W2 and FliS-W2, as well as cultured W2 and ITG, diluted in whole blood down to 10-40 pg/mL HRP2, depending on the protein tested. uRDT specificity was 100% against 123 archived frozen whole blood samples. Rapid test cross-reactivity with HRP3 was investigated using pfhrp2 gene deletion strains D10 and Dd2, pfhrp3 gene deletion strain HB3, and controls pfhrp2 and pfhrp3 double deletion strain 3BD5 and pfhrp2 and pfhrp3 competent strain ITG. The commercial Standard Diagnostics, Inc. BIOLINE Malaria Ag P.f RDT (SD-RDT) and uRDT detected pfhrp2 positive strains down to 49 and 3.13 p/µL, respectively. The pfhrp2 deletion strains were detected down to 98 p/µL by both tests. The performance of the uRDT was variable depending on the protein, but overall showed a greater than 10-fold improvement over the SD-RDT. The uRDT also exhibited excellent specificity and showed the same cross-reactivity with HRP3 as the SD-RDT. Together, the results support the uRDT as a more sensitive HRP2 test that could be a potentially effective tool in elimination campaigns. Further clinical evaluations for this purpose are merited.

Circularized Chromosome with a Large Palindromic Structure in Streptomyces griseus Mutants

PubMed Central

Uchida, Tetsuya; Ishihara, Naoto; Zenitani, Hiroyuki; Hiratsu, Keiichiro; Kinashi, Haruyasu

2004-01-01

Streptomyces linear chromosomes display various types of rearrangements after telomere deletion, including circularization, arm replacement, and amplification. We analyzed the new chromosomal deletion mutants Streptomyces griseus 301-22-L and 301-22-M. In these mutants, chromosomal arm replacement resulted in long terminal inverted repeats (TIRs) at both ends; different sizes were deleted again and recombined inside the TIRs, resulting in a circular chromosome with an extremely large palindrome. Short palindromic sequences were found in parent strain 2247, and these sequences might have played a role in the formation of this unique structure. Dynamic structural changes of Streptomyces linear chromosomes shown by this and previous studies revealed extraordinary strategies of members of this genus to keep a functional chromosome, even if it is linear or circular. PMID:15150216

Identification of novel deletions of 15q11q13 in Angelman syndrome by array-CGH: molecular characterization and genotype-phenotype correlations.

PubMed

Sahoo, Trilochan; Bacino, Carlos A; German, Jennifer R; Shaw, Chad A; Bird, Lynne M; Kimonis, Virginia; Anselm, Irinia; Waisbren, Susan; Beaudet, Arthur L; Peters, Sarika U

2007-09-01

Angelman syndrome (AS) is a neurodevelopmental disorder characterized by mental retardation, absent speech, ataxia, and a happy disposition. Deletions of the 15q11q13 region are found in approximately 70% of AS patients. The deletions are sub-classified into class I and class II based on their sizes of approximately 6.8 and approximately 6.0, respectively, with two different proximal breakpoints and a common distal breakpoint. Utilizing a chromosome 15-specific comparative genomic hybridization genomic microarray (array-CGH), we have identified, determined the deletion sizes, and mapped the breakpoints in a cohort of 44 cases, to relate those breakpoints to the genomic architecture and derive more precise genotype-phenotype correlations. Interestingly four patients of the 44 studied (9.1%) had novel and unusually large deletions, and are reported here. This is the first report of very large deletions of 15q11q13 resulting in AS; the largest deletion being >10.6 Mb. These novel deletions involve three different distal breakpoints, two of which have been earlier shown to be involved in the generation of isodicentric 15q chromosomes (idic15). Additionally, precise determination of the deletion breakpoints reveals the presence of directly oriented low-copy repeats (LCRs) flanking the recurrent and novel breakpoints. The LCRs are adequate in size, orientation, and homology to enable abnormal recombination events leading to deletions and duplications. This genomic organization provides evidence for a common mechanism for the generation of both common and rare deletion types. Larger deletions result in a loss of several genes outside the common Angelman syndrome-Prader-Willi syndrome (AS-PWS) critical interval, and a more severe phenotype.

[Chromosomal large fragment deletion induced by CRISPR/Cas9 gene editing system].

PubMed

Cheng, L H; Liu, Y; Niu, T

2017-05-14

Objective: Using CRISPR-Cas9 gene editing technology to achieve a number of genes co-deletion on the same chromosome. Methods: CRISPR-Cas9 lentiviral plasmid that could induce deletion of Aloxe3-Alox12b-Alox8 cluster genes located on mouse 11B3 chromosome was constructed via molecular clone. HEK293T cells were transfected to package lentivirus of CRISPR or Cas9 cDNA, then mouse NIH3T3 cells were infected by lentivirus and genomic DNA of these cells was extracted. The deleted fragment was amplified by PCR, TA clone, Sanger sequencing and other techniques were used to confirm the deletion of Aloxe3-Alox12b-Alox8 cluster genes. Results: The CRISPR-Cas9 lentiviral plasmid, which could induce deletion of Aloxe3-Alox12b-Alox8 cluster genes, was successfully constructed. Deletion of target chromosome fragment (Aloxe3-Alox12b-Alox8 cluster genes) was verified by PCR. The deletion of Aloxe3-Alox12b-Alox8 cluster genes was affirmed by TA clone, Sanger sequencing, and the breakpoint junctions of the CRISPR-Cas9 system mediate cutting events were accurately recombined, insertion mutation did not occur between two cleavage sites at all. Conclusion: Large fragment deletion of Aloxe3-Alox12b-Alox8 cluster genes located on mouse chromosome 11B3 was successfully induced by CRISPR-Cas9 gene editing system.

Hypervirulent Chlamydia trachomatis Clinical Strain Is a Recombinant between Lymphogranuloma Venereum (L2) and D Lineages

PubMed Central

Somboonna, Naraporn; Wan, Raymond; Ojcius, David M.; Pettengill, Matthew A.; Joseph, Sandeep J.; Chang, Alexander; Hsu, Ray; Read, Timothy D.; Dean, Deborah

2011-01-01

ABSTRACT Chlamydia trachomatis is an obligate intracellular bacterium that causes a diversity of severe and debilitating diseases worldwide. Sporadic and ongoing outbreaks of lymphogranuloma venereum (LGV) strains among men who have sex with men (MSM) support the need for research on virulence factors associated with these organisms. Previous analyses have been limited to single genes or genomes of laboratory-adapted reference strain L2/434 and outbreak strain L2b/UCH-1/proctitis. We characterized an unusual LGV strain, termed L2c, isolated from an MSM with severe hemorrhagic proctitis. L2c developed nonfusing, grape-like inclusions and a cytotoxic phenotype in culture, unlike the LGV strains described to date. Deep genome sequencing revealed that L2c was a recombinant of L2 and D strains with conserved clustered regions of genetic exchange, including a 78-kb region and a partial, yet functional, toxin gene that was lost with prolonged culture. Indels (insertions/deletions) were discovered in an ftsK gene promoter and in the tarp and hctB genes, which encode key proteins involved in replication, inclusion formation, and histone H1-like protein activity, respectively. Analyses suggest that these indels affect gene and/or protein function, supporting the in vitro and disease phenotypes. While recombination has been known to occur for C. trachomatis based on gene sequence analyses, we provide the first whole-genome evidence for recombination between a virulent, invasive LGV strain and a noninvasive common urogenital strain. Given the lack of a genetic system for producing stable C. trachomatis mutants, identifying naturally occurring recombinants can clarify gene function and provide opportunities for discovering avenues for genomic manipulation. PMID:21540364

A simple and effective strategy for solving the problem of inclusion bodies in recombinant protein technology: His-tag deletions enhance soluble expression.

PubMed

Zhu, Shaozhou; Gong, Cuiyu; Ren, Lu; Li, Xingzhou; Song, Dawei; Zheng, Guojun

2013-01-01

The formation of inclusion bodies (IBs) in recombinant protein biotechnology has become one of the most frequent undesirable occurrences in both research and industrial applications. So far, the pET System is the most powerful system developed for the production of recombinant proteins when Escherichia coli is used as the microbial cell factory. Also, using fusion tags to facilitate detection and purification of the target protein is a commonly used tactic. However, there is still a large fraction of proteins that cannot be produced in E. coli in a soluble (and hence functional) form. Intensive research efforts have tried to address this issue, and numerous parameters have been modulated to avoid the formation of inclusion bodies. However, hardly anyone has noticed that adding fusion tags to the recombinant protein to facilitate purification is a key factor that affects the formation of inclusion bodies. To test this idea, the industrial biocatalysts uridine phosphorylase from Aeropyrum pernix K1 and (+)-γ-lactamase and (-)-γ-lactamase from Bradyrhizobium japonicum USDA 6 were expressed in E. coli by using the pET System and then examined. We found that using a histidine tag as a fusion partner for protein expression did affect the formation of inclusion bodies in these examples, suggesting that removing the fusion tag can promote the solubility of heterologous proteins. The production of soluble and highly active uridine phosphorylase, (+)-γ-lactamase, and (-)-γ-lactamase in our results shows that the traditional process needs to be reconsidered. Accordingly, a simple and efficient structure-based strategy for the production of valuable soluble recombinant proteins in E. coli is proposed.

Epstein-Barr virus recombinants from overlapping cosmid fragments.

PubMed

Tomkinson, B; Robertson, E; Yalamanchili, R; Longnecker, R; Kieff, E

1993-12-01

Five overlapping type 1 Epstein-Barr virus (EBV) DNA fragments constituting a complete replication- and transformation-competent genome were cloned into cosmids and transfected together into P3HR-1 cells, along with a plasmid encoding the Z immediate-early activator of EBV replication. P3HR-1 cells harbor a type 2 EBV which is unable to transform primary B lymphocytes because of a deletion of DNA encoding EBNA LP and EBNA 2, but the P3HR-1 EBV can provide replication functions in trans and can recombine with the transfected cosmids. EBV recombinants which have the type 1 EBNA LP and 2 genes from the transfected EcoRI-A cosmid DNA were selectively and clonally recovered by exploiting the unique ability of the recombinants to transform primary B lymphocytes into lymphoblastoid cell lines. PCR and immunoblot analyses for seven distinguishing markers of the type 1 transfected DNAs identified cell lines infected with EBV recombinants which had incorporated EBV DNA fragments beyond the transformation marker-rescuing EcoRI-A fragment. Approximately 10% of the transforming virus recombinants had markers mapping at 7, 46 to 52, 93 to 100, 108 to 110, 122, and 152 kbp from the 172-kbp transfected genome. These recombinants probably result from recombination among the transfected cosmid-cloned EBV DNA fragments. The one recombinant virus examined in detail by Southern blot analysis has all the polymorphisms characteristic of the transfected type 1 cosmid DNA and none characteristic of the type 2 P3HR-1 EBV DNA. This recombinant was wild type in primary B-lymphocyte infection, growth transformation, and lytic replication. Overall, the type 1 EBNA 3A gene was incorporated into 26% of the transformation marker-rescued recombinants, a frequency which was considerably higher than that observed in previous experiments with two-cosmid EBV DNA cotransfections into P3HR-1 cells (B. Tomkinson and E. Kieff, J. Virol. 66:780-789, 1992). Of the recombinants which had incorporated the marker-rescuing cosmid DNA fragment and the fragment encoding the type 1 EBNA 3A gene, most had incorporated markers from at least two other transfected cosmid DNA fragments, indicating a propensity for multiple homologous recombinations. The frequency of incorporation of the nonselected transfected type 1 EBNA 3C gene, which is near the end of two of the transfected cosmids, was 26% overall, versus 3% in previous experiments using transfections with two EBV DNA cosmids. In contrast, the frequency of incorporation of a 12-kb EBV DNA deletion which was near the end of two of the transfected cosmids was only 13%.(ABSTRACT TRUNCATED AT 400 WORDS)

The frequency of previously undetectable deletions involving 3' Exons of the PMS2 gene.

PubMed

Vaughn, Cecily P; Baker, Christine L; Samowitz, Wade S; Swensen, Jeffrey J

2013-01-01

Lynch syndrome is characterized by mutations in one of four mismatch repair genes, MLH1, MSH2, MSH6, or PMS2. Clinical mutation analysis of these genes includes sequencing of exonic regions and deletion/duplication analysis. However, detection of deletions and duplications in PMS2 has previously been confined to Exons 1-11 due to gene conversion between PMS2 and the pseudogene PMS2CL in the remaining 3' exons (Exons 12-15). We have recently described an MLPA-based method that permits detection of deletions of PMS2 Exons 12-15; however, the frequency of such deletions has not yet been determined. To address this question, we tested for 3' deletions in 58 samples that were reported to be negative for PMS2 mutations using previously available methods. All samples were from individuals whose tumors exhibited loss of PMS2 immunohistochemical staining without concomitant loss of MLH1 immunostaining. We identified seven samples in this cohort with deletions in the 3' region of PMS2, including three previously reported samples with deletions of Exons 13-15 (two samples) and Exons 14-15. Also detected were deletions of Exons 12-15, Exon 13, and Exon 14 (two samples). Breakpoint analysis of the intragenic deletions suggests they occurred through Alu-mediated recombination. Our results indicate that ∼12% of samples suspected of harboring a PMS2 mutation based on immunohistochemical staining, for which mutations have not yet been identified, would benefit from testing using the new methodology. Copyright © 2012 Wiley Periodicals, Inc.

Recombined sequences between the non-coding control regions of JC and BK viruses found in the urine of a renal transplantation patient.

PubMed

Liaw, Yu-Ching; Chen, Cheng-Hsu; Shu, Kuo-Hsiung; Fang, Chiung-Yao; Ou, Wei-Chih; Chen, Pei-Lain; Shen, Cheng-Huang; Lin, Mien-Chun; Chang, Deching; Wang, Meilin

2012-12-01

Kidney cells are the common host for JC virus (JCV) and BK virus (BKV). Reactivation of JCV and/or BKV in patients after organ transplantation, such as renal transplantation, may cause hemorrhagic cystitis and polyomavirus-associated nephropathy. Furthermore, JCV and BKV may be shed in the urine after reactivation in the kidney. Rearranged as well as archetypal non-coding control regions (NCCRs) of JCV and BKV have been frequently identified in human samples. In this study, three JC/BK recombined NCCR sequences were identified in the urine of a patient who had undergone renal transplantation. They were designated as JC-BK hybrids 1, 2, and 3. The three JC/BK recombinant NCCRs contain up-stream JCV as well as down-stream BKV sequences. Deletions of both JCV and BKV sequences were found in these recombined NCCRs. Recombination of DNA sequences between JCV and BKV may occur during co-infection due to the relatively high homology of the two viral genomes.

SPAR1/RTEL1 maintains genomic stability by suppressing homologous recombination

PubMed Central

Barber, Louise J.; Youds, Jillian L.; Ward, Jordan D.; McIlwraith, Michael J.; O’Neil, Nigel J.; Petalcorin, Mark I.R.; Martin, Julie S.; Collis, Spencer J.; Cantor, Sharon B.; Auclair, Melissa; Tissenbaum, Heidi; West, Stephen C.; Rose, Ann M.; Boulton, Simon J.

2013-01-01

SUMMARY Inappropriate homologous recombination (HR) can cause gross chromosomal rearrangements that in mammalian cells may lead to tumorigenesis. In yeast, the Srs2 protein is an anti-recombinase that eliminates inappropriate recombination events, but the functional equivalent of Srs2 in higher eukaryotes has proven to be elusive. In this work, we identify C. elegans SPAR-1 as a functional analogue of Srs2 and describe its vertebrate counterpart, SPAR1/RTEL1, which is required for genome stability and tumour avoidance. We find that spar-1 mutant worms and SPAR1 knockdown human cells share characteristic phenotypes with yeast srs2 mutants, including inviability upon deletion of the sgs1/BLM homologue, hyper-recombination, and DNA damage sensitivity. In vitro, purified human SPAR1 antagonises HR by promoting the disassembly of D loop recombination intermediates in a reaction dependent upon ATP hydrolysis. We propose that loss of HR control following deregulation of SPAR1/RTEL1 may be a critical event that drives genome instability and cancer. PMID:18957201

Primary growth hormone insensitivity (Laron syndrome) and acquired hypothyroidism: a case report

PubMed Central

2011-01-01

Introduction Primary growth hormone resistance or growth hormone insensitivity syndrome, also known as Laron syndrome, is a hereditary disease caused by deletions or different types of mutations in the growth hormone receptor gene or by post-receptor defects. This disorder is characterized by a clinical appearance of severe growth hormone deficiency with high levels of circulating growth hormone in contrast to low serum insulin-like growth factor 1 values. Case presentation We report the case of a 15-year-old Caucasian girl who was diagnosed with Silver-Russell syndrome at the age of four and a half years. Recombinant growth hormone was administered for 18 months without an appropriate increase in growth velocity. At the age of seven years, her serum growth hormone levels were high, and an insulin-like growth factor 1 generation test did not increase insulin-like growth factor 1 levels (baseline insulin-like growth factor 1 levels, 52 μg/L; reference range, 75 μg/L to 365 μg/L; and peak, 76 μg/L and 50 μg/L after 12 and 84 hours, respectively, from baseline). The genetic analysis showed that the patient was homozygous for the R217X mutation in the growth hormone receptor gene, which is characteristic of Laron syndrome. On the basis of these results, the diagnosis of primary growth hormone insensitivity syndrome was made, and recombinant insulin-like growth factor 1 therapy was initiated. The patient's treatment was well tolerated, but unexplained central hypothyroidism occurred at the age of 12.9 years. At the age of 15 years, when the patient's sexual development was almost completed and her menstrual cycle occurred irregularly, her height was 129.8 cm, which is 4.71 standard deviations below the median for normal girls her age. Conclusion The most important functional tests for the diagnosis of growth hormone insensitivity are the insulin-like growth factor 1 generation test and genetic analysis. Currently, the only effective treatment is daily administration of recombinant insulin-like growth factor 1 starting from early childhood. However, these patients show a dramatically impaired final height. In our case, unexplained central hypothyroidism occurred during treatment. PMID:21745362

Primary growth hormone insensitivity (Laron syndrome) and acquired hypothyroidism: a case report.

PubMed

Cotta, Oana R; Santarpia, Libero; Curtò, Lorenzo; Aimaretti, Gianluca; Corneli, Ginevra; Trimarchi, Francesco; Cannavò, Salvatore

2011-07-11

Primary growth hormone resistance or growth hormone insensitivity syndrome, also known as Laron syndrome, is a hereditary disease caused by deletions or different types of mutations in the growth hormone receptor gene or by post-receptor defects. This disorder is characterized by a clinical appearance of severe growth hormone deficiency with high levels of circulating growth hormone in contrast to low serum insulin-like growth factor 1 values. We report the case of a 15-year-old Caucasian girl who was diagnosed with Silver-Russell syndrome at the age of four and a half years. Recombinant growth hormone was administered for 18 months without an appropriate increase in growth velocity. At the age of seven years, her serum growth hormone levels were high, and an insulin-like growth factor 1 generation test did not increase insulin-like growth factor 1 levels (baseline insulin-like growth factor 1 levels, 52 μg/L; reference range, 75 μg/L to 365 μg/L; and peak, 76 μg/L and 50 μg/L after 12 and 84 hours, respectively, from baseline). The genetic analysis showed that the patient was homozygous for the R217X mutation in the growth hormone receptor gene, which is characteristic of Laron syndrome. On the basis of these results, the diagnosis of primary growth hormone insensitivity syndrome was made, and recombinant insulin-like growth factor 1 therapy was initiated. The patient's treatment was well tolerated, but unexplained central hypothyroidism occurred at the age of 12.9 years. At the age of 15 years, when the patient's sexual development was almost completed and her menstrual cycle occurred irregularly, her height was 129.8 cm, which is 4.71 standard deviations below the median for normal girls her age. The most important functional tests for the diagnosis of growth hormone insensitivity are the insulin-like growth factor 1 generation test and genetic analysis. Currently, the only effective treatment is daily administration of recombinant insulin-like growth factor 1 starting from early childhood. However, these patients show a dramatically impaired final height. In our case, unexplained central hypothyroidism occurred during treatment.

Detection of isotype switch rearrangement in bulk culture by PCR.

PubMed

Max, E E; Mills, F C; Chu, C

2001-05-01

When a B lymphocyte changes from synthesizing IgM to synthesizing IgG, IgA, or IgE, this isotype switch is generally accompanied by a unique DNA rearrangement. The protocols in this unit describe two polymerase chain reaction (PCR)-based strategies for detecting switch rearrangements in bulk culture. The first involves direct PCR across the switch junctions, providing the opportunity for characterizing the recombination products by nucleotide sequence analysis; however, because of characteristics inherent to the PCR methodology this strategy cannot easily be used as a quantitative assay for recombination. A support protocol details the preparation of the 5' Su PCR probe for this protocol. The second basic protocol describes a method known as digestion-circularization PCR (DCPCR) that is more amenable to quantitation but yields no information on structure of the recombination products. Both techniques should be capable of detecting reciprocal deletion circles as well as functional recombination products remaining on the expressed chromosome.

A role for recombination junctions in the segregation of mitochondrial DNA in yeast.

PubMed

Lockshon, D; Zweifel, S G; Freeman-Cook, L L; Lorimer, H E; Brewer, B J; Fangman, W L

1995-06-16

In S. cerevisiae, mitochondrial DNA (mtDNA) molecules, in spite of their high copy number, segregate as if there were a small number of heritable units. The rapid segregation of mitochondrial genomes can be analyzed using mtDNA deletion variants. These small, amplified genomes segregate preferentially from mixed zygotes relative to wild-type mtDNA. This segregation advantage is abolished by mutations in a gene, MGT1, that encodes a recombination junction-resolving enzyme. We show here that resolvase deficiency causes a larger proportion of molecules to be linked together by recombination junctions, resulting in the aggregation of mtDNA into a small number of cytological structures. This change in mtDNA structure can account for the increased mitotic loss of mtDNA and the altered pattern of mtDNA segregation from zygotes. We propose that the level of unresolved recombination junctions influences the number of heritable units of mtDNA.

Sequence Stability of PRRSV Chimeras After Passage in Swine

USDA-ARS?s Scientific Manuscript database

Recombinant chimeric porcine reproductive and respiratory syndrome virus (PRRSV), generated from parental strains MN184 and a licensed modified live vaccine (Ingelvac® PRRS MLV), and a MN184 nsp2 deletion mutant were used to elucidate the mechanisms of attenuation and/or protective immunity to heter...

Various 30 and 69 bp deletion variants of the Epstein-Barr virus LMP1 may arise by homologous recombination in nasopharyngeal carcinoma of Tunisian patients.

PubMed

Hadhri-Guiga, Boutheina; Khabir, Abdel-Majid; Mokdad-Gargouri, Raja; Ghorbel, Abdel-Monem; Drira, Mohamed; Daoud, Jamel; Frikha, Mounir; Jlidi, Rachid; Gargouri, Ali

2006-01-01

Nasopharyngeal carcinoma (NPC) occurs with a striking geographic distribution, it is endemic in certain areas of Southeast Asia and North Africa. NPC is tightly linked to Epstein-Barr virus (EBV), however, only a small subset of EBV genes are expressed, among them the latent membrane protein 1 (LMP1). LMP1 is considered as the main EBV oncoprotein and its 30 bp deleted-variant has been reported to be more prevalent in biopsies of NPC. We have assessed the 30 bp deletion and the XhoI polymorphisms of the BNLF1 gene in 30 peripheral bloods of NPC patients and 62 nasopharyngeal biopsies, 42 being confirmed as undifferentiated nasopharyngeal carcinoma and 20 are normal nasopharyngeal epithelium cells. Our results show that 100% of individuals retained the XhoI restriction site. A rare NPC variant, having a 69 bp deletion in the C-terminus region of the BNLF1 gene, covering the 30 bp deletion, was found in two NPC biopsies. The deleted 30 and 69 bp deleted-variants are significantly (p = 0.006) more frequent in NPC (71.42%) than in control biopsies (52%). In peripheral blood of NPC patients, the deleted-variants (47%) are also lower than in tumor tissues (p = 0.0004), suggesting that the deletion could be associated with a risk of tumor genesis. Direct repeats, located at the extremities of the 30 and 69 bp deletions, should be involved in this process. We propose that other deletions could be found since another similar direct repeat is present at the vicinity of the former ones.

Genome engineering using a synthetic gene circuit in Bacillus subtilis.

PubMed

Jeong, Da-Eun; Park, Seung-Hwan; Pan, Jae-Gu; Kim, Eui-Joong; Choi, Soo-Keun

2015-03-31

Genome engineering without leaving foreign DNA behind requires an efficient counter-selectable marker system. Here, we developed a genome engineering method in Bacillus subtilis using a synthetic gene circuit as a counter-selectable marker system. The system contained two repressible promoters (B. subtilis xylA (Pxyl) and spac (Pspac)) and two repressor genes (lacI and xylR). Pxyl-lacI was integrated into the B. subtilis genome with a target gene containing a desired mutation. The xylR and Pspac-chloramphenicol resistant genes (cat) were located on a helper plasmid. In the presence of xylose, repression of XylR by xylose induced LacI expression, the LacIs repressed the Pspac promoter and the cells become chloramphenicol sensitive. Thus, to survive in the presence of chloramphenicol, the cell must delete Pxyl-lacI by recombination between the wild-type and mutated target genes. The recombination leads to mutation of the target gene. The remaining helper plasmid was removed easily under the chloramphenicol absent condition. In this study, we showed base insertion, deletion and point mutation of the B. subtilis genome without leaving any foreign DNA behind. Additionally, we successfully deleted a 2-kb gene (amyE) and a 38-kb operon (ppsABCDE). This method will be useful to construct designer Bacillus strains for various industrial applications. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Survey of molecular chaperone requirement for the biosynthesis of hamster polyomavirus VP1 protein in Saccharomyces cerevisiae.

PubMed

Valaviciute, Monika; Norkiene, Milda; Goda, Karolis; Slibinskas, Rimantas; Gedvilaite, Alma

2016-07-01

A number of viruses utilize molecular chaperones during various stages of their life cycle. It has been shown that members of the heat-shock protein 70 (Hsp70) chaperone family assist polyomavirus capsids during infection. However, the molecular chaperones that assist the formation of recombinant capsid viral protein 1 (VP1)-derived virus-like particles (VLPs) in yeast remain unclear. A panel of yeast strains with single chaperone gene deletions were used to evaluate the chaperones required for biosynthesis of recombinant hamster polyomavirus capsid protein VP1. The impact of deletion or mild overexpression of chaperone genes was determined in live cells by flow cytometry using enhanced green fluorescent protein (EGFP) fused with VP1. Targeted genetic analysis demonstrated that VP1-EGFP fusion protein levels were significantly higher in yeast strains in which the SSZ1 or ZUO1 genes encoding ribosome-associated complex components were deleted. The results confirmed the participation of cytosolic Hsp70 chaperones and suggested the potential involvement of the Ydj1 and Caj1 co-chaperones and the endoplasmic reticulum chaperones in the biosynthesis of VP1 VLPs in yeast. Likewise, the markedly reduced levels of VP1-EGFP in Δhsc82 and Δhsp82 yeast strains indicated that both Hsp70 and Hsp90 chaperones might assist VP1 VLPs during protein biosynthesis.

Apurinic/Apyrimidinic Endonuclease 1 Is the Essential Nuclease during Immunoglobulin Class Switch Recombination

PubMed Central

Masani, Shahnaz; Han, Li

2013-01-01

Immunoglobulin (Ig) class switch recombination (CSR) is initiated by activation-induced cytidine deaminase (AID) that catalyzes numerous DNA cytosine deaminations within switch regions. The resulting uracils are processed by uracil base excision and/or mismatch repair enzymes that ultimately generate switch region DNA double-strand breaks (DSBs). Uracil glycosylase 2 (UNG2) is required for CSR, most likely by removing uracils to generate abasic sites. Although it is presumed that the apurinic/apyrimidinic endonuclease 1 (APE1) generates DNA strand incisions (a prerequisite for CSR) at these abasic sites, a direct test of the requirement for APE1 in CSR has been difficult because of the embryonic lethality of APE1 ablation in mice. Here, we report the successful deletion of the APE1 gene in a mouse B cell line (CH12F3) capable of robust CSR in vitro. In contrast to the general assumption that APE1 is essential for cellular viability, deletion of APE1 in CH12F3 cells has no apparent effect on cell viability or growth. Moreover, CSR in APE1-null CH12F3 cells is drastically reduced, providing direct evidence for an essential role for APE1 in switch region cleavage and CSR. Finally, deletion of AP endonuclease 2 (APE2) has no effect on CSR in either APE1-proficient or -deficient cells. PMID:23382073

Method for introducing unidirectional nested deletions

DOEpatents

Dunn, J.J.; Quesada, M.A.; Randesi, M.

1999-07-27

Disclosed is a method for the introduction of unidirectional deletions in a cloned DNA segment. More specifically, the method comprises providing a recombinant DNA construct comprising a DNA segment of interest inserted in a cloning vector. The cloning vector has an f1 endonuclease recognition sequence adjacent to the insertion site of the DNA segment of interest. The recombinant DNA construct is then contacted with the protein pII encoded by gene II of phage f1 thereby generating a single-stranded nick. The nicked DNA is then contacted with E. coli Exonuclease III thereby expanding the single-stranded nick into a single-stranded gap. The single-stranded gapped DNA is then contacted with a single-strand-specific endonuclease thereby producing a linearized DNA molecule containing a double-stranded deletion corresponding in size to the single-stranded gap. The DNA treated in this manner is then incubated with DNA ligase under conditions appropriate for ligation. Also disclosed is a method for producing single-stranded DNA probes. In this embodiment, single-stranded gapped DNA, produced as described above, is contacted with a DNA polymerase in the presence of labeled nucleotides to fill in the gap. This DNA is then linearized by digestion with a restriction enzyme which cuts outside the DNA segment of interest. The product of this digestion is then denatured to produce a labeled single-stranded nucleic acid probe. 1 fig.

Molecular characterization of Brucella abortus chromosome II recombination.

PubMed

Tsoktouridis, Georgios; Merz, Christian A; Manning, Simon P; Giovagnoli-Kurtz, Renée; Williams, Leanne E; Mujer, Cesar V; Hagius, Sue; Elzer, Philip; Redkar, Rajendra J; Patra, Guy; DelVecchio, Vito G

2003-10-01

Large-scale genomic rearrangements including inversions, deletions, and duplications are significant in bacterial evolution. The recently completed Brucella melitensis 16M and Brucella suis 1330 genomes have facilitated the investigation of such events in the Brucella spp. Suppressive subtractive hybridization (SSH) was employed in identifying genomic differences between B. melitensis 16M and Brucella abortus 2308. Analysis of 45 SSH clones revealed several deletions on chromosomes of B. abortus and B. melitensis that encoded proteins of various metabolic pathways. A 640-kb inversion on chromosome II of B. abortus has been reported previously (S. Michaux Charachon, G. Bourg, E. Jumas Bilak, P. Guigue Talet, A. Allardet Servent, D. O'Callaghan, and M. Ramuz, J. Bacteriol. 179:3244-3249, 1997) and is further described in this study. One end of the inverted region is located on a deleted TATGC site between open reading frames BMEII0292 and BMEII0293. The other end inserted at a GTGTC site of the cyclic-di-GMP phosphodiesterase A (PDEA) gene (BMEII1009), dividing PDEA into two unequal DNA segments of 160 and 977 bp. As a consequence of inversion, the 160-bp segment that encodes the N-terminal region of PDEA was relocated at the opposite end of the inverted chromosomal region. The splitting of the PDEA gene most likely inactivated the function of this enzyme. A recombination mechanism responsible for this inversion is proposed.

Molecular Characterization of Brucella abortus Chromosome II Recombination

PubMed Central

Tsoktouridis, Georgios; Merz, Christian A.; Manning, Simon P.; Giovagnoli-Kurtz, Renée; Williams, Leanne E.; Mujer, Cesar V.; Hagius, Sue; Elzer, Philip; Redkar, Rajendra J.; Patra, Guy; DelVecchio, Vito G.

2003-01-01

Large-scale genomic rearrangements including inversions, deletions, and duplications are significant in bacterial evolution. The recently completed Brucella melitensis 16M and Brucella suis 1330 genomes have facilitated the investigation of such events in the Brucella spp. Suppressive subtractive hybridization (SSH) was employed in identifying genomic differences between B. melitensis 16M and Brucella abortus 2308. Analysis of 45 SSH clones revealed several deletions on chromosomes of B. abortus and B. melitensis that encoded proteins of various metabolic pathways. A 640-kb inversion on chromosome II of B. abortus has been reported previously (S. Michaux Charachon, G. Bourg, E. Jumas Bilak, P. Guigue Talet, A. Allardet Servent, D. O'Callaghan, and M. Ramuz, J. Bacteriol. 179:3244-3249, 1997) and is further described in this study. One end of the inverted region is located on a deleted TATGC site between open reading frames BMEII0292 and BMEII0293. The other end inserted at a GTGTC site of the cyclic-di-GMP phosphodiesterase A (PDEA) gene (BMEII1009), dividing PDEA into two unequal DNA segments of 160 and 977 bp. As a consequence of inversion, the 160-bp segment that encodes the N-terminal region of PDEA was relocated at the opposite end of the inverted chromosomal region. The splitting of the PDEA gene most likely inactivated the function of this enzyme. A recombination mechanism responsible for this inversion is proposed. PMID:14526025

Method for introducing unidirectional nested deletions

DOEpatents

Dunn, John J.; Quesada, Mark A.; Randesi, Matthew

1999-07-27

Disclosed is a method for the introduction of unidirectional deletions in a cloned DNA segment. More specifically, the method comprises providing a recombinant DNA construct comprising a DNA segment of interest inserted in a cloning vector, the cloning vector having an f1 endonuclease recognition sequence adjacent to the insertion site of the DNA segment of interest. The recombinant DNA construct is then contacted with the protein pII encoded by gene II of phage f1 thereby generating a single-stranded nick. The nicked DNA is then contacted with E. coli Exonuclease III thereby expanding the single-stranded nick into a single-stranded gap. The single-stranded gapped DNA is then contacted with a single-strand-specific endonuclease thereby producing a linearized DNA molecule containing a double-stranded deletion corresponding in size to the single-stranded gap. The DNA treated in this manner is then incubated with DNA ligase under conditions appropriate for ligation. Also disclosed is a method for producing single-stranded DNA probes. In this embodiment, single-stranded gapped DNA, produced as described above, is contacted with a DNA polymerase in the presence of labeled nucleotides to fill in the gap. This DNA is then linearized by digestion with a restriction enzyme which cuts outside the DNA segment of interest. The product of this digestion is then denatured to produce a labeled single-stranded nucleic acid probe.

Identification of a Recurrent Microdeletion at 17q23.1q23.2 Flanked by Segmental Duplications Associated with Heart Defects and Limb Abnormalities

PubMed Central

Ballif, Blake C.; Theisen, Aaron; Rosenfeld, Jill A.; Traylor, Ryan N.; Gastier-Foster, Julie; Thrush, Devon Lamb; Astbury, Caroline; Bartholomew, Dennis; McBride, Kim L.; Pyatt, Robert E.; Shane, Kate; Smith, Wendy E.; Banks, Valerie; Gallentine, William B.; Brock, Pamela; Rudd, M. Katharine; Adam, Margaret P.; Keene, Julia A.; Phillips, John A.; Pfotenhauer, Jean P.; Gowans, Gordon C.; Stankiewicz, Pawel; Bejjani, Bassem A.; Shaffer, Lisa G.

2010-01-01

Segmental duplications, which comprise ∼5%–10% of the human genome, are known to mediate medically relevant deletions, duplications, and inversions through nonallelic homologous recombination (NAHR) and have been suggested to be hot spots in chromosome evolution and human genomic instability. We report seven individuals with microdeletions at 17q23.1q23.2, identified by microarray-based comparative genomic hybridization (aCGH). Six of the seven deletions are ∼2.2 Mb in size and flanked by large segmental duplications of >98% sequence identity and in the same orientation. One of the deletions is ∼2.8 Mb in size and is flanked on the distal side by a segmental duplication, whereas the proximal breakpoint falls between segmental duplications. These characteristics suggest that NAHR mediated six out of seven of these rearrangements. These individuals have common features, including mild to moderate developmental delay (particularly speech delay), microcephaly, postnatal growth retardation, heart defects, and hand, foot, and limb abnormalities. Although all individuals had at least mild dysmorphic facial features, there was no characteristic constellation of features that would elicit clinical suspicion of a specific disorder. The identification of common clinical features suggests that microdeletions at 17q23.1q23.2 constitute a novel syndrome. Furthermore, the inclusion in the minimal deletion region of TBX2 and TBX4, transcription factors belonging to a family of genes implicated in a variety of developmental pathways including those of heart and limb, suggests that these genes may play an important role in the phenotype of this emerging syndrome. PMID:20206336

A Simple and Universal System for Gene Manipulation in Aspergillus fumigatus: In Vitro-Assembled Cas9-Guide RNA Ribonucleoproteins Coupled with Microhomology Repair Templates.

PubMed

Al Abdallah, Qusai; Ge, Wenbo; Fortwendel, Jarrod R

2017-01-01

CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 is a novel genome-editing system that has been successfully established in Aspergillus fumigatus . However, the current state of the technology relies heavily on DNA-based expression cassettes for delivering Cas9 and the guide RNA (gRNA) to the cell. Therefore, the power of the technology is limited to strains that are engineered to express Cas9 and gRNA. To overcome such limitations, we developed a simple and universal CRISPR-Cas9 system for gene deletion that works across different genetic backgrounds of A. fumigatus . The system employs in vitro assembly of dual Cas9 ribonucleoproteins (RNPs) for targeted gene deletion. Additionally, our CRISPR-Cas9 system utilizes 35 to 50 bp of flanking regions for mediating homologous recombination at Cas9 double-strand breaks (DSBs). As a proof of concept, we first tested our system in the Δ akuB (Δ akuB ku80 ) laboratory strain and generated high rates (97%) of gene deletion using 2 µg of the repair template flanked by homology regions as short as 35 bp. Next, we inspected the portability of our system across other genetic backgrounds of A. fumigatus , namely, the wild-type strain Af293 and a clinical isolate, A. fumigatus DI15-102. In the Af293 strain, 2 µg of the repair template flanked by 35 and 50 bp of homology resulted in highly efficient gene deletion (46% and 74%, respectively) in comparison to classical gene replacement systems. Similar deletion efficiencies were also obtained in the clinical isolate DI15-102. Taken together, our data show that in vitro -assembled Cas9 RNPs coupled with microhomology repair templates are an efficient and universal system for gene manipulation in A. fumigatus . IMPORTANCE Tackling the multifactorial nature of virulence and antifungal drug resistance in A. fumigatus requires the mechanistic interrogation of a multitude of genes, sometimes across multiple genetic backgrounds. Classical fungal gene replacement systems can be laborious and time-consuming and, in wild-type isolates, are impeded by low rates of homologous recombination. Our simple and universal CRISPR-Cas9 system for gene manipulation generates efficient gene targeting across different genetic backgrounds of A. fumigatus . We anticipate that our system will simplify genome editing in A. fumigatus , allowing for the generation of single- and multigene knockout libraries. In addition, our system will facilitate the delineation of virulence factors and antifungal drug resistance genes in different genetic backgrounds of A. fumigatus .

Nonhomologous Recombination between Defective Poliovirus and Coxsackievirus Genomes Suggests a New Model of Genetic Plasticity for Picornaviruses

PubMed Central

Holmblat, Barbara; Jégouic, Sophie; Muslin, Claire; Blondel, Bruno; Joffret, Marie-Line

2014-01-01

ABSTRACT Most of the circulating vaccine-derived polioviruses (cVDPVs) implicated in poliomyelitis outbreaks in Madagascar have been shown to be recombinants between the type 2 poliovirus (PV) strain of the oral polio vaccine (Sabin 2) and another species C human enterovirus (HEV-C), such as type 17 coxsackie A virus (CA17) in particular. We studied intertypic genetic exchanges between PV and non-PV HEV-C by developing a recombination model, making it possible to rescue defective type 2 PV RNA genomes with a short deletion at the 3′ end by the cotransfection of cells with defective or infectious CA17 RNAs. We isolated over 200 different PV/CA17 recombinants, using murine cells expressing the human PV receptor (PVR) and selecting viruses with PV capsids. We found some homologous (H) recombinants and, mostly, nonhomologous (NH) recombinants presenting duplications of parental sequences preferentially located in the regions encoding proteins 2A, 2B, and 3A. Short duplications appeared to be stable, whereas longer duplications were excised during passaging in cultured cells or after multiplication in PVR-transgenic mice, generating H recombinants with diverse sites of recombination. This suggests that NH recombination events may be a transient, intermediate step in the generation and selection of the fittest H recombinants. In addition to the classical copy-choice mechanism of recombination thought to generate mostly H recombinants, there may also be a modular mechanism of recombination, involving NH recombinant precursors, shaping the genomes of recombinant enteroviruses and other picornaviruses. PMID:25096874

BDNF is required for taste axon regeneration following unilateral chorda tympani nerve section.

PubMed

Meng, Lingbin; Huang, Tao; Sun, Chengsan; Hill, David L; Krimm, Robin

2017-07-01

Taste nerves readily regenerate to reinnervate denervated taste buds; however, factors required for regeneration have not yet been identified. When the chorda tympani nerve is sectioned, expression of brain-derived neurotrophic factor (BDNF) remains high in the geniculate ganglion and lingual epithelium, despite the loss of taste buds. These observations suggest that BDNF is present in the taste system after nerve section and may support taste nerve regeneration. To test this hypothesis, we inducibly deleted Bdnf during adulthood in mice. Shortly after Bdnf gene recombination, the chorda tympani nerve was unilaterally sectioned causing a loss of both taste buds and neurons, irrespective of BDNF levels. Eight weeks after nerve section, however, regeneration was differentially affected by Bdnf deletion. In control mice, there was regeneration of the chorda tympani nerve and taste buds reappeared with innervation. In contrast, few taste buds were reinnervated in mice lacking normal Bdnf expression such that taste bud number remained low. In all genotypes, taste buds that were reinnervated were normal-sized, but non-innervated taste buds remained small and atrophic. On the side of the tongue contralateral to the nerve section, taste buds for some genotypes became larger and all taste buds remained innervated. Our findings suggest that BDNF is required for nerve regeneration following gustatory nerve section. Copyright © 2017 Elsevier Inc. All rights reserved.

The C-terminal domain of TRPV4 is essential for plasma membrane localization.

PubMed

Becker, Daniel; Müller, Margarethe; Leuner, Kristina; Jendrach, Marina

2008-02-01

Many members of the TRP superfamily oligomerize in the ER before trafficking to the plasma membrane. For membrane localization of the non-selective cation channel TRPV4 specific domains in the N-terminus are required, but the role of the C-terminus in the oligomerization and trafficking process has been not determined until now. Therefore, the localization of recombinant TRPV4 in two cell models was analyzed: HaCaT keratinocytes that express TRPV4 endogenously were compared to CHO cells that are devoid of endogenous TRPV4. When deletions were introduced in the C-terminal domain three states of TRPV4 localization were defined: a truncated TRPV4 protein of 855 amino acids was exported to the plasma membrane like the full-length channel (871 aa) and was also functional. Mutants with a length of 828 to 844 amino acids remained in the ER of CHO cells, but in HaCaT cells plasma membrane localization was partially rescued by oligomerization with endogenous TRPV4. This was confirmed by coexpression of recombinant full-length TRPV4 together with these deletion mutants, which resulted in an almost complete plasma membrane localization of both proteins and significant FRET in the plasma membrane and the ER. All deletions upstream of amino acid 828 resulted in total ER retention that could not rescued by coexpression with the full-length protein. However, these deletion mutants did not impair export of full-length TRPV4, implying that no oligomerization took place. These data indicate that the C-terminus of TRPV4 is required for oligomerization, which takes place in the ER and precedes plasma membrane trafficking.

Deletion of Brca2 exon 27 causes hypersensitivity to DNA crosslinks, chromosomal instability, and reduced life span in mice

NASA Technical Reports Server (NTRS)

Donoho, Greg; Brenneman, Mark A.; Cui, Tracy X.; Donoviel, Dorit; Vogel, Hannes; Goodwin, Edwin H.; Chen, David J.; Hasty, Paul

2003-01-01

The Brca2 tumor-suppressor gene contributes to genomic stability, at least in part by a role in homologous recombinational repair. BRCA2 protein is presumed to function in homologous recombination through interactions with RAD51. Both exons 11 and 27 of Brca2 code for domains that interact with RAD51; exon 11 encodes eight BRC motifs, whereas exon 27 encodes a single, distinct interaction domain. Deletion of all RAD51-interacting domains causes embryonic lethality in mice. A less severe phenotype is seen with BRAC2 truncations that preserve some, but not all, of the BRC motifs. These mice can survive beyond weaning, but are runted and infertile, and die very young from cancer. Cells from such mice show hypersensitivity to some genotoxic agents and chromosomal instability. Here, we have analyzed mice and cells with a deletion of only the RAD51-interacting region encoded by exon 27. Mice homozygous for this mutation (called brca2(lex1)) have a shorter life span than that of control littermates, possibly because of early onsets of cancer and sepsis. No other phenotype was observed in these animals; therefore, the brca2(lex1) mutation is less severe than truncations that delete some BRC motifs. However, at the cellular level, the brca2(lex1) mutation causes reduced viability, hypersensitivity to the DNA interstrand crosslinking agent mitomycin C, and gross chromosomal instability, much like more severe truncations. Thus, the extreme carboxy-terminal region encoded by exon 27 is important for BRCA2 function, probably because it is required for a fully functional interaction between BRCA2 and RAD51. Copyright 2003 Wiley-Liss, Inc.

Mapping of IgE-binding regions on recombinant Cyn d 1, a major allergen from Bermuda Grass Pollen (BGP).

PubMed

Tiwari, Ruby; Bhalla, Prem L; Singh, Mohan B

2009-02-02

Bermuda grass (Cynodon dactylon; subfamily Chloridoideae) is an important source of seasonal aeroallergens in warm tropical and sub-tropical areas worldwide. Improved approaches to diagnosis and therapy of allergic diseases require a thorough understanding of the structure and epitopes on the allergen molecule that are crucial for the antigen-antibody interaction. This study describes the localization of the human IgE-binding regions of the major group 1 pollen allergen Cyn d 1 from Bermuda grass. A cDNA library was constructed from Bermuda grass pollen (BGP) using a Lambda gt11 expression vector. The gene encoding the Cyn d 1 allergen was isolated by screening the library with a mouse monoclonal antibody raised against grass group 1 allergen. In order to characterize the IgE epitopes on Cyn d 1, seven overlapping fragments and three deletion mutants were cloned and over-expressed in E. coli. The recombinant fragments and deletion mutants were evaluated for their comparative IgE reactivity with sera of non atopic individuals and grass pollen allergic patients by ELISA and a dot-blot assay. Analysis of IgE binding regions by overlapping fragments and deletion mutants identified two major allergenic regions corresponding to amino acids 120-170 and 224-244. Deletion of either or both regions led to a significant reduction in IgE binding, emphasizing the importance of the C-terminal region on Cyn d 1 in epitope-IgE interaction. Anti-Cyn d 1 IgE antibodies from allergic human sera recognize two epitopes located at the C-terminal end of the molecule. These data will enable the design of improved diagnostic and therapeutic approaches for BGP hypersensitivity.

Factors Limiting SOS Expression in Log-Phase Cells of Escherichia coli

PubMed Central

Massoni, Shawn C.; Leeson, Michael C.; Long, Jarukit Edward; Gemme, Kristin; Mui, Alice

2012-01-01

In Escherichia coli, RecA–single-stranded DNA (RecA-ssDNA) filaments catalyze DNA repair, recombination, and induction of the SOS response. It has been shown that, while many (15 to 25%) log-phase cells have RecA filaments, few (about 1%) are induced for SOS. It is hypothesized that RecA's ability to induce SOS expression in log-phase cells is repressed because of the potentially detrimental effects of SOS mutagenesis. To test this, mutations were sought to produce a population where the number of cells with SOS expression more closely equaled the number of RecA filaments. Here, it is shown that deleting radA (important for resolution of recombination structures) and increasing recA transcription 2- to 3-fold with a recAo1403 operator mutation act independently to minimally satisfy this condition. This allows 24% of mutant cells to have elevated levels of SOS expression, a percentage similar to that of cells with RecA-green fluorescent protein (RecA-GFP) foci. In an xthA (exonuclease III gene) mutant where there are 3-fold more RecA loading events, recX (a destabilizer of RecA filaments) must be additionally deleted to achieve a population of cells where the percentage having elevated SOS expression (91%) nearly equals the percentage with at least one RecA-GFP focus (83%). It is proposed that, in the xthA mutant, there are three independent mechanisms that repress SOS expression in log-phase cells. These are the rapid processing of RecA filaments by RadA, maintaining the concentration of RecA below a critical level, and the destabilizing of RecA filaments by RecX. Only the first two mechanisms operate independently in a wild-type cell. PMID:22843848

Factors limiting SOS expression in log-phase cells of Escherichia coli.

PubMed

Massoni, Shawn C; Leeson, Michael C; Long, Jarukit Edward; Gemme, Kristin; Mui, Alice; Sandler, Steven J

2012-10-01

In Escherichia coli, RecA-single-stranded DNA (RecA-ssDNA) filaments catalyze DNA repair, recombination, and induction of the SOS response. It has been shown that, while many (15 to 25%) log-phase cells have RecA filaments, few (about 1%) are induced for SOS. It is hypothesized that RecA's ability to induce SOS expression in log-phase cells is repressed because of the potentially detrimental effects of SOS mutagenesis. To test this, mutations were sought to produce a population where the number of cells with SOS expression more closely equaled the number of RecA filaments. Here, it is shown that deleting radA (important for resolution of recombination structures) and increasing recA transcription 2- to 3-fold with a recAo1403 operator mutation act independently to minimally satisfy this condition. This allows 24% of mutant cells to have elevated levels of SOS expression, a percentage similar to that of cells with RecA-green fluorescent protein (RecA-GFP) foci. In an xthA (exonuclease III gene) mutant where there are 3-fold more RecA loading events, recX (a destabilizer of RecA filaments) must be additionally deleted to achieve a population of cells where the percentage having elevated SOS expression (91%) nearly equals the percentage with at least one RecA-GFP focus (83%). It is proposed that, in the xthA mutant, there are three independent mechanisms that repress SOS expression in log-phase cells. These are the rapid processing of RecA filaments by RadA, maintaining the concentration of RecA below a critical level, and the destabilizing of RecA filaments by RecX. Only the first two mechanisms operate independently in a wild-type cell.

Targeting skeletal endothelium to ameliorate bone loss.

PubMed

Xu, Ren; Yallowitz, Alisha; Qin, An; Wu, Zhuhao; Shin, Dong Yeon; Kim, Jung-Min; Debnath, Shawon; Ji, Gang; Bostrom, Mathias P; Yang, Xu; Zhang, Chao; Dong, Han; Kermani, Pouneh; Lalani, Sarfaraz; Li, Na; Liu, Yifang; Poulos, Michael G; Wach, Amanda; Zhang, Yi; Inoue, Kazuki; Di Lorenzo, Annarita; Zhao, Baohong; Butler, Jason M; Shim, Jae-Hyuck; Glimcher, Laurie H; Greenblatt, Matthew B

2018-06-01

Recent studies have identified a specialized subset of CD31 hi endomucin hi (CD31 hi EMCN hi ) vascular endothelium that positively regulates bone formation. However, it remains unclear how CD31 hi EMCN hi endothelium levels are coupled to anabolic bone formation. Mice with an osteoblast-specific deletion of Shn3, which have markedly elevated bone formation, demonstrated an increase in CD31 hi EMCN hi endothelium. Transcriptomic analysis identified SLIT3 as an osteoblast-derived, SHN3-regulated proangiogenic factor. Genetic deletion of Slit3 reduced skeletal CD31 hi EMCN hi endothelium, resulted in low bone mass because of impaired bone formation and partially reversed the high bone mass phenotype of Shn3 -/- mice. This coupling between osteoblasts and CD31 hi EMCN hi endothelium is essential for bone healing, as shown by defective fracture repair in SLIT3-mutant mice and enhanced fracture repair in SHN3-mutant mice. Finally, administration of recombinant SLIT3 both enhanced bone fracture healing and counteracted bone loss in a mouse model of postmenopausal osteoporosis. Thus, drugs that target the SLIT3 pathway may represent a new approach for vascular-targeted osteoanabolic therapy to treat bone loss.

Molecular and clinical characterization of two patients with Prader-Willi syndrome and atypical deletions of proximal chromosome 15q.

PubMed

Calounova, Gabriela; Hedvicakova, Petra; Silhanova, Eva; Kreckova, Gabriela; Sedlacek, Zdenek

2008-08-01

Prader-Willi syndrome (PWS) is caused by the disturbed expression of genes from the imprinted region of 15q11-q13, but the specific contributions of individual genes remain unknown. Most paternal PWS deletions are bracketed by recurrent breakpoints BP1 or BP2 and BP3. Atypical deletions are very rare. In the present work, we describe the molecular analysis of two patients with atypical deletions using microsatellite analysis, methylation-specific MLPA, and microarray CGH. A deletion of about 2 Mb in Patient 1 started at BP2 and ended in the middle of the typically deleted region within the UBE3A gene. The deletion in Patient 2 started 1.3 Mb distal from BP2 within the C15ORF2 gene, extended over 9.5 Mb, and ended within the AVEN gene in proximal 15q14. In Patient 1 both deletion breakpoints involved repetitive regions, which precluded cloning of the junction and pointed to non-allelic homologous recombination as a possible mechanism of this rearrangement. The breakpoints in Patient 2 were sequenced, and their structure suggested non-homologous end joining as the most likely cause of this deletion. The phenotype of both patients did not depart significantly from the typical clinical picture of PWS, although some symptoms in Patient 2 were also reminiscent of the phenotype of individuals with the recently described 15q13.3 microdeletion syndrome. Our findings support previous observations of relatively mild phenotypic effects resulting from deletions that extend distally from the PWS region and observations of the modest effects of different types of genetic defects on the spectrum and severity of symptoms in PWS. Copyright 2008 Wiley-Liss, Inc.

Genome-Wide Estimates of Transposable Element Insertion and Deletion Rates in Drosophila Melanogaster

PubMed Central

Adrion, Jeffrey R.; Song, Michael J.; Schrider, Daniel R.; Hahn, Matthew W.

2017-01-01

Abstract Knowing the rate at which transposable elements (TEs) insert and delete is critical for understanding their role in genome evolution. We estimated spontaneous rates of insertion and deletion for all known, active TE superfamilies present in a set of Drosophila melanogaster mutation-accumulation (MA) lines using whole genome sequence data. Our results demonstrate that TE insertions far outpace TE deletions in D. melanogaster. We found a significant effect of background genotype on TE activity, with higher rates of insertions in one MA line. We also found significant rate heterogeneity between the chromosomes, with both insertion and deletion rates elevated on the X relative to the autosomes. Further, we identified significant associations between TE activity and chromatin state, and tested for associations between TE activity and other features of the local genomic environment such as TE content, exon content, GC content, and recombination rate. Our results provide the most detailed assessment of TE mobility in any organism to date, and provide a useful benchmark for both addressing theoretical predictions of TE dynamics and for exploring large-scale patterns of TE movement in D. melanogaster and other species. PMID:28338986

RAG-mediated recombination is the predominant driver of oncogenic rearrangement in ETV6-RUNX1 acute lymphoblastic leukemia

PubMed Central

Papaemmanuil, Elli; Rapado, Inmaculada; Li, Yilong; Potter, Nicola E; Wedge, David C; Tubio, Jose; Alexandrov, Ludmil B; Van Loo, Peter; Cooke, Susanna L; Marshall, John; Martincorena, Inigo; Hinton, Jonathan; Gundem, Gunes; van Delft, Frederik W; Nik-Zainal, Serena; Jones, David R; Ramakrishna, Manasa; Titley, Ian; Stebbings, Lucy; Leroy, Catherine; Menzies, Andrew; Gamble, John; Robinson, Ben; Mudie, Laura; Raine, Keiran; O’Meara, Sarah; Teague, Jon W; Butler, Adam P; Cazzaniga, Giovanni; Biondi, Andrea; Zuna, Jan; Kempski, Helena; Muschen, Markus; Ford, Anthony M; Stratton, Michael R; Greaves, Mel; Campbell, Peter J

2014-01-01

The ETV6-RUNX1 fusion gene, found in 25% of childhood acute lymphoblastic leukemia (ALL), is acquired in utero but requires additional somatic mutations for overt leukemia. We used exome and low-coverage whole-genome sequencing to characterize secondary events associated with leukemic transformation. RAG-mediated deletions emerge as the dominant mutational process, characterized by recombination signal sequence motifs near the breakpoints; incorporation of non-templated sequence at the junction; ~30-fold enrichment at promoters and enhancers of genes actively transcribed in B-cell development and an unexpectedly high ratio of recurrent to non-recurrent structural variants. Single cell tracking shows that this mechanism is active throughout leukemic evolution with evidence of localized clustering and re-iterated deletions. Integration of point mutation and rearrangement data identifies ATF7IP and MGA as two new tumor suppressor genes in ALL. Thus, a remarkably parsimonious mutational process transforms ETV6-RUNX1 lymphoblasts, targeting the promoters, enhancers and first exons of genes that normally regulate B-cell differentiation. PMID:24413735

An avian influenza H5N1 virus vaccine candidate based on the extracellular domain produced in yeast system as subviral particles protects chickens from lethal challenge.

PubMed

Pietrzak, Maria; Macioła, Agnieszka; Zdanowski, Konrad; Protas-Klukowska, Anna Maria; Olszewska, Monika; Śmietanka, Krzysztof; Minta, Zenon; Szewczyk, Bogusław; Kopera, Edyta

2016-09-01

Highly pathogenic avian influenza is an on-going problem in poultry and a potential human pandemic threat. Pandemics occur suddenly and vaccine production must be fast and effective to be of value in controlling the spread of the virus. In this study we evaluated the potential of a recombinant protein from the extracellular domain of an H5 hemagglutinin protein produced in a yeast expression system to act as an effective vaccine. Protein production was efficient, with up to 200 mg purified from 1 L of culture medium. We showed that the deletion of the multibasic cleavage site from the protein improves oligomerization and, consequentially, its immunogenicity. We also showed that immunization with this deleted protein protected chickens from challenge with a highly pathogenic avian influenza H5N1 virus. Our results suggest that this recombinant protein produced in yeast may be an effective vaccine against H5N1 virus in poultry. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Recombinant structures expand and contract inter and intragenic diversification at the KIR locus

PubMed Central

2013-01-01

Background The human KIR genes are arranged in at least six major gene-content haplotypes, all of which are combinations of four centromeric and two telomeric motifs. Several less frequent or minor haplotypes also exist, including insertions, deletions, and hybridization of KIR genes derived from the major haplotypes. These haplotype structures and their concomitant linkage disequilibrium among KIR genes suggest that more meaningful correlative data from studies of KIR genetics and complex disease may be achieved by measuring haplotypes of the KIR region in total. Results Towards that end, we developed a KIR haplotyping method that reports unambiguous combinations of KIR gene-content haplotypes, including both phase and copy number for each KIR. A total of 37 different gene content haplotypes were detected from 4,512 individuals and new sequence data was derived from haplotypes where the detailed structure was not previously available. Conclusions These new structures suggest a number of specific recombinant events during the course of KIR evolution, and add to an expanding diversity of potential new KIR haplotypes derived from gene duplication, deletion, and hybridization. PMID:23394822

Correlated Template-Switching Events during Minus-Strand DNA Synthesis: a Mechanism for High Negative Interference during Retroviral Recombination

PubMed Central

Anderson, Jeffrey A.; Teufel, Ronald J.; Yin, Philip D.; Hu, Wei-Shau

1998-01-01

Two models for the mechanism of retroviral recombination have been proposed: forced copy choice (minus-strand recombination) and strand displacement-assimilation (plus-strand recombination). Each minus-strand recombination event results in one template switch, whereas each plus-strand recombination event results in two template switches. Recombinant proviruses with one and more than one template switches were previously observed. Recombinants with one template switch were generated by minus-strand recombination, while recombinants containing more than one template switch may have been generated by plus-strand recombination or by correlated minus-strand recombination. We recently observed that retroviral recombination exhibits high negative interference whereby the frequency of recombinants containing multiple template-switching events is higher than expected. To delineate the mechanism that generates recombinants with more than one template switch, we devised a system that permits only minus-strand recombination. Two highly homologous vectors, WH204 and WH221, containing eight different restriction site markers were used. The primer binding site (PBS) of WH221 was deleted; although reverse transcription cannot initiate from WH221 RNA, it can serve as a template for DNA synthesis in heterozygotic virions. After one round of retroviral replication, the structures of the recombinant proviruses were examined. Recombinants containing two, three, four, and five template switches were observed at 1.4-, 10-, 65-, and 50-fold-higher frequencies, respectively, than expected. This indicates that minus-strand recombination events are correlated and can generate proviruses with multiple template switches efficiently. The frequencies of recombinants containing multiple template switches were similar to those observed in the previous system, which allowed both minus- and plus-strand recombination. Thus, the previously reported high negative interference during retroviral recombination can be caused by correlated template switches during minus-strand DNA synthesis. In addition, all examined recombinants contained an intact PBS, indicating that most of the plus-strand DNA transfer occurs after completion of the strong-stop DNA. PMID:9445017

DNA-Binding Protein HU Coordinates Pathogenicity in Vibrio parahaemolyticus.

PubMed

Phan, Ngoc Quang; Uebanso, Takashi; Shimohata, Takaaki; Nakahashi, Mutsumi; Mawatari, Kazuaki; Takahashi, Akira

2015-09-01

HU is one of the most abundant nucleoid-associated proteins in bacterial cells and regulates the expression of many genes involved in growth, motility, metabolism, and virulence. It is known that Vibrio parahaemolyticus pathogenicity is related to its characteristic rapid growth and that type III secretion system 1 (T3SS1) contributes to its cytotoxicity. However, it is not known if HU plays a role in the pathogenicity of V. parahaemolyticus. In the present study, we investigated the effect of HU proteins HU-2 (HUα) (V. parahaemolyticus 2911 [vp2911]) and HUβ (vp0920) on the pathogenicity of V. parahaemolyticus. We found that a deletion of both HU subunits (yielding the ΔHUs [Δvp0920 Δvp2911] strain), but not single deletions, led to a reduction of the growth rate. In addition, expression levels of T3SS1-related genes, including exsA (positive regulator), exsD (negative regulator), vp1680 (cytotoxic effector), and vp1671 (T3SS1 apparatus), were reduced in the ΔHUs strain compared to the wild type (WT). As a result, cytotoxicity to HeLa cells was decreased in the ΔHUs strain. The additional deletion of exsD in the ΔHUs strain restored T3SS1-related gene expression levels and cytotoxicity but not the growth rate. These results suggest that the HU protein regulates the levels of T3SS1 gene expression and cytotoxicity in a growth rate-independent manner. Nucleoid-binding protein HU regulates cellular behaviors, including nucleoid structuring, general recombination, transposition, growth, replication, motility, metabolism, and virulence. It is thought that both the number of bacteria and the number of virulence factors may affect the pathogenicity of bacteria. In the present study, we investigated which factor(s) has a dominant role during infection in one of the most rapidly growing bacterial species, Vibrio parahaemolyticus. We found that V. parahaemolyticus cytotoxicity is regulated, in a growth rate-independent manner, by the HU proteins through regulation of a number of virulence factors, including T3SS1 gene expression. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Level of tissue differentiation influences the activation of a heat-inducible flower-specific system for genetic containment in poplar (Populus tremula L.).

PubMed

Hoenicka, Hans; Lehnhardt, Denise; Nunna, Suneetha; Reinhardt, Richard; Jeltsch, Albert; Briones, Valentina; Fladung, Matthias

2016-02-01

Differentiation level but not transgene copy number influenced activation of a gene containment system in poplar. Heat treatments promoted CRE gene body methylation. The flower-specific transgene deletion was confirmed. Gene flow between genetic modified trees and their wild relatives is still motive of concern. Therefore, approaches for gene containment are required. In this study, we designed a novel strategy for achieving an inducible and flower-specific transgene removal from poplar trees but still expressing the transgene in the plant body. Hence, pollen carrying transgenes could be used for breeding purposes under controlled conditions in a first phase, and in the second phase genetic modified poplars developing transgene-free pollen grains could be released. This approach is based on the recombination systems CRE/loxP and FLP/frt. Both gene constructs contained a heat-inducible CRE/loxP-based spacer sequence for in vivo assembling of the flower-specific FLP/frt system. This allowed inducible activation of gene containment. The FLP/frt system was under the regulation of a flower-specific promoter, either CGPDHC or PTD. Our results confirmed complete CRE/loxP-based in vivo assembling of the flower-specific transgene excision system after heat treatment in all cells for up to 30 % of regenerants derived from undifferentiated tissue cultures. Degradation of HSP::CRE/loxP spacer after recombination but also persistence as extrachromosomal DNA circles were detected in sub-lines obtained after heat treatments. Furthermore, heat treatment promoted methylation of the CRE gene body. A lower methylation level was detected at CpG sites in transgenic sub-lines showing complete CRE/loxP recombination and persistence of CRE/loxP spacer, compared to sub-lines with incomplete recombination. However, our results suggest that low methylation might be necessary but not sufficient for recombination. The flower-specific FLP/frt-based transgene deletion was confirmed in 6.3 % of flowers.

Mob/oriT, a mobilizable site-specific recombination system for unmarked genetic manipulation in Bacillus thuringiensis and Bacillus cereus.

PubMed

Wang, Pengxia; Zhu, Yiguang; Zhang, Yuyang; Zhang, Chunyi; Xu, Jianyi; Deng, Yun; Peng, Donghai; Ruan, Lifang; Sun, Ming

2016-06-10

Bacillus thuringiensis and Bacillus cereus are two important species in B. cereus group. The intensive study of these strains at the molecular level and construction of genetically modified bacteria requires the development of efficient genetic tools. To insert genes into or delete genes from bacterial chromosomes, marker-less manipulation methods were employed. We present a novel genetic manipulation method for B. thuringiensis and B. cereus strains that does not leave selection markers. Our approach takes advantage of the relaxase Mob02281 encoded by plasmid pBMB0228 from Bacillus thuringiensis. In addition to its mobilization function, this Mob protein can mediate recombination between oriT sites. The Mob02281 mobilization module was associated with a spectinomycin-resistance gene to form a Mob-Spc cassette, which was flanked by the core 24-bp oriT sequences from pBMB0228. A strain in which the wild-type chromosome was replaced with the modified copy containing the Mob-Spc cassette at the target locus was obtained via homologous recombination. Thus, the spectinomycin-resistance gene can be used to screen for Mob-Spc cassette integration mutants. Recombination between the two oriT sequences mediated by Mob02281, encoded by the Mob-Spc cassette, resulted in the excision of the Mob-Spc cassette, producing the desired chromosomal alteration without introducing unwanted selection markers. We used this system to generate an in-frame deletion of a target gene in B. thuringiensis as well as a gene located in an operon of B. cereus. Moreover, we demonstrated that this system can be used to introduce a single gene or an expression cassette of interest in B. thuringiensis. The Mob/oriT recombination system provides an efficient method for unmarked genetic manipulation and for constructing genetically modified bacteria of B. thuringiensis and B. cereus. Our method extends the available genetic tools for B. thuringiensis and B. cereus strains.

Sequences outside that of residues 93-102 of 3A protein can contribute to the ability of foot-and-mouth disease virus (FMDV) to replicate in bovine-derived cells.

PubMed

Ma, Xueqing; Li, Pinghua; Bai, Xingwen; Sun, Pu; Bao, Huifang; Lu, Zengjun; Cao, Yimei; Li, Dong; Chen, Yingli; Qiao, Zilin; Liu, Zaixin

2014-10-13

Foot-and-mouth disease (FMD) is a highly contagious and economically devastating disease of cloven-hoofed animals. During 2010 and 2011, there was an epidemic of the Mya-98 lineage of the Southeast Asia (SEA) topotype in East Asia, including China. Changes in the FMDV 3A protein have been previously reported to be associated with the inability of FMDV to grow in bovine cells and cause disease in cattle. In this paper, we report the generation of a full-length infectious cDNA clone of FMDV O/SEA/Mya-98 strain O/GZSB/2011 for the first time along with two genetically modified viruses with deletion at positions 93-102 and 133-143 in 3A based on the established infectious clone. All the recombinant viruses grew well and displayed growth properties and plaque phenotypes similar to those of the parental virus in baby hamster kidney (BHK-21) cells, porcine kidney (PK-15) cells, and primary fetal porcine kidney (FPK) cells. While the recombinant viruses rvGZSB and rvSBΔ133-143 exhibited similar growth properties and plaque phenotypes with the parental virus in primary fetal bovine kidney (FBK) cells, the recombinant virus rvSBΔ93-102, containing deletion at positions 93-102 in 3A, grew at a slower rate and had a smaller plaque size phenotype in FBK cells than that of the parental virus. Therefore, the results suggest that the deletion at positions 93-102 of 3A protein does not affect FMDV replication efficiency in BHK-21, PK-15 and FPK cells, but affects virus replication efficiency in FBK cells, although, cannot alone account for the inability to replicate in bovine cells. Copyright © 2014 Elsevier B.V. All rights reserved.

Two novel types of contiguous gene deletion of the AVPR2 and ARHGAP4 genes in unrelated Japanese kindreds with nephrogenic diabetes insipidus.

PubMed

Demura, Masashi; Takeda, Yoshiyu; Yoneda, Takashi; Furukawa, Kenji; Usukura, Mikiya; Itoh, Yuji; Mabuchi, Hiroshi

2002-01-01

Study of two families containing individuals with nephrogenic diabetes insipidus (NDI) indicated different types of 21.3 kb and 26.3 kb deletions involving the AVPR2 and ARHGAP4 (RhoGAP C1) genes. In the case of the 21.3 kb deletion, the deletion consensus motif (5'-TGAAGG-3') and polypurine runs, known as the arrest site of polymerase alpha, were detected in the vicinity of the deletion junction. Inverted repeats (7/8 matches), believed to potentiate DNA loop formation, flank the deletion breakpoint. We propose this deletion to be the result of slipped mispairing during DNA replication. In the case of the 26.3 kb deletion, the 12,945 bp inverted region with the 10,003 bp internal deletion was accompanied with the 2,509 bp deletion in the 5'-side and the 13,785 bp deletion in the 3'-side. We defined three deletion junctions in this rearrangement (DJ1, DJ2, and DJ3) from the 5'-side. The surrounding sequence of DJ1 (5'-CCC-3') closely resembled that of DJ3 (5'-AGGG-3') (DJ1; 5'-cCCCgaggg-3', DJ3; 5'-ccccAGGG-3'), and DJ1 was located in the 5'-side of DJ3 without any overlapping in sequence. The immunoglobulin class switch (ICS) motif (5'-TGGGG-3') was found around the complementary sequence of DJ3. There was a 10-base palindrome (5'-aGACAtgtct-3') in the alignment of the DJ2 (5'-GACA-3') region. From these findings, we propose a novel mutation process with the rearrangement probably resulting from stem-loop induced non-homologous recombination in an ICS-like fashion. Both patients, despite lacking ARHGAP4, had no morphological, clinical, or laboratory abnormalities except for those usually found in patients with NDI. Copyright 2001 Wiley-Liss, Inc.

Construction of Various γ34.5 Deleted Fluorescent-Expressing Oncolytic herpes Simplex type 1 (oHSV) for Generation and Isolation of HSV-Based Vectors

PubMed

Abdoli, Shahriyar; Roohvand, Farzin; Teimoori-Toolabi, Ladan; Shokrgozar, Mohammad Ali; Bahrololoumi, Mina; Azadmanesh, Kayhan

2017-07-01

Oncolytic herpes simplex virus (oHSV)-based vectors lacking γ34.5 gene, are considered as ideal templates to construct efficient vectors for (targeted) cancer gene therapy. Herein, we reported the construction of three single/dually-flourescence labeled and γ34.5-deleted, recombinant HSV-1 vectors for rapid generation and easy selection/isolation of different HSV-Based vectors. Generation of recombinant viruses was performed with conventional homologous recombination methods using green fluorescent protein (GFP) and BleCherry harboring shuttle vectors. Viruses were isolated by direct fluorescence observation and standard plaque purifying methods and confirmed by PCR and sequencing and flow cytometry. XTT and plaque assay titration were performed on Vero, U87MG, and T98 GBM cell lines. We generated three recombinant viruses, HSV-GFP, HSV-GR (Green-Red), and HSV-Red. The HSV-GFP showed two log higher titer (1010 PFU) than wild type (108 PFU). In contrast, HSV-GR and HSV-Red showed one log lower titer (107 PFU) than parental HSV. Cytotoxicity analysis showed that HSV-GR and HSV-Red can lyse target tumor cells at multiplicity of infection of 10 and 1 (P<0.001). Moreover, HSV-GFP showed higher infection potency (98%) in comparison with HSV-GR (82%). Our oHSVs provide a simple and an efficient platform for construction and rapid isolation of 2nd and 3rd generation oHSVs by replacing the inserted dyes with transgenes and also for rapid identification via fluorescence activated cell sorting. These vectors can also be used for tracing the efficacy of therapeutic agents on target cells, imaging of neural or tumoral cells in vitro/in vivo and as oncolytic agents in cancer therapy.

Improved Yield of High Molecular Weight Hyaluronic Acid Production in a Stable Strain of Streptococcus zooepidemicus via the Elimination of the Hyaluronidase-Encoding Gene.

PubMed

Pourzardosht, Navid; Rasaee, Mohammad Javad

2017-06-01

Despite the significant potential of Streptococcus zooepidemicus for hyaluronic acid (HA) production with high molecular weight (MW), the HA degrading properties of hyaluronidase prevents the bacteria to achieve enhanced HA yield with high MW. In the present study, we aim to knockout the hyaluronidase enzyme and assess its effects on the yield and MW of the produced HA. The kanamycin resistance gene between the left and right arms of hyaluronidase gene was inserted into pUC18 plasmid to construct pUC18-L-kana r -R as a recombinant suicide plasmid. The construct was then transferred into S. zooepidemicus to induce the homologous recombination between the hyaluronidase gene and the kanamycin resistance gene. Gene deletion was confirmed by PCR and enzyme assay. The product was cultured on selectable medium in which the MW of HA was increased from 1.5 to 3.8 MDa. The yield of HA production using the mutant strain was higher in all different concentrations of glucose from 40 to 120 g/l. Moreover, glucose increase results in higher HA production within both wild-type and recombinant strains. However, the growth rate of HA concentration (the slope of the plot), as a consequence of increased glucose concentration, is always higher for the recombinant strain. Unlike the wild-type strain, there was no sharp HA production drop approaching the 6 g/l HA concentration. In conclusion, hyaluronidase activity and HA concentration and MW exhibited a mutual control on each other. Based on our results, deletion of the hyaluronidase gene positively affects the yield and MW of HA.

The Aspartate-Semialdehyde Dehydrogenase of Edwardsiella ictaluri and Its Use as Balanced-Lethal System in Fish Vaccinology

PubMed Central

Santander, Javier; Xin, Wei; Yang, Zhao; Curtiss, Roy

2010-01-01

asdA mutants of Gram-negative bacteria have an obligate requirement for diaminopimelic acid (DAP), which is an essential constituent of the peptidoglycan layer of the cell wall of these organisms. In environments deprived of DAP, i.e., animal tissues, they will undergo lysis. Deletion of the asdA gene has previously been exploited to develop antibiotic-sensitive strains of live attenuated recombinant bacterial vaccines. Introduction of an Asd+ plasmid into a ΔasdA mutant makes the bacterial strain plasmid-dependent. This dependence on the Asd+ plasmid vector creates a balanced-lethal complementation between the bacterial strain and the recombinant plasmid. E. ictaluri is an enteric Gram-negative fish pathogen that causes enteric septicemia in catfish. Because E. ictaluri is a nasal/oral invasive intracellular pathogen, this bacterium is a candidate to develop a bath/oral live recombinant attenuated Edwardsiella vaccine (RAEV) for the catfish aquaculture industry. As a first step to develop an antibiotic-sensitive RAEV strain, we characterized and deleted the E. ictaluri asdA gene. E. ictaluri ΔasdA01 mutants exhibit an absolute requirement for DAP to grow. The asdA gene of E. ictaluri was complemented by the asdA gene from Salmonella. Several Asd+ expression vectors with different origins of replication were transformed into E. ictaluri ΔasdA01. Asd+ vectors were compatible with the pEI1 and pEI2 E. ictaluri native plasmids. The balanced-lethal system was satisfactorily evaluated in vivo. Recombinant GFP, PspA, and LcrV proteins were synthesized by E. ictaluri ΔasdA01 harboring Asd+ plasmids. Here we constructed a balanced-lethal system, which is the first step to develop an antibiotic-sensitive RAEV for the aquaculture industry. PMID:21209920

The aspartate-semialdehyde dehydrogenase of Edwardsiella ictaluri and its use as balanced-lethal system in fish vaccinology.

PubMed

Santander, Javier; Xin, Wei; Yang, Zhao; Curtiss, Roy

2010-12-29

asdA mutants of gram-negative bacteria have an obligate requirement for diaminopimelic acid (DAP), which is an essential constituent of the peptidoglycan layer of the cell wall of these organisms. In environments deprived of DAP, i.e., animal tissues, they will undergo lysis. Deletion of the asdA gene has previously been exploited to develop antibiotic-sensitive strains of live attenuated recombinant bacterial vaccines. Introduction of an Asd(+) plasmid into a ΔasdA mutant makes the bacterial strain plasmid-dependent. This dependence on the Asd(+) plasmid vector creates a balanced-lethal complementation between the bacterial strain and the recombinant plasmid. E. ictaluri is an enteric gram-negative fish pathogen that causes enteric septicemia in catfish. Because E. ictaluri is a nasal/oral invasive intracellular pathogen, this bacterium is a candidate to develop a bath/oral live recombinant attenuated Edwardsiella vaccine (RAEV) for the catfish aquaculture industry. As a first step to develop an antibiotic-sensitive RAEV strain, we characterized and deleted the E. ictaluri asdA gene. E. ictaluri ΔasdA01 mutants exhibit an absolute requirement for DAP to grow. The asdA gene of E. ictaluri was complemented by the asdA gene from Salmonella. Several Asd(+) expression vectors with different origins of replication were transformed into E. ictaluri ΔasdA01. Asd(+) vectors were compatible with the pEI1 and pEI2 E. ictaluri native plasmids. The balanced-lethal system was satisfactorily evaluated in vivo. Recombinant GFP, PspA, and LcrV proteins were synthesized by E. ictaluri ΔasdA01 harboring Asd(+) plasmids. Here we constructed a balanced-lethal system, which is the first step to develop an antibiotic-sensitive RAEV for the aquaculture industry.

Rad6–Bre1-mediated H2B ubiquitination regulates telomere replication by promoting telomere-end resection

PubMed Central

Wu, Zhenfang; Liu, Jun; Zhang, Qiong-Di; Lv, De-Kang; Wu, Nian-Feng

2017-01-01

Abstract Rad6 and Bre1, ubiquitin-conjugating E2 and E3 enzymes respectively, are responsible for histone H2B lysine 123 mono-ubiquitination (H2Bub1) in Saccharomyces cerevisiae. Previous studies have shown that Rad6 and Bre1 regulate telomere length and recombination. However, the underlying molecular mechanism remains largely unknown. Here we report that H2BK123 mutation results in telomere shortening, while inactivation of Ubp8 and/or Ubp10, deubiquitinases of H2Bub1, leads to telomere lengthening in Rad6–Bre1-dependent manner. In telomerase-deficient cells, inactivation of Rad6–Bre1 pathway retards telomere shortening rate and the onset of senescence, while deletion of UBP8 and/or UBP10 accelerates senescence. Thus, Rad6–Bre1 pathway regulates both telomere length and recombination through its role in H2Bub1. Additionally, inactivation of both Rad6–Bre1–H2Bub1 and Mre11–Rad50–Xrs2 (MRX) pathways causes synthetic growth defects and telomere shortening in telomerase-proficient cells, and significantly accelerates senescence and eliminates type II telomere recombination in telomerase-deficient cells. Furthermore, RAD6 or BRE1 deletion, or H2BK123R mutation decreases the accumulation of ssDNA at telomere ends. These results support the model that Rad6–Bre1–H2Bub1 cooperates with MRX to promote telomere-end resection and thus positively regulates both telomerase- and recombination-dependent telomere replication. This study provides a mechanistic link between histone H2B ubiquitination and telomere replication. PMID:28180293

Molecular basis and consequences of a deletion in the amelogenin gene, analyzed by capture PCR

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lagerstroem-Fermer, M.; Pettersson, U.; Landegren, U.

1993-07-01

A mutation that disrupts the gene for one of the major proteins in tooth enamel has been investigated. The mutation is located in the amelogenin gene and causes X-linked amelogenesis imperfecta, characterized by defective mineralization of tooth enamel. The authors have isolated the breakpoints of a 5-kb deletion in the amelogenin gene on the basis of nucleotide sequence information located upstream of the lesion, using a technique termed capture PCR. The deletion removes five of the seven exons, spanning from the second intron to the last exon. Only the first two codons for the mature protein remain, consistent with themore » relatively severe phenotype of affected individuals in the present family. The mutation appears to have arisen as an illegitimate recombination event since of 11 nucleotide positions immediately surrounding the two breakpoints, 9 are identical. 17 refs., 3 figs., 1 tab.« less

Stability of Yellow Fever Virus under Recombinatory Pressure as Compared with Chikungunya Virus

PubMed Central

McGee, Charles E.; Tsetsarkin, Konstantin A.; Guy, Bruno; Lang, Jean; Plante, Kenneth; Vanlandingham, Dana L.; Higgs, Stephen

2011-01-01

Recombination is a mechanism whereby positive sense single stranded RNA viruses exchange segments of genetic information. Recent phylogenetic analyses of naturally occurring recombinant flaviviruses have raised concerns regarding the potential for the emergence of virulent recombinants either post-vaccination or following co-infection with two distinct wild-type viruses. To characterize the conditions and sequences that favor RNA arthropod-borne virus recombination we constructed yellow fever virus (YFV) 17D recombinant crosses containing complementary deletions in the envelope protein coding sequence. These constructs were designed to strongly favor recombination, and the detection conditions were optimized to achieve high sensitivity recovery of putative recombinants. Full length recombinant YFV 17D virus was never detected under any of the experimental conditions examined, despite achieving estimated YFV replicon co-infection levels of ∼2.4×106 in BHK-21 (vertebrate) cells and ∼1.05×105 in C710 (arthropod) cells. Additionally YFV 17D superinfection resistance was observed in vertebrate and arthropod cells harboring a primary infection with wild-type YFV Asibi strain. Furthermore recombination potential was also evaluated using similarly designed chikungunya virus (CHIKV) replicons towards validation of this strategy for recombination detection. Non-homologus recombination was observed for CHIKV within the structural gene coding sequence resulting in an in-frame duplication of capsid and E3 gene. Based on these data, it is concluded that even in the unlikely event of a high level acute co-infection of two distinct YFV genomes in an arthropod or vertebrate host, the generation of viable flavivirus recombinants is extremely unlikely. PMID:21826243

Stability of yellow fever virus under recombinatory pressure as compared with chikungunya virus.

PubMed

McGee, Charles E; Tsetsarkin, Konstantin A; Guy, Bruno; Lang, Jean; Plante, Kenneth; Vanlandingham, Dana L; Higgs, Stephen

2011-01-01

Recombination is a mechanism whereby positive sense single stranded RNA viruses exchange segments of genetic information. Recent phylogenetic analyses of naturally occurring recombinant flaviviruses have raised concerns regarding the potential for the emergence of virulent recombinants either post-vaccination or following co-infection with two distinct wild-type viruses. To characterize the conditions and sequences that favor RNA arthropod-borne virus recombination we constructed yellow fever virus (YFV) 17D recombinant crosses containing complementary deletions in the envelope protein coding sequence. These constructs were designed to strongly favor recombination, and the detection conditions were optimized to achieve high sensitivity recovery of putative recombinants. Full length recombinant YFV 17D virus was never detected under any of the experimental conditions examined, despite achieving estimated YFV replicon co-infection levels of ∼2.4 x 10⁶ in BHK-21 (vertebrate) cells and ∼1.05 x 10⁵ in C₇10 (arthropod) cells. Additionally YFV 17D superinfection resistance was observed in vertebrate and arthropod cells harboring a primary infection with wild-type YFV Asibi strain. Furthermore recombination potential was also evaluated using similarly designed chikungunya virus (CHIKV) replicons towards validation of this strategy for recombination detection. Non-homologus recombination was observed for CHIKV within the structural gene coding sequence resulting in an in-frame duplication of capsid and E3 gene. Based on these data, it is concluded that even in the unlikely event of a high level acute co-infection of two distinct YFV genomes in an arthropod or vertebrate host, the generation of viable flavivirus recombinants is extremely unlikely.

Incorporation of a lambda phage recombination system and EGFP detection to simplify mutagenesis of Herpes simplex virus bacterial artificial chromosomes

PubMed Central

Schmeisser, Falko; Weir, Jerry P

2007-01-01

Background Targeted mutagenesis of the herpesvirus genomes has been facilitated by the use of bacterial artificial chromosome (BAC) technology. Such modified genomes have potential uses in understanding viral pathogenesis, gene identification and characterization, and the development of new viral vectors and vaccines. We have previously described the construction of a herpes simplex virus 2 (HSV-2) BAC and the use of an allele replacement strategy to construct HSV-2 recombinants. While the BAC mutagenesis procedure is a powerful method to generate HSV-2 recombinants, particularly in the absence of selective marker in eukaryotic culture, the mutagenesis procedure is still difficult and cumbersome. Results Here we describe the incorporation of a phage lambda recombination system into an allele replacement vector. This strategy enables any DNA fragment containing the phage attL recombination sites to be efficiently inserted into the attR sites of the allele replacement vector using phage lambda clonase. We also describe how the incorporation of EGFP into the allele replacement vector can facilitate the selection of the desired cross-over recombinant BACs when the allele replacement reaction is a viral gene deletion. Finally, we incorporate the lambda phage recombination sites directly into an HSV-2 BAC vector for direct recombination of gene cassettes using the phage lambda clonase-driven recombination reaction. Conclusion Together, these improvements to the techniques of HSV BAC mutagenesis will facilitate the construction of recombinant herpes simplex viruses and viral vectors. PMID:17501993

Quantitative PCR analysis reveals a high incidence of large intragenic deletions in the FANCA gene in Spanish Fanconi anemia patients.

PubMed

Callén, E; Tischkowitz, M D; Creus, A; Marcos, R; Bueren, J A; Casado, J A; Mathew, C G; Surrallés, J

2004-01-01

Fanconi anaemia is an autosomal recessive disease characterized by chromosome fragility, multiple congenital abnormalities, progressive bone marrow failure and a high predisposition to develop malignancies. Most of the Fanconi anaemia patients belong to complementation group FA-A due to mutations in the FANCA gene. This gene contains 43 exons along a 4.3-kb coding sequence with a very heterogeneous mutational spectrum that makes the mutation screening of FANCA a difficult task. In addition, as the FANCA gene is rich in Alu sequences, it was reported that Alu-mediated recombination led to large intragenic deletions that cannot be detected in heterozygous state by conventional PCR, SSCP analysis, or DNA sequencing. To overcome this problem, a method based on quantitative fluorescent multiplex PCR was proposed to detect intragenic deletions in FANCA involving the most frequently deleted exons (exons 5, 11, 17, 21 and 31). Here we apply the proposed method to detect intragenic deletions in 25 Spanish FA-A patients previously assigned to complementation group FA-A by FANCA cDNA retroviral transduction. A total of eight heterozygous deletions involving from one to more than 26 exons were detected. Thus, one third of the patients carried a large intragenic deletion that would have not been detected by conventional methods. These results are in agreement with previously published data and indicate that large intragenic deletions are one of the most frequent mutations leading to Fanconi anaemia. Consequently, this technology should be applied in future studies on FANCA to improve the mutation detection rate. Copyright 2003 S. Karger AG, Basel

Corrective recombination of mouse immunoglobulin kappa alleles in Abelson murine leukemia virus-transformed pre-B cells.

PubMed Central

Feddersen, R M; Van Ness, B G

1990-01-01

Previous characterization of mouse immunoglobulin kappa gene rearrangement products cloned from murine plasmacytomas has indicated that two recombination events can take place on a single kappa allele (R. M. Feddersen and B. G. Van Ness, Proc. Natl. Acad. Sci. USA 82:4792-4797, 1985; M. A. Shapiro and M. Weigert, J. Immunol. 139:3834-3839, 1987). To determine whether multiple recombinations on a single kappa allele can contribute to the formation of productive V-J genes through corrective recombinations, we have examined several Abelson murine leukemia virus-transformed pre-B-cell clones which rearrange the kappa locus during cell culture. Clonal cell lines which had rearranged one kappa allele nonproductively while maintaining the other allele in the germ line configuration were grown, and secondary subclones, which subsequently expressed kappa protein, were isolated and examined for further kappa rearrangement. A full spectrum of rearrangement patterns was observed in this sequential cloning, including productive and nonproductive recombinations of the germ line allele and secondary recombinations of the nonproductive allele. The results show that corrective V-J recombinations, with displacement of the nonproductive kappa gene, occur with a significant frequency (6 of 17 kappa-producing subclones). Both deletion and maintenance of the primary (nonfunctional) V-J join, as a reciprocal product, were observed. Images PMID:2153918

Saccharomyces cerevisiae MSH2-MSH3 and MSH2-MSH6 complexes display distinct requirements for DNA binding Domain I in mismatch recognition.

PubMed Central

Lee, Susan D.; Surtees, Jennifer A.; Alani, Eric

2007-01-01

In eukaryotic mismatch repair (MMR) MSH2-MSH6 initiates the repair of base-base and small insertion/deletion mismatches while MSH2-MSH3 repairs larger insertion/deletion mismatches. In this study we showed that the msh2Δ1 mutation, containing a complete deletion of the conserved mismatch recognition Domain I of MSH2, conferred a separation of function phenotype with respect to MSH2-MSH3 and MSH2-MSH6 functions. Strains bearing the msh2Δ1 mutation were nearly wild-type in MSH2-MSH6-mediated MMR and in suppressing recombination between DNA sequences predicted to form mismatches recognized by MSH2-MSH6. However, these strains were completely defective in MSH2-MSH3-mediated MMR and recombination functions. This information encouraged us to analyze the contributions of Domain I to the mismatch binding specificity of MSH2-MSH3 in genetic and biochemical assays. We found that Domain I in MSH2 contributed a non-specific DNA binding activity while Domain I of MSH3 appeared important for mismatch binding specificity and for suppressing non-specific DNA-binding. These observations reveal distinct requirements for the MSH2 DNA binding Domain I in the repair of DNA mismatches and suggest that the binding of MSH2-MSH3 to mismatch DNA involves protein-DNA contacts that appear very different from those required for MSH2-MSH6 mismatch binding. PMID:17157869

Saccharomyces cerevisiae MSH2-MSH3 and MSH2-MSH6 complexes display distinct requirements for DNA binding domain I in mismatch recognition.

PubMed

Lee, Susan D; Surtees, Jennifer A; Alani, Eric

2007-02-09

In eukaryotic mismatch repair (MMR) MSH2-MSH6 initiates the repair of base-base and small insertion/deletion mismatches while MSH2-MSH3 repairs larger insertion/deletion mismatches. Here, we show that the msh2Delta1 mutation, containing a complete deletion of the conserved mismatch recognition domain I of MSH2, conferred a separation of function phenotype with respect to MSH2-MSH3 and MSH2-MSH6 functions. Strains bearing the msh2Delta1 mutation were nearly wild-type in MSH2-MSH6-mediated MMR and in suppressing recombination between DNA sequences predicted to form mismatches recognized by MSH2-MSH6. However, these strains were completely defective in MSH2-MSH3-mediated MMR and recombination functions. This information encouraged us to analyze the contributions of domain I to the mismatch binding specificity of MSH2-MSH3 in genetic and biochemical assays. We found that domain I in MSH2 contributed a non-specific DNA binding activity while domain I of MSH3 appeared important for mismatch binding specificity and for suppressing non-specific DNA binding. These observations reveal distinct requirements for the MSH2 DNA binding domain I in the repair of DNA mismatches and suggest that the binding of MSH2-MSH3 to mismatch DNA involves protein-DNA contacts that appear very different from those required for MSH2-MSH6 mismatch binding.

Improved multiplex ligation-dependent probe amplification analysis identifies a deleterious PMS2 allele generated by recombination with crossover between PMS2 and PMS2CL.

PubMed

Wernstedt, Annekatrin; Valtorta, Emanuele; Armelao, Franco; Togni, Roberto; Girlando, Salvatore; Baudis, Michael; Heinimann, Karl; Messiaen, Ludwine; Staehli, Noemie; Zschocke, Johannes; Marra, Giancarlo; Wimmer, Katharina

2012-09-01

Heterozygous PMS2 germline mutations are associated with Lynch syndrome. Up to one third of these mutations are genomic deletions. Their detection is complicated by a pseudogene (PMS2CL), which--owing to extensive interparalog sequence exchange--closely resembles PMS2 downstream of exon 12. A recently redesigned multiplex ligation-dependent probe amplification (MLPA) assay identifies PMS2 copy number alterations with improved reliability when used with reference DNAs containing equal numbers of PMS2- and PMS2CL-specific sequences. We selected eight such reference samples--all publicly available--and used them with this assay to study 13 patients with PMS2-defective colorectal tumors. Three presented deleterious alterations: an Alu-mediated exon deletion; a 125-kb deletion encompassing PMS2 and four additional genes (two with tumor-suppressing functions); and a novel deleterious hybrid PMS2 allele produced by recombination with crossover between PMS2 and PMS2CL, with the breakpoint in intron 10 (the most 5' breakpoint of its kind reported thus far). We discuss mechanisms that might generate this allele in different chromosomal configurations (and their diagnostic implications) and describe an allele-specific PCR assay that facilitates its detection. Our data indicate that the redesigned PMS2 MLPA assay is a valid first-line option. In our series, it identified roughly a quarter of all PMS2 mutations. Copyright © 2012 Wiley Periodicals, Inc.

Improved Multiplex Ligation-Dependent Probe Amplification Analysis Identifies a Deleterious PMS2 Allele Generated by Recombination with Crossover Between PMS2 and PMS2CL

PubMed Central

Wernstedt, Annekatrin; Valtorta, Emanuele; Armelao, Franco; Togni, Roberto; Girlando, Salvatore; Baudis, Michael; Heinimann, Karl; Messiaen, Ludwine; Staehli, Noemie; Zschocke, Johannes; Marra, Giancarlo; Wimmer, Katharina

2012-01-01

Heterozygous PMS2 germline mutations are associated with Lynch syndrome. Up to one third of these mutations are genomic deletions. Their detection is complicated by a pseudogene (PMS2CL), which – owing to extensive interparalog sequence exchange – closely resembles PMS2 downstream of exon 12. A recently redesigned multiplex ligation-dependent probe amplification (MLPA) assay identifies PMS2 copy number alterations with improved reliability when used with reference DNAs containing equal numbers of PMS2- and PMS2CL-specific sequences. We selected eight such reference samples – all publicly available – and used them with this assay to study 13 patients with PMS2-defective colorectal tumors. Three presented deleterious alterations: an Alu-mediated exon deletion; a 125-kb deletion encompassing PMS2 and four additional genes (two with tumor-suppressing functions); and a novel deleterious hybrid PMS2 allele produced by recombination with crossover between PMS2 and PMS2CL, with the breakpoint in intron 10 (the most 5′ breakpoint of its kind reported thus far). We discuss mechanisms that might generate this allele in different chromosomal configurations (and their diagnostic implications) and describe an allele-specific PCR assay that facilitates its detection. Our data indicate that the redesigned PMS2 MLPA assay is a valid first-line option. In our series, it identified roughly a quarter of all PMS2 mutations. © 2012 Wiley Periodicals, Inc. PMID:22585707

A versatile toolkit for high throughput functional genomics with Trichoderma reesei

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schuster, Andre; Bruno, Kenneth S.; Collett, James R.

2012-01-02

The ascomycete fungus, Trichoderma reesei (anamorph of Hypocrea jecorina), represents a biotechnological workhorse and is currently one of the most proficient cellulase producers. While strain improvement was traditionally accomplished by random mutagenesis, a detailed understanding of cellulase regulation can only be gained using recombinant technologies. RESULTS: Aiming at high efficiency and high throughput methods, we present here a construction kit for gene knock out in T. reesei. We provide a primer database for gene deletion using the pyr4, amdS and hph selection markers. For high throughput generation of gene knock outs, we constructed vectors using yeast mediated recombination and thenmore » transformed a T. reesei strain deficient in non-homologous end joining (NHEJ) by spore electroporation. This NHEJ-defect was subsequently removed by crossing of mutants with a sexually competent strain derived from the parental strain, QM9414.CONCLUSIONS:Using this strategy and the materials provided, high throughput gene deletion in T. reesei becomes feasible. Moreover, with the application of sexual development, the NHEJ-defect can be removed efficiently and without the need for additional selection markers. The same advantages apply for the construction of multiple mutants by crossing of strains with different gene deletions, which is now possible with considerably less hands-on time and minimal screening effort compared to a transformation approach. Consequently this toolkit can considerably boost research towards efficient exploitation of the resources of T. reesei for cellulase expression and hence second generation biofuel production.« less

Aberrant interchromosomal exchanges are the predominant cause of the 22q11.2 deletion

PubMed Central

Saitta, Sulagna C.; Harris, Stacy E.; Gaeth, Ann P.; Driscoll, Deborah A.; McDonald-McGinn, Donna M.; Maisenbacher, Melissa K.; Yersak, Jill M.; Chakraborty, Prabir K.; Hacker, April M.; Zackai, Elaine H.; Ashley, Terry; Emanuel, Beverly S.

2010-01-01

Chromosome 22q11.2 deletions are found in almost 90% of patients with DiGeorge/velocardiofacial syndrome (DGS/VCFS). Large, chromosome-specific low copy repeats (LCRs), flanking and within the deletion interval, are presumed to lead to misalignment and aberrant recombination in meiosis resulting in this frequent microdeletion syndrome. We traced the grandparental origin of regions flanking de novo 3 Mb deletions in 20 informative three-generation families. Haplotype reconstruction showed an unexpectedly high number of proximal interchromosomal exchanges between homologs, occurring in 19/20 families. Instead, the normal chromosome 22 in these probands showed interchromosomal exchanges in 2/15 informative meioses, a rate consistent with the genetic distance. Meiotic exchanges, visualized as MLH1 foci, localize to the distal long arm of chromosome 22 in 75% of human spermatocytes tested, also reflecting the genetic map. Additionally, we found no effect of proband gender or parental age on the crossover frequency. Parental origin studies in 65 de novo 3 Mb deletions (including these 20 patients) demonstrated no bias. Unlike Williams syndrome, we found no chromosomal inversions flanked by LCRs in 22 sets of parents of 22q11 deleted patients, or in eight non-deleted patients with a DGS/VCFS phenotype using FISH. Our data are consistent with significant aberrant interchromosomal exchange events during meiosis I in the proximal region of the affected chromosome 22 as the likely etiology for the deletion. This type of exchange occurs more often than is described for deletions of chromosomes 7q11, 15q11, 17p11 and 17q11, implying a difference in the meiotic behavior of chromosome 22. PMID:14681306

Observation of a parental inversion variant in a rare Williams–Beuren syndrome family with two affected children

PubMed Central

Scherer, Stephen W.; Gripp, Karen W.; Lucena, Jaume; Nicholson, Linda; Bonnefont, Jean-Paul; Pérez-Jurado, Luis A.

2010-01-01

The Williams–Beuren syndrome (WBS) region at 7q11.23 is subject to several genomic rearrangements, one of which, the WBSinv-1 variant, is an inversion polymorphism. The WBSinv-1 chromosome has been shown to occur frequently in parents of individuals with WBS, implying that it predisposes the region to the WBS deletion. Here we investigate two WBS families with multiple affected children, and show that in one family, both siblings have a deletion on a WBSinv-1 chromosome background that arose due to interchromosomal recombination. These results suggest that the two WBS deletions in this family were independent events, and that there is likely a significant increase in the risk of deletion of the WBS region associated with the WBSinv-1 chromosome. The rarity of multiplex WBS families would suggest that the overall risk of having a child with WBS is still relatively low; however, families with an existing member with WBS may choose to opt for WBSinv-1 testing and genetic counseling. PMID:15933846

Reduction of Aspergillus niger Virulence in Apple Fruits by Deletion of the Catalase Gene cpeB.

PubMed

Zhang, Meng-Ke; Tang, Jun; Huang, Zhong-Qin; Hu, Kang-Di; Li, Yan-Hong; Han, Zhuo; Chen, Xiao-Yan; Hu, Lan-Ying; Yao, Gai-Fang; Zhang, Hua

2018-05-30

Aspergillus niger, a common saprophytic fungus, causes rot in many fruits. We studied the role of a putative catalase-peroxidase-encoding gene, cpeB, in oxidative stress and virulence in fruit. The cpeB gene was deleted in A. niger by homologous recombination, and the Δ cpeB mutant showed decreased CAT activity compared with that of the wild type. The cpeB gene deletion caused increased sensitivity to H 2 O 2 stress, and spore germination was significantly reduced; in addition, the reactive-oxygen-species (ROS) metabolites superoxide anions (·O 2 - ), hydrogen peroxide (H 2 O 2 ), and malondialdehyde (MDA) accumulated in the Δ cpeB mutant during H 2 O 2 stress. Furthermore, ROS metabolism in A. niger infected apples was determined, and our results showed that the Δ cpeB mutant induced an attenuated response in apple fruit during the fruit-pathogen interaction; the cpeB gene deletion significantly reduced the development of lesions, suggesting that the cpeB gene in A. niger is essential for full virulence in apples.

Nested Inversion Polymorphisms Predispose Chromosome 22q11.2 to Meiotic Rearrangements.

PubMed

Demaerel, Wolfram; Hestand, Matthew S; Vergaelen, Elfi; Swillen, Ann; López-Sánchez, Marcos; Pérez-Jurado, Luis A; McDonald-McGinn, Donna M; Zackai, Elaine; Emanuel, Beverly S; Morrow, Bernice E; Breckpot, Jeroen; Devriendt, Koenraad; Vermeesch, Joris R

2017-10-05

Inversion polymorphisms between low-copy repeats (LCRs) might predispose chromosomes to meiotic non-allelic homologous recombination (NAHR) events and thus lead to genomic disorders. However, for the 22q11.2 deletion syndrome (22q11.2DS), the most common genomic disorder, no such inversions have been uncovered as of yet. Using fiber-FISH, we demonstrate that parents transmitting the de novo 3 Mb LCR22A-D 22q11.2 deletion, the reciprocal duplication, and the smaller 1.5 Mb LCR22A-B 22q11.2 deletion carry inversions of LCR22B-D or LCR22C-D. Hence, the inversions predispose chromosome 22q11.2 to meiotic rearrangements and increase the individual risk for transmitting rearrangements. Interestingly, the inversions are nested or flanking rather than coinciding with the deletion or duplication sizes. This finding raises the possibility that inversions are a prerequisite not only for 22q11.2 rearrangements but also for all NAHR-mediated genomic disorders. Copyright © 2017. Published by Elsevier Inc.

Construction of Poxviruses as Cloning Vectors: Insertion of the Thymidine Kinase Gene from Herpes Simplex Virus into the DNA of Infectious Vaccinia Virus

NASA Astrophysics Data System (ADS)

Panicali, Dennis; Paoletti, Enzo

1982-08-01

We have constructed recombinant vaccinia viruses containing the thymidine kinase gene from herpes simplex virus. The gene was inserted into the genome of a variant of vaccinia virus that had undergone spontaneous deletion as well as into the 120-megadalton genome of the large prototypic vaccinia variant. This was accomplished via in vivo recombination by contransfection of eukaryotic tissue culture cells with cloned BamHI-digested thymidine kinase gene from herpes simplex virus containing flanking vaccinia virus DNA sequences and infectious rescuing vaccinia virus. Pure populations of the recombinant viruses were obtained by replica filter techniques or by growth of the recombinant virus in biochemically selective medium. The herpes simplex virus thymidine kinase gene, as an insert in vaccinia virus, is transcribed in vivo and in vitro, and the fidelity of in vivo transcription into a functional gene product was detected by the phosphorylation of 5-[125I]iodo-2'-deoxycytidine.

CEP63 deficiency promotes p53-dependent microcephaly and reveals a role for the centrosome in meiotic recombination.

PubMed

Marjanović, Marko; Sánchez-Huertas, Carlos; Terré, Berta; Gómez, Rocío; Scheel, Jan Frederik; Pacheco, Sarai; Knobel, Philip A; Martínez-Marchal, Ana; Aivio, Suvi; Palenzuela, Lluís; Wolfrum, Uwe; McKinnon, Peter J; Suja, José A; Roig, Ignasi; Costanzo, Vincenzo; Lüders, Jens; Stracker, Travis H

2015-07-09

CEP63 is a centrosomal protein that facilitates centriole duplication and is regulated by the DNA damage response. Mutations in CEP63 cause Seckel syndrome, a human disease characterized by microcephaly and dwarfism. Here we demonstrate that Cep63-deficient mice recapitulate Seckel syndrome pathology. The attrition of neural progenitor cells involves p53-dependent cell death, and brain size is rescued by the deletion of p53. Cell death is not the result of an aberrant DNA damage response but is triggered by centrosome-based mitotic errors. In addition, Cep63 loss severely impairs meiotic recombination, leading to profound male infertility. Cep63-deficient spermatocytes display numerical and structural centrosome aberrations, chromosome entanglements and defective telomere clustering, suggesting that a reduction in centrosome-mediated chromosome movements underlies recombination failure. Our results provide novel insight into the molecular pathology of microcephaly and establish a role for the centrosome in meiotic recombination.

CEP63 deficiency promotes p53-dependent microcephaly and reveals a role for the centrosome in meiotic recombination

PubMed Central

Marjanović, Marko; Sánchez-Huertas, Carlos; Terré, Berta; Gómez, Rocío; Scheel, Jan Frederik; Pacheco, Sarai; Knobel, Philip A.; Martínez-Marchal, Ana; Aivio, Suvi; Palenzuela, Lluís; Wolfrum, Uwe; McKinnon, Peter J.; Suja, José A.; Roig, Ignasi; Costanzo, Vincenzo; Lüders, Jens; Stracker, Travis H.

2015-01-01

CEP63 is a centrosomal protein that facilitates centriole duplication and is regulated by the DNA damage response. Mutations in CEP63 cause Seckel syndrome, a human disease characterized by microcephaly and dwarfism. Here we demonstrate that Cep63 deficient mice recapitulate Seckel syndrome pathology. The attrition of neural progenitor cells involves p53-dependent cell death and brain size is rescued by the deletion of p53. Cell death is not the result of an aberrant DNA damage response but is triggered by centrosome-based mitotic errors. In addition, Cep63 loss severely impairs meiotic recombination, leading to profound male infertility. Cep63 deficient spermatocytes display numerical and structural centrosome aberrations, chromosome entanglements and defective telomere clustering, suggesting that a reduction in centrosome-mediated chromosome movements underlies recombination failure. Our results provide novel insight into the molecular pathology of microcephaly and establish a role for the centrosome in meiotic recombination. PMID:26158450

LCR-initiated rearrangements at the IDS locus, completed with Alu-mediated recombination or non-homologous end joining.

PubMed

Oshima, Junko; Lee, Jennifer A; Breman, Amy M; Fernandes, Priscilla H; Babovic-Vuksanovic, Dusica; Ward, Patricia A; Wolfe, Lynne A; Eng, Christine M; Del Gaudio, Daniela

2011-07-01

Mucopolysaccharidosis type II (MPS II) is caused by mutations in the IDS gene, which encodes the lysosomal enzyme iduronate-2-sulfatase. In ∼20% of MPS II patients the disorder is caused by gross IDS structural rearrangements. We identified two male cases harboring complex rearrangements involving the IDS gene and the nearby pseudogene, IDSP1, which has been annotated as a low-copy repeat (LCR). In both cases the rearrangement included a partial deletion of IDS and an inverted insertion of the neighboring region. In silico analyses revealed the presence of repetitive elements as well as LCRs at the junctions of rearrangements. Our models illustrate two alternative consequences of rearrangements initiated by non-allelic homologous recombination of LCRs: resolution by a second recombination event (that is, Alu-mediated recombination), or resolution by non-homologous end joining repair. These complex rearrangements have the potential to be recurrent and may be present among those MSP II cases with previously uncharacterized aberrations involving IDS.

RTEL1 maintains genomic stability by suppressing homologous recombination.

PubMed

Barber, Louise J; Youds, Jillian L; Ward, Jordan D; McIlwraith, Michael J; O'Neil, Nigel J; Petalcorin, Mark I R; Martin, Julie S; Collis, Spencer J; Cantor, Sharon B; Auclair, Melissa; Tissenbaum, Heidi; West, Stephen C; Rose, Ann M; Boulton, Simon J

2008-10-17

Homologous recombination (HR) is an important conserved process for DNA repair and ensures maintenance of genome integrity. Inappropriate HR causes gross chromosomal rearrangements and tumorigenesis in mammals. In yeast, the Srs2 helicase eliminates inappropriate recombination events, but the functional equivalent of Srs2 in higher eukaryotes has been elusive. Here, we identify C. elegans RTEL-1 as a functional analog of Srs2 and describe its vertebrate counterpart, RTEL1, which is required for genome stability and tumor avoidance. We find that rtel-1 mutant worms and RTEL1-depleted human cells share characteristic phenotypes with yeast srs2 mutants: lethality upon deletion of the sgs1/BLM homolog, hyperrecombination, and DNA damage sensitivity. In vitro, purified human RTEL1 antagonizes HR by promoting the disassembly of D loop recombination intermediates in a reaction dependent upon ATP hydrolysis. We propose that loss of HR control after deregulation of RTEL1 may be a critical event that drives genome instability and cancer.

Pathogenic role of mtDNA duplications in mitochondrial diseases associated with mtDNA deletions.

PubMed

Odoardi, Francesca; Rana, Michele; Broccolini, Aldobrando; Mirabella, Massimiliano; Modoni, Anna; D'Amico, Adele; Papacci, Manuela; Tonali, Pietro; Servidei, Serenella; Silvestri, Gabriella

2003-04-30

We estimated the frequency of multiple mtDNA rearrangements by Southern blot in 32 patients affected by mitochondrial disorders associated with single deletions in order to assess genotype-phenotype correlations and elucidate the pathogenic significance of mtDNA duplications. Muscle in situ hybridization studies were performed in patients showing mtDNA duplications at Southern blot. We found multiple rearrangements in 12/32 (37.5%) patients; in particular, mtDNA duplications were detected in 4/4 Kearns-Sayre syndrome (KSS), in 1 Pearson's syndrome, in 1/3 encephalomyopathies with progressive external ophthalmoplegia (PEO), and in 2/23 PEO. In situ studies documented an exclusive accumulation of deleted mtDNAs in cytochrome c oxidase negative fibers of patients with mtDNA duplications. The presence of mtDNA duplications significantly correlated with onset of symptoms before age 15 and occurrence of clinical multisystem involvement. Analysis of biochemical data documented a predominant reduction of complex III in patients without duplications compared to patients with mtDNA duplications. Our data indicate that multiple mtDNA rearrangements are detectable in a considerable proportion of patients with single deletions and that mtDNA duplications do not cause any oxidative impairment. They more likely play a pathogenic role in the determination of clinical expression of mitochondrial diseases associated with single mtDNA deletions, possibly generating deleted mtDNAs in embryonic tissues by homologous recombination. Copyright 2003 Wiley-Liss, Inc.

Efficacy of a recombinant Rift Valley fever virus MP-12 with NSm deletion as a vaccine candidate in sheep

USDA-ARS?s Scientific Manuscript database

Rift Valley fever virus (RVFV), a mosquito-borne virus in the Bunyaviridae family and Phlebovirus genus, causes RVF, a disease of ruminants and man, endemic in Sub-Saharan African countries. However, outbreaks in Yemen and Saudi Arabia demonstrate the ability for RVFV to spread into virgin territory...

Simultaneous Deletion of the 9GL and UK Genes from the African Swine Fever Virus Georgia 2007 Isolate Offers Increased Safety and Protection against Homologous Challenge

PubMed Central

O'Donnell, Vivian; Risatti, Guillermo R.; Holinka, Lauren G.; Krug, Peter W.; Carlson, Jolene; Velazquez-Salinas, Lauro; Azzinaro, Paul A.; Gladue, Douglas P.

2016-01-01

ABSTRACT African swine fever virus (ASFV) is the etiological agent of a contagious and often lethal viral disease of domestic pigs that has significant economic consequences for the swine industry. The control of African swine fever (ASF) has been hampered by the unavailability of vaccines. Successful experimental vaccines have been derived from naturally occurring, cell culture-adapted, or genetically modified live attenuated ASFV. Recombinant viruses harboring engineered deletions of specific virulence-associated genes induce solid protection against challenge with parental viruses. Deletion of the 9GL (B119L) gene in the highly virulent ASFV isolates Malawi Lil-20/1 (Mal) and Pretoriuskop/96/4 (Δ9GL viruses) resulted in complete protection when challenged with parental isolates. When similar deletions were created within the ASFV Georgia 2007 (ASFV-G) genome, attenuation was achieved but the protective and lethal doses were too similar. To enhance attenuation of ASFV-G, we deleted another gene, UK (DP96R), which was previously shown to be involved in attenuation of the ASFV E70 isolate. Here, we report the construction of a double-gene-deletion recombinant virus, ASFV-G-Δ9GL/ΔUK. When administered intramuscularly (i.m.) to swine, there was no induction of disease, even at high doses (106 HAD50). Importantly, animals infected with 104 50% hemadsorbing doses (HAD50) of ASFV-G-Δ9GL/ΔUK were protected as early as 14 days postinoculation when challenged with ASFV-G. The presence of protection correlates with the appearance of serum anti-ASFV antibodies, but not with virus-specific circulating ASFV-specific gamma interferon (IFN-γ)-producing cells. ASFV-G-Δ9GL/ΔUK is the first rationally designed experimental ASFV vaccine that protects against the highly virulent ASFV Georgia 2007 isolate as early as 2 weeks postvaccination. IMPORTANCE Currently, there is no commercially available vaccine against African swine fever. Outbreaks of the disease are devastating to the swine industry and are caused by circulating strains of African swine fever virus. Here, we report a putative vaccine derived from a currently circulating strain but containing two deletions in two separate areas of the virus, allowing increased safety. Using this genetically modified virus, we were able to vaccinate swine and protect them from developing ASF. We were able to achieve protection from disease as early as 2 weeks after vaccination, even when the pigs were exposed to a higher than normal concentration of ASFV. PMID:27795430

Simultaneous Deletion of the 9GL and UK Genes from the African Swine Fever Virus Georgia 2007 Isolate Offers Increased Safety and Protection against Homologous Challenge.

PubMed

O'Donnell, Vivian; Risatti, Guillermo R; Holinka, Lauren G; Krug, Peter W; Carlson, Jolene; Velazquez-Salinas, Lauro; Azzinaro, Paul A; Gladue, Douglas P; Borca, Manuel V

2017-01-01

African swine fever virus (ASFV) is the etiological agent of a contagious and often lethal viral disease of domestic pigs that has significant economic consequences for the swine industry. The control of African swine fever (ASF) has been hampered by the unavailability of vaccines. Successful experimental vaccines have been derived from naturally occurring, cell culture-adapted, or genetically modified live attenuated ASFV. Recombinant viruses harboring engineered deletions of specific virulence-associated genes induce solid protection against challenge with parental viruses. Deletion of the 9GL (B119L) gene in the highly virulent ASFV isolates Malawi Lil-20/1 (Mal) and Pretoriuskop/96/4 (Δ9GL viruses) resulted in complete protection when challenged with parental isolates. When similar deletions were created within the ASFV Georgia 2007 (ASFV-G) genome, attenuation was achieved but the protective and lethal doses were too similar. To enhance attenuation of ASFV-G, we deleted another gene, UK (DP96R), which was previously shown to be involved in attenuation of the ASFV E70 isolate. Here, we report the construction of a double-gene-deletion recombinant virus, ASFV-G-Δ9GL/ΔUK. When administered intramuscularly (i.m.) to swine, there was no induction of disease, even at high doses (10 6 HAD 50 ). Importantly, animals infected with 10 4 50% hemadsorbing doses (HAD 50 ) of ASFV-G-Δ9GL/ΔUK were protected as early as 14 days postinoculation when challenged with ASFV-G. The presence of protection correlates with the appearance of serum anti-ASFV antibodies, but not with virus-specific circulating ASFV-specific gamma interferon (IFN-γ)-producing cells. ASFV-G-Δ9GL/ΔUK is the first rationally designed experimental ASFV vaccine that protects against the highly virulent ASFV Georgia 2007 isolate as early as 2 weeks postvaccination. Currently, there is no commercially available vaccine against African swine fever. Outbreaks of the disease are devastating to the swine industry and are caused by circulating strains of African swine fever virus. Here, we report a putative vaccine derived from a currently circulating strain but containing two deletions in two separate areas of the virus, allowing increased safety. Using this genetically modified virus, we were able to vaccinate swine and protect them from developing ASF. We were able to achieve protection from disease as early as 2 weeks after vaccination, even when the pigs were exposed to a higher than normal concentration of ASFV. Copyright © 2016 American Society for Microbiology.

Nonreplicative RNA Recombination of an Animal Plus-Strand RNA Virus in the Absence of Efficient Translation of Viral Proteins

PubMed Central

Kleine Büning, Maximiliane; Meyer, Denise; Austermann-Busch, Sophia; Roman-Sosa, Gleyder; Rümenapf, Tillmann

2017-01-01

RNA recombination is a major driving force for the evolution of RNA viruses and is significantly implicated in the adaptation of viruses to new hosts, changes of virulence, as well as in the emergence of new viruses including drug-resistant and escape mutants. However, the molecular details of recombination in animal RNA viruses are only poorly understood. In order to determine whether viral RNA recombination depends on translation of viral proteins, a nonreplicative recombination system was established which is based on cotransfection of cells with synthetic bovine viral diarrhea virus (family Flaviviridae) RNA genome fragments either lacking the internal ribosome entry site required for cap-independent translation or lacking almost the complete polyprotein coding region. The emergence of a number of recombinant viruses demonstrated that IRES-mediated translation of viral proteins is dispensable for efficient recombination and suggests that RNA recombination can occur in the absence of viral proteins. Analyses of 58 independently emerged viruses led to the detection of recombinant genomes with duplications, deletions and insertions in the 5′ terminal region of the open reading frame, leading to enlarged core fusion proteins detectable by Western blot analysis. This demonstrates a remarkable flexibility of the pestivirus core protein. Further experiments with capped and uncapped genome fragments containing a luciferase gene for monitoring the level of protein translation revealed that even a ∼1,000-fold enhancement of translation of viral proteins did not increase the frequency of RNA recombination. Taken together, this study highlights that nonreplicative RNA recombination does not require translation of viral proteins. PMID:28338950

[Asymmetric confusability effect in recognition memory of cats pictures].

PubMed

Ando, M; Hakoda, Y

1999-06-01

Performance superiority of the addition of features in the stimuli over the deletion on recognition (asymmetric confusability effect) has been shown in previous studies (Pezdek, Maki, Valencia-Laver, Whetstone, Stoeckert, & Dougherty, 1988; Ando & Hakoda, 1998). We investigated the same effect by using a familiar living thing (cat) as a stimulus. Ten subjects were given a recognition task using pictures of cats with feature changes (additions, deletions, or no change). Results showed that the picture with deletions were easier to recognize than those with additions, which was opposite to the previous studies. Then, we examined the possibility that performance superiority of the deletions over the additions was mediated by the factor of impression. Another group of 18 subjects was asked to rate the impression scales consisting of a "typicality-reality factor", a "stability-balance factor", and a "grotesque-disgust factor". Results showed that there was a significant difference in impression ratings for each factor between the additions and the deletions, and that impression ratings predicted recognition performance well. It was concluded that performance superiority of the deletions over the additions was mediated by the factor of impression.

IRF4 controls the positioning of mature B cells in the lymphoid microenvironments by regulating NOTCH2 expression and activity

PubMed Central

Simonetti, Giorgia; Carette, Amanda; Silva, Kathryn; Wang, Haowei; De Silva, Nilushi S.; Heise, Nicole; Siebel, Christian W.; Shlomchik, Mark J.

2013-01-01

The transcription factor interferon regulatory factor-4 (IRF4) is expressed in B cells at most developmental stages. In antigen-activated B cells, IRF4 controls germinal center formation, class-switch recombination, and the generation of plasma cells. Here we describe a novel function for IRF4 in the homeostasis of mature B cells. Inducible deletion of irf4 specifically in B cells in vivo led to the aberrant accumulation of irf4-deleted follicular B cells in the marginal zone (MZ) area. IRF4-deficient B cells showed elevated protein expression and activation of NOTCH2, a transmembrane receptor and transcriptional regulator known to be required for MZ B cell development. Administration of a NOTCH2-inhibitory antibody abolished nuclear translocation of NOTCH2 in B cells within 12 h and caused a rapid and progressive disintegration of the MZ that was virtually complete 48 h after injection. The disappearance of the MZ was accompanied by a transient increase of MZ-like B cells in the blood rather than increased B cell apoptosis, demonstrating that continued NOTCH2 activation is critical for the retention of B cells in the MZ. Our results suggest that IRF4 controls the positioning of mature B cells in the lymphoid microenvironments by regulating NOTCH2 expression. These findings may have implications for the understanding of B cell malignancies with dysregulated IRF4 and NOTCH2 activity. PMID:24323359

Recurrent proximal 18p monosomy and 18q trisomy in a family due to a pericentric inversion.

PubMed

Zamani, Ayse Gul; Acar, Aynur; Durakbasi-Dursun, Gul; Yildirim, M Selman; Ceylaner, Serdar; Tuncez, Ebru

2014-05-01

Here, we report on a family with pericentric inversion of chromosome 18 [inv(18)(p11.2q21)] and two recombinants with a duplication of q21 → qter and a deletion of p11.2 → pter regions in a four-generation family. This chromosomal abnormality was inherited in our first patient from the father, while it was transmitted to the second patient from the mother. Array-CGH analysis were used to better characterize duplicated and deleted chromosomal regions and showed no genomic copy number variation (CNV) differences between these two relatives. We discussed genotype-phenotype correlations including previously reported. © 2014 Wiley Periodicals, Inc.

UCP2 and 3 deletion screening and distribution in 15 pig breeds.

PubMed

Li, Yanhua; Li, Hanjie; Zhao, Xingbo; Li, Ning; Wu, Changxin

2007-02-01

The uncoupling protein family is a mitochondrial anion carrier family. It plays an important role in the biological traits of animal body weight, basal metabolic rate and energy conversion. Using PCR and PCR-SSCP, we scanned the porcine uncoupling protein 2 gene (UCP2) and uncoupling protein 3 gene (UCP3) and found seven deletion sites, three in UCP2 and four in UCP3. The deletions in 15 pig breeds showed that deletion influenced weight. The genotype compounding of seven deletion sites in 15 pig breeds had significant effects on performance traits of the pig, such as body weight. We predicted the potential protein factor binding sites using the transcription factor analysis tool TFSearch version 1.3 online. Two deletions (1830 nt and 3219 nt) in UCP3 were found to change the transcriptional factor sites. The 16 bp deletion in 1830 nt added a SP1 site and a 6 bp deletion in 3219 nt removed two MZF1 sites. Seven deletion polymorphisms were covered in introns of linkage genes of UCP2 and UCP3, showing that UCPs have conservation and genetic reliability.

Deacetylase activity of histone deacetylase 3 is required for productive VDJ recombination and B-cell development

PubMed Central

Stengel, Kristy R.; Barnett, Kelly R.; Wang, Jing; Liu, Qi; Hodges, Emily; Hiebert, Scott W.; Bhaskara, Srividya

2017-01-01

Histone deacetylase 3 (HDAC3) is the catalytic component of NCoR/SMRT corepressor complexes that mediate the actions of transcription factors implicated in the regulation of B-cell development and function. We crossed Hdac3 conditional knockout mice with Mb1-Cre knockin animals to delete Hdac3 in early progenitor B cells. The spleens of Hdac3F/−Mb1-Cre+/− mice were virtually devoid of mature B cells, and B220+CD43+ B-cell progenitors accumulated within the bone marrow. Quantitative deep sequencing of the Ig heavy chain locus from B220+CD43+ populations identified a defect in VHDJH recombination with a severe reduction in productive rearrangements, which directly corresponded to the loss of pre-B cells from Hdac3Δ/− bone marrow. For Hdac3Δ/− B cells that did show productive VDJ rearrangement, there was significant skewing toward the incorporation of proximal VH gene segments and a corresponding reduction in distal VH gene segment use. Although transcriptional effects within these loci were modest, Hdac3Δ/− progenitor cells displayed global changes in chromatin structure that likely hindered effective distal V-DJ recombination. Reintroduction of wild-type Hdac3 restored normal B-cell development, whereas an Hdac3 point mutant lacking deacetylase activity failed to complement this defect. Thus, the deacetylase activity of Hdac3 is required for the generation of mature B cells. PMID:28739911

Studies on the Nucleotide Sequence, Transcription and Deletion Analysis of the BmNPV Protein Kinase Gene.

PubMed

Zhang, Chuan-Xi; Hu, Cui; Wu, Xiang-Fu

1998-01-01

The coding region of BmvPK-1 gene of Bombyx mori NPV (Strain ZJ8) is 828 nt long and encodes a 276 aa polypeptide with predicted molecular mass of 32 kD. Dot blot analysis showed its mRNA to be gene is first detectable at 18 h p.i. and reaching the highest transcriptional level at 48 h p.i. The result suggested that BmvPK-1 gene is a late or very late gene. The most conserved 365 bp of the BmvPK-1 gene was deleted in a transfer vector (pUCPK-lac), and a report gene (lacZ) was inserted in the deleted position. Cotransfection of BmN cells with pUCPK-lac DNA and BmNPV DNA resulted in the recombinant virus which expressed detectable product of lacZ gene. But the virus with the deleted BmvPK-1 gene could not be isolated from the wild BmNPV by plaque purification method. The result showed that the BmvPK-1 gene deleted virus can multiply only with the help of the product of this gene from the wild type virus, and the gene is necessary for the virus to finish its life cycle in the cultured cells.

Laron syndrome. First report from Greece.

PubMed

Galli-Tsinopoulou, Assimina; Nousia-Arvanitakis, Sanda; Tsinopoulos, Ioannis; Bechlivanides, Christos; Shevah, Orit; Laron, Zvi

2003-01-01

Laron-type dwarfism is an autosomal recessive disorder caused by deletions or mutations of the growth hormone receptor gene. It is characterized by high circulating levels of growth hormone (GH) and low levels of insulin-like growth factor I (IGF-I). Patients are refractory to both endogenous and exogenous GH, and present severe growth retardation and obesity. Therapy with recombinant human insulin-like growth factor-I (rhIGF-I) accelerates linear growth. We describe a 2-year old girl with Laron syndrome, who presented with postnatal growth failure and hypoglycaemic seizures. Her evaluation disclosed high GH values during a glucagon test (peak GH value 170 ng/ml) and very low IGF I value (0.1 ng/ml) with no rise following GH administration. The growth velocity improved considerably with the administration of IGF I. Molecular analysis showed a heterozygous mutation on exon 4 of the GH receptor gene, inherited from the mother, a rather puzzling finding considering the clinical findings in mother and infant. This case constitutes the first report of Laron syndrome from Greece.

CD200/BTLA deletions in pediatric precursor B-cell acute lymphoblastic leukemia treated according to the EORTC-CLG 58951 protocol

PubMed Central

Ghazavi, Farzaneh; Clappier, Emmanuelle; Lammens, Tim; Suciu, Stefan; Caye, Aurélie; Zegrari, Samira; Bakkus, Marleen; Grardel, Nathalie; Benoit, Yves; Bertrand, Yves; Minckes, Odile; Costa, Vitor; Ferster, Alina; Mazingue, Françoise; Plat, Geneviève; Plouvier, Emmanuel; Poirée, Marilyne; Uyttebroeck, Anne; van der Werff-ten Bosch, Jutte; Yakouben, Karima; Helsmoortel, Hetty; Meul, Magali; Van Roy, Nadine; Philippé, Jan; Speleman, Frank; Cavé, Hélène; Van Vlierberghe, Pieter; De Moerloose, Barbara

2015-01-01

DNA copy number analysis has been instrumental for the identification of genetic alterations in B-cell precursor acute lymphoblastic leukemia. Notably, some of these genetic defects have been associated with poor treatment outcome and might be relevant for future risk stratification. In this study, we characterized recurrent deletions of CD200 and BTLA genes, mediated by recombination-activating genes, and used breakpoint-specific polymerase chain reaction assay to screen a cohort of 1154 cases of B-cell precursor acute lymphoblastic leukemia uniformly treated according to the EORTC-CLG 58951 protocol. CD200/BTLA deletions were identified in 56 of the patients (4.8%) and were associated with an inferior 8-year event free survival in this treatment protocol [70.2% ± 1.2% for patients with deletions versus 83.5% ± 6.4% for non-deleted cases (hazard ratio 2.02; 95% confidence interval 1.23–3.32; P=0.005)]. Genetically, CD200/BTLA deletions were strongly associated with ETV6-RUNX1-positive leukemias (P<0.0001), but were also identified in patients who did not have any genetic abnormality that is currently used for risk stratification. Within the latter population of patients, the presence of CD200/BTLA deletions was associated with inferior event-free survival and overall survival. Moreover, the multivariate Cox model indicated that these deletions had independent prognostic impact on event-free survival when adjusting for conventional risk criteria. All together, these findings further underscore the rationale for copy number profiling as an important tool for risk stratification in human B-cell precursor acute lymphoblastic leukemia. This trial was registered at www.ClinicalTrials.gov as #NCT00003728. PMID:26137961

CD200/BTLA deletions in pediatric precursor B-cell acute lymphoblastic leukemia treated according to the EORTC-CLG 58951 protocol.

PubMed

Ghazavi, Farzaneh; Clappier, Emmanuelle; Lammens, Tim; Suciu, Stefan; Caye, Aurélie; Zegrari, Samira; Bakkus, Marleen; Grardel, Nathalie; Benoit, Yves; Bertrand, Yves; Minckes, Odile; Costa, Vitor; Ferster, Alina; Mazingue, Françoise; Plat, Geneviève; Plouvier, Emmanuel; Poirée, Marilyne; Uyttebroeck, Anne; van der Werff-Ten Bosch, Jutte; Yakouben, Karima; Helsmoortel, Hetty; Meul, Magali; Van Roy, Nadine; Philippé, Jan; Speleman, Frank; Cavé, Hélène; Van Vlierberghe, Pieter; De Moerloose, Barbara

2015-10-01

DNA copy number analysis has been instrumental for the identification of genetic alterations in B-cell precursor acute lymphoblastic leukemia. Notably, some of these genetic defects have been associated with poor treatment outcome and might be relevant for future risk stratification. In this study, we characterized recurrent deletions of CD200 and BTLA genes, mediated by recombination-activating genes, and used breakpoint-specific polymerase chain reaction assay to screen a cohort of 1154 cases of B-cell precursor acute lymphoblastic leukemia uniformly treated according to the EORTC-CLG 58951 protocol. CD200/BTLA deletions were identified in 56 of the patients (4.8%) and were associated with an inferior 8-year event free survival in this treatment protocol [70.2% ± 1.2% for patients with deletions versus 83.5% ± 6.4% for non-deleted cases (hazard ratio 2.02; 95% confidence interval 1.23-3.32; P=0.005)]. Genetically, CD200/BTLA deletions were strongly associated with ETV6-RUNX1-positive leukemias (P<0.0001), but were also identified in patients who did not have any genetic abnormality that is currently used for risk stratification. Within the latter population of patients, the presence of CD200/BTLA deletions was associated with inferior event-free survival and overall survival. Moreover, the multivariate Cox model indicated that these deletions had independent prognostic impact on event-free survival when adjusting for conventional risk criteria. All together, these findings further underscore the rationale for copy number profiling as an important tool for risk stratification in human B-cell precursor acute lymphoblastic leukemia. This trial was registered at www.ClinicalTrials.gov as #NCT00003728. Copyright© Ferrata Storti Foundation.

Chromosomal instability in Streptomyces avermitilis: major deletion in the central region and stable circularized chromosome

PubMed Central

2010-01-01

Background The chromosome of Streptomyces has been shown to be unstable, frequently undergoing gross chromosomal rearrangements. However, the mechanisms underlying this phenomenon remain unclear, with previous studies focused on two chromosomal ends as targets for rearrangements. Here we investigated chromosomal instability of Streptomyces avermitilis, an important producer of avermectins, and characterized four gross chromosomal rearrangement events, including a major deletion in the central region. The present findings provide a valuable contribution to the mechanistic study of genetic instability in Streptomyces. Results Thirty randomly-selected "bald" mutants derived from the wild-type strain all contained gross chromosomal rearrangements of various types. One of the bald mutants, SA1-8, had the same linear chromosomal structure as the high avermectin-producing mutant 76-9. Chromosomes of both strains displayed at least three independent chromosomal rearrangements, including chromosomal arm replacement to form new 88-kb terminal inverted repeats (TIRs), and two major deletions. One of the deletions eliminated the 36-kb central region of the chromosome, but surprisingly did not affect viability of the cells. The other deletion (74-kb) was internal to the right chromosomal arm. The chromosome of another bald mutant, SA1-6, was circularized with deletions at both ends. No obvious homology was found in all fusion sequences. Generational stability analysis showed that the chromosomal structure of SA1-8 and SA1-6 was stable. Conclusions Various chromosomal rearrangements, including chromosomal arm replacement, interstitial deletions and chromosomal circularization, occurred in S. avermitilis by non-homologous recombination. The finding of an inner deletion involving in the central region of S. avermitilis chromosome suggests that the entire Streptomyces chromosome may be the target for rearrangements, which are not limited, as previously reported, to the two chromosomal ends. PMID:20653985

Phenotypic Characterization of a Novel Virulence-Factor Deletion Strain of Burkholderia mallei That Provides Partial Protection against Inhalational Glanders in Mice.

PubMed

Bozue, Joel A; Chaudhury, Sidhartha; Amemiya, Kei; Chua, Jennifer; Cote, Christopher K; Toothman, Ronald G; Dankmeyer, Jennifer L; Klimko, Christopher P; Wilhelmsen, Catherine L; Raymond, Jolynn W; Zavaljevski, Nela; Reifman, Jaques; Wallqvist, Anders

2016-01-01

Burkholderia mallei (Bm) is a highly infectious intracellular pathogen classified as a category B biological agent by the Centers for Disease Control and Prevention. After respiratory exposure, Bm establishes itself within host macrophages before spreading into major organ systems, which can lead to chronic infection, sepsis, and death. Previously, we combined computational prediction of host-pathogen interactions with yeast two-hybrid experiments and identified novel virulence factor genes in Bm, including BMAA0553, BMAA0728 (tssN), and BMAA1865. In the present study, we used recombinant allelic exchange to construct deletion mutants of BMAA0553 and tssN (ΔBMAA0553 and ΔTssN, respectively) and showed that both deletions completely abrogated virulence at doses of >100 times the LD50 of the wild-type Bm strain. Analysis of ΔBMAA0553- and ΔTssN-infected mice showed starkly reduced bacterial dissemination relative to wild-type Bm, and subsequent in vitro experiments characterized pathogenic phenotypes with respect to intracellular growth, macrophage uptake and phagosomal escape, actin-based motility, and multinucleated giant cell formation. Based on observed in vitro and in vivo phenotypes, we explored the use of ΔTssN as a candidate live-attenuated vaccine. Mice immunized with aerosolized ΔTssN showed a 21-day survival rate of 67% after a high-dose aerosol challenge with the wild-type Bm ATCC 23344 strain, compared to a 0% survival rate for unvaccinated mice. However, analysis of histopathology and bacterial burden showed that while the surviving vaccinated mice were protected from acute infection, Bm was still able to establish a chronic infection. Vaccinated mice showed a modest IgG response, suggesting a limited potential of ΔTssN as a vaccine candidate, but also showed prolonged elevation of pro-inflammatory cytokines, underscoring the role of cellular and innate immunity in mitigating acute infection in inhalational glanders.

Phenotypic Characterization of a Novel Virulence-Factor Deletion Strain of Burkholderia mallei That Provides Partial Protection against Inhalational Glanders in Mice

PubMed Central

Bozue, Joel A.; Chaudhury, Sidhartha; Amemiya, Kei; Chua, Jennifer; Cote, Christopher K.; Toothman, Ronald G.; Dankmeyer, Jennifer L.; Klimko, Christopher P.; Wilhelmsen, Catherine L.; Raymond, Jolynn W.; Zavaljevski, Nela; Reifman, Jaques; Wallqvist, Anders

2016-01-01

Burkholderia mallei (Bm) is a highly infectious intracellular pathogen classified as a category B biological agent by the Centers for Disease Control and Prevention. After respiratory exposure, Bm establishes itself within host macrophages before spreading into major organ systems, which can lead to chronic infection, sepsis, and death. Previously, we combined computational prediction of host-pathogen interactions with yeast two-hybrid experiments and identified novel virulence factor genes in Bm, including BMAA0553, BMAA0728 (tssN), and BMAA1865. In the present study, we used recombinant allelic exchange to construct deletion mutants of BMAA0553 and tssN (ΔBMAA0553 and ΔTssN, respectively) and showed that both deletions completely abrogated virulence at doses of >100 times the LD50 of the wild-type Bm strain. Analysis of ΔBMAA0553- and ΔTssN-infected mice showed starkly reduced bacterial dissemination relative to wild-type Bm, and subsequent in vitro experiments characterized pathogenic phenotypes with respect to intracellular growth, macrophage uptake and phagosomal escape, actin-based motility, and multinucleated giant cell formation. Based on observed in vitro and in vivo phenotypes, we explored the use of ΔTssN as a candidate live-attenuated vaccine. Mice immunized with aerosolized ΔTssN showed a 21-day survival rate of 67% after a high-dose aerosol challenge with the wild-type Bm ATCC 23344 strain, compared to a 0% survival rate for unvaccinated mice. However, analysis of histopathology and bacterial burden showed that while the surviving vaccinated mice were protected from acute infection, Bm was still able to establish a chronic infection. Vaccinated mice showed a modest IgG response, suggesting a limited potential of ΔTssN as a vaccine candidate, but also showed prolonged elevation of pro-inflammatory cytokines, underscoring the role of cellular and innate immunity in mitigating acute infection in inhalational glanders. PMID:26955620

A 1.5 Mb submicroscopic deletion in 17p11.2-p12 is frequently observed in Italian families with hereditary neuropathy with liability to pressure palsies

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lorenzetti, D.; Roa, B.B.; Abbas, N.E.

1994-09-01

Hereditary neuropathy with liability to pressure palsies (HNPP) is an autosomal dominant disorder characterized by recurrent mononeuropathies that was recently associated with a 1.5 Mb deletion in chromosome 17p11.2-p12. Duplication of the same region is known to be associated with Charcot-Marie-Tooth disease type 1A (CMT1A), a more severe peripheral neuropathy characterized by symmetrically slowed nerve conduction velocity. The CMT1A duplication and HNPP deletion are reciprocal recombination products involving a repeat element (CMT1A-REP) which flanks the 1.5 Mb region involved in the duplication/deletion. Patients from 9 unrelated HNPP Italian families were clinically, electrophysiologically and histologically evaluated. Families were typed with amore » polymorphic (CA){sub n} repeat and with RFLPs corresponding to loci D17S122, D17S125 and D17S61, which all map within the deleted region. Lack of allelic transmission from affected parent to affected offspring was observed in four informative families, suggesting the presence of deletion. Southern blot analysis of EcoRI digested genomic DNA from HNPP patients and control subjects was performed using a probe mapping within the CMT1A-REP elements. A reduced hybridization signal of a 6.0 kb EcoRI fragment, mapping within the distal CMT1A-REP, was observed in all HNPP patients suggesting the loss of one copy of this fragment in the HNPP-deleted chromosome. PFGE analysis of SacII digested genomic DNA from selected HNPP subjects showed the presence of a junction fragment which has previously been found in association with the 1.5 Mb HNPP deletion. Evidence for deletion could be demonstrated in all 9 families suggesting that the 17p11.2-p12 deletion is commonly associated with HNPP.« less

A 1.5-Mb deletion in 17p11.2-p12 is frequently observed in Italian families with hereditary neuropathy with liability to pressure palsies

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lorenzetti, D.; Pandolfo, M.; Pareyson, D.

1995-01-01

Hereditary neuropathy with liability to pressure palsies (HNPP) is an autosomal dominant disorder characterized by recurrent mononeuropathies. A 1.5-Mb deletion in chromosome 17p11.2-p12 has been associated with HNPP. Duplication of the same 1.5-Mb region is known to be associated with Charcot-Marie-Tooth disease type 1 (CMT1A), a more severe peripheral neuropathy characterized by symmetrically slowed nerve conduction velocity (NCV). The CMT1A duplication and HNPP deletion appear to be the reciprocal products of a recombination event involving a repeat element (CMT1A-REP) that flanks the 1.5-Mb region involved in the duplication/deletion. Patients from nine unrelated Italian families who were diagnosed with HNPP onmore » the basis of clinical, electrophysiological, and histological evaluations were analyzed by molecular methods for DNA deletion on chromosome 17p. In all nine families, Southern analysis using a CMT1A-REP probe detected a reduced hybridization signal of a 6.0-kb EcoRI fragment mapping within the distal CMT1A-REP, indicating deletion of one copy of CMT1A-REP in these HNPP patients. Families were also typed with a polymorphic (CA){sub n} repeat and with RFLPs corresponding to loci D17S122, D17S125, and D17S61, which all map within the deleted region. Lack of allelic transmission from affected parent to affected offspring was observed in four informative families, providing an independent indication for deletion. Furthermore, pulsed-field gel electrophoresis analysis of SacII-digested genomic DNA detected junction fragments specific to the 1.5-Mb HNPP deletion in seven of nine Italian families included in this study. These findings suggest that a 1.5-Mb deletion on 17p11.2-p12 is the most common mutation associated with HNPP. 51 refs., 5 figs., 1 tab.« less

Deleting the para-nitrophenyl phosphatase (pNPPase), PHO13, in recombinant Saccharomyces cerevisiae improves growth and ethanol production on D-xylose

Treesearch

Jennifer Van Vleet; Thomas W. Jeffries; Lisbeth Olsson

2008-01-01

Overexpression of D-xylulokinase in Saccharomyces cerevisiae engineered for assimilation of xylose results in growth inhibition that is more pronounced at higher xylose concentrations. Mutants deficient in the para-nitrophenyl phosphatase, PHO13, resist growth inhibition on xylose. We studied this inhibition under aerobic growth conditions in well-controlled...

Autographa californica multiple nucleopolyhedrovirus ac53 plays a role in nucleocapsid assembly

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu Chao; Li Zhaofei; Wu Wenbi

2008-12-05

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) orf53 (ac53) is a highly conserved gene existing in all sequenced Lepidoptera and Hymenoptera baculoviruses, but its function remains unknown. To investigate its role in the baculovirus life cycle, an ac53 deletion virus (vAc{sup ac53KO-PH-GFP}) was generated through homologous recombination in Escherichia coli. Fluorescence and light microscopy and titration analysis revealed that vAc{sup ac53KO-PH-GFP} could not produce infectious budded virus in infected Sf9 cells. Real-time PCR demonstrated that the ac53 deletion did not affect the levels of viral DNA replication. Electron microscopy showed that many lucent tubular shells devoid of the nucleoprotein core are presentmore » in the virogenic stroma and ring zone, indicating that the ac53 knockout affected nucleocapsid assembly. With a recombinant virus expressing an Ac53-GFP fusion protein, we observed that Ac53 was distributed within the cytoplasm and nucleus at 24 h post-infection, but afterwards accumulated predominantly near the nucleus-cytoplasm boundary. These data demonstrate that ac53 is involved in nucleocapsid assembly and is an essential gene for virus production.« less

Bacterial genome reduction using the progressive clustering of deletions via yeast sexual cycling

DOE PAGES

Suzuki, Yo; Assad-Garcia, Nacyra; Kostylev, Maxim; ...

2015-02-05

The availability of genetically tractable organisms with simple genomes is critical for the rapid, systems-level understanding of basic biological processes. Mycoplasma bacteria, with the smallest known genomes among free-living cellular organisms, are ideal models for this purpose, but the natural versions of these cells have genome complexities still too great to offer a comprehensive view of a fundamental life form. Here in this paper we describe an efficient method for reducing genomes from these organisms by identifying individually deletable regions using transposon mutagenesis and progressively clustering deleted genomic segments using meiotic recombination between the bacterial genomes harbored in yeast. Mycoplasmalmore » genomes subjected to this process and transplanted into recipient cells yielded two mycoplasma strains. The first simultaneously lacked eight singly deletable regions of the genome, representing a total of 91 genes and ~10%of the original genome. The second strain lacked seven of the eight regions, representing 84 genes. Growth assay data revealed an absence of genetic interactions among the 91 genes under tested conditions. Despite predicted effects of the deletions on sugar metabolism and the proteome, growth rates were unaffected by the gene deletions in the seven-deletion strain. These results support the feasibility of using single-gene disruption data to design and construct viable genomes lacking multiple genes, paving the way toward genome minimization. The progressive clustering method is expected to be effective for the reorganization of any mega-sized DNA molecules cloned in yeast, facilitating the construction of designer genomes in microbes as well as genomic fragments for genetic engineering of higher eukaryotes.« less

Bacterial genome reduction using the progressive clustering of deletions via yeast sexual cycling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Suzuki, Yo; Assad-Garcia, Nacyra; Kostylev, Maxim

The availability of genetically tractable organisms with simple genomes is critical for the rapid, systems-level understanding of basic biological processes. Mycoplasma bacteria, with the smallest known genomes among free-living cellular organisms, are ideal models for this purpose, but the natural versions of these cells have genome complexities still too great to offer a comprehensive view of a fundamental life form. Here in this paper we describe an efficient method for reducing genomes from these organisms by identifying individually deletable regions using transposon mutagenesis and progressively clustering deleted genomic segments using meiotic recombination between the bacterial genomes harbored in yeast. Mycoplasmalmore » genomes subjected to this process and transplanted into recipient cells yielded two mycoplasma strains. The first simultaneously lacked eight singly deletable regions of the genome, representing a total of 91 genes and ~10%of the original genome. The second strain lacked seven of the eight regions, representing 84 genes. Growth assay data revealed an absence of genetic interactions among the 91 genes under tested conditions. Despite predicted effects of the deletions on sugar metabolism and the proteome, growth rates were unaffected by the gene deletions in the seven-deletion strain. These results support the feasibility of using single-gene disruption data to design and construct viable genomes lacking multiple genes, paving the way toward genome minimization. The progressive clustering method is expected to be effective for the reorganization of any mega-sized DNA molecules cloned in yeast, facilitating the construction of designer genomes in microbes as well as genomic fragments for genetic engineering of higher eukaryotes.« less

Application of the FLP/FRT system for conditional gene deletion in yeast Saccharomyces cerevisiae.

PubMed

Park, Yang-Nim; Masison, Daniel; Eisenberg, Evan; Greene, Lois E

2011-09-01

The yeast Saccharomyces cerevisiae has proved to be an excellent model organism to study the function of proteins. One of the many advantages of yeast is the many genetic tools available to manipulate gene expression, but there are still limitations. To complement the many methods used to control gene expression in yeast, we have established a conditional gene deletion system by using the FLP/FRT system on yeast vectors to conditionally delete specific yeast genes. Expression of Flp recombinase, which is under the control of the GAL1 promoter, was induced by galactose, which in turn excised FRT sites flanked genes. The efficacy of this system was examined using the FRT site-flanked genes HSP104, URA3 and GFP. The pre-excision frequency of this system, which might be caused by the basal activity of the GAL1 promoter or by spontaneous recombination between FRT sites, was detected ca. 2% under the non-selecting condition. After inducing expression of Flp recombinase, the deletion efficiency achieved ca. 96% of cells in a population within 9 h. After conditional deletion of the specific gene, protein degradation and cell division then diluted out protein that was expressed from this gene prior to its excision. Most importantly, the specific protein to be deleted could be expressed under its own promoter, so that endogenous levels of protein expression were maintained prior to excision by the Flp recombinase. Therefore, this system provides a useful tool for the conditional deletion of genes in yeast. Published in 2011 by John Wiley & Sons, Ltd.

Orpheus recombination : a comprehensive bacteriophage system for murine targeting vector construction by transplacement.

PubMed

Woltjen, Knut; Ito, Kenichi; Tsuzuki, Teruhisa; Rancourt, Derrick E

2008-01-01

In recent years, methods to address the simplification of targeting vector (TV) construction have been developed and validated. Based on in vivo recombination in Escherichia coli, these protocols have reduced dependence on restriction endonucleases, allowing the fabrication of complex TV constructs with relative ease. Using a methodology based on phage-plasmid recombination, we have developed a comprehensive TV construction protocol dubbed Orpheus recombination (ORE). The ORE system addresses all necessary requirements for TV construction; from the isolation of genespecific regions of homology to the deposition of selection/disruption cassettes. ORE makes use of a small recombination plasmid, which bears positive and negative selection markers and a cloned homologous "probe" region. This probe plasmid may be introduced into and excised from phage-borne murine genomic clones by two rounds of single crossover recombination. In this way, desired clones can be specifically isolated from a heterogeneous library of phage. Furthermore, if the probe region contains a designed mutation, it may be deposited seamlessly into the genomic clone. The complete removal of operational sequences allows unlimited repetition of the procedure to customize and finalize TVs within a few weeks. Successful gene-specific clone isolation, point mutations, large deletions, cassette insertions, and finally coincident clone isolation and mutagenesis have all been demonstrated with this method.

The yeast MSH1 gene is not involved in DNA repair or recombination during meiosis.

PubMed

Sia, Elaine A; Kirkpatrick, David T

2005-02-03

Six strong homologs of the bacterial MutS DNA mismatch repair (MMR) gene have been identified in the yeast Saccharomyces cerevisiae. With the exception of the MSH1 gene, the involvement of each homolog in DNA repair and recombination during meiosis has been determined previously. Five of the homologs have been demonstrated to act in meiotic DNA repair (MSH2, MSH3, MSH6 and MSH4) and/or meiotic recombination (MSH4 and MSH5). Unfortunately the loss of mitochondrial function that results from deletion of MSH1 disrupts meiotic progression, precluding an analysis of MSH1 function in meiotic DNA repair and recombination. However, the recent identification of two separation-of-function alleles of MSH1 that interfere with protein function but still maintain functional mitochondria allow the meiotic activities of MSH1 to be determined. We show that the G776D and F105A alleles of MSH1 exhibit no defects in meiotic recombination, repair base-base mismatches and large loop mismatches efficiently during meiosis, and have high levels of spore viability. These data indicate that the MSH1 protein, unlike other MutS homologs in yeast, plays no role in DNA repair or recombination during meiosis.

AluY-mediated germline deletion, duplication and somatic stem cell reversion in UBE2T defines a new subtype of Fanconi anemia.

PubMed

Virts, Elizabeth L; Jankowska, Anna; Mackay, Craig; Glaas, Marcel F; Wiek, Constanze; Kelich, Stephanie L; Lottmann, Nadine; Kennedy, Felicia M; Marchal, Christophe; Lehnert, Erik; Scharf, Rüdiger E; Dufour, Carlo; Lanciotti, Marina; Farruggia, Piero; Santoro, Alessandra; Savasan, Süreyya; Scheckenbach, Kathrin; Schipper, Jörg; Wagenmann, Martin; Lewis, Todd; Leffak, Michael; Farlow, Janice L; Foroud, Tatiana M; Honisch, Ellen; Niederacher, Dieter; Chakraborty, Sujata C; Vance, Gail H; Pruss, Dmitry; Timms, Kirsten M; Lanchbury, Jerry S; Alpi, Arno F; Hanenberg, Helmut

2015-09-15

Fanconi anemia (FA) is a rare inherited disorder clinically characterized by congenital malformations, progressive bone marrow failure and cancer susceptibility. At the cellular level, FA is associated with hypersensitivity to DNA-crosslinking genotoxins. Eight of 17 known FA genes assemble the FA E3 ligase complex, which catalyzes monoubiquitination of FANCD2 and is essential for replicative DNA crosslink repair. Here, we identify the first FA patient with biallelic germline mutations in the ubiquitin E2 conjugase UBE2T. Both mutations were aluY-mediated: a paternal deletion and maternal duplication of exons 2-6. These loss-of-function mutations in UBE2T induced a cellular phenotype similar to biallelic defects in early FA genes with the absence of FANCD2 monoubiquitination. The maternal duplication produced a mutant mRNA that could encode a functional protein but was degraded by nonsense-mediated mRNA decay. In the patient's hematopoietic stem cells, the maternal allele with the duplication of exons 2-6 spontaneously reverted to a wild-type allele by monoallelic recombination at the duplicated aluY repeat, thereby preventing bone marrow failure. Analysis of germline DNA of 814 normal individuals and 850 breast cancer patients for deletion or duplication of UBE2T exons 2-6 identified the deletion in only two controls, suggesting aluY-mediated recombinations within the UBE2T locus are rare and not associated with an increased breast cancer risk. Finally, a loss-of-function germline mutation in UBE2T was detected in a high-risk breast cancer patient with wild-type BRCA1/2. Cumulatively, we identified UBE2T as a bona fide FA gene (FANCT) that also may be a rare cancer susceptibility gene. © The Author 2015. Published by Oxford University Press.

Role of Bunyamwera Orthobunyavirus NSs protein in infection of mosquito cells.

PubMed

Szemiel, Agnieszka M; Failloux, Anna-Bella; Elliott, Richard M

2012-01-01

Bunyamwera orthobunyavirus is both the prototype and study model of the Bunyaviridae family. The viral NSs protein seems to contribute to the different outcomes of infection in mammalian and mosquito cell lines. However, only limited information is available on the growth of Bunyamwera virus in cultured mosquito cells other than the Aedes albopictus C6/36 line. To determine potential functions of the NSs protein in mosquito cells, replication of wild-type virus and a recombinant NSs deletion mutant was compared in Ae. albopictus C6/36, C7-10 and U4.4 cells, and in Ae. aegypti Ae cells by monitoring N protein production and virus yields at various times post infection. Both viruses established persistent infections, with the exception of NSs deletion mutant in U4.4 cells. The NSs protein was nonessential for growth in C6/36 and C7-10 cells, but was important for productive replication in U4.4 and Ae cells. Fluorescence microscopy studies using recombinant viruses expressing green fluorescent protein allowed observation of three stages of infection, early, acute and late, during which infected cells underwent morphological changes. In the absence of NSs, these changes were less pronounced. An RNAi response efficiently reduced virus replication in U4.4 cells transfected with virus specific dsRNA, but not in C6/36 or C7/10 cells. Lastly, Ae. aegypti mosquitoes were exposed to blood-meal containing either wild-type or NSs deletion virus, and at various times post-feeding, infection and disseminated infection rates were measured. Compared to wild-type virus, infection rates by the mutant virus were lower and more variable. If the NSs deletion virus was able to establish infection, it was detected in salivary glands at 6 days post-infection, 3 days later than wild-type virus. Bunyamwera virus NSs is required for efficient replication in certain mosquito cell lines and in Ae. aegypti mosquitoes.

Susceptibility of proliferating cells to benzo[a]pyrene-induced homologous recombination in mice.

PubMed

Bishop, A J; Kosaras, B; Carls, N; Sidman, R L; Schiestl, R H

2001-04-01

The pink-eyed unstable mutation, p(un), is the result of a 70 kb tandem duplication within the murine pink-eyed, p, gene. Deletion of one copy of the duplicated region by homologous deletion/recombination occurs spontaneously in embryos and results in pigmented spots in the fur and eye. Such deletion events are inducible by a variety of DNA damaging agents, as we have observed previously with both fur- and eye-spot assays. Here we describe a study of the effect of exposure to benzo[a]pyrene (B[a]P) at different times of development on reversion induction in the eye. Previously we, among others, have reported that the retinal pigment epithelium (RPE) displays a position effect variegation phenotype in the pattern of pink-eyed unstable reversions. Following an acute exposure to B[a]P or X-rays on the tenth day of gestation an increased frequency of reversion events was detected in a distinct region of the adult RPE. Examining exposure at different times of eye development reveals that both B[a]P and X-rays result in an increased frequency of reversion events, though the increase was only significant following B[a]P exposure, similar to our previous report limited to exposure on the tenth day of gestation. Examination of B[a]P-exposed RPE in the present study revealed distinct regions where the induced events lie and that the positions of these regions are found at increasing distances from the optic nerve the later the time of exposure. This position effect directly reflects the previously observed developmental pattern of the RPE, namely that cells in the regions most distal from the optic nerve are proliferating most vigorously. The numbers and positions of RPE cells displaying the transformed (pigmented) phenotype strongly advocate the proposal that dividing cells are at highest risk to deletions induced by carcinogens.

Atm knock-in mice harboring an in-frame deletion corresponding to the human ATM 7636del9 common mutation exhibit a variant phenotype.

PubMed

Spring, K; Cross, S; Li, C; Watters, D; Ben-Senior, L; Waring, P; Ahangari, F; Lu, S L; Chen, P; Misko, I; Paterson, C; Kay, G; Smorodinsky, N I; Shiloh, Y; Lavin, M F

2001-06-01

ATM, the gene mutated in the human immunodeficiency disorder ataxia-telangiectasia (A-T), plays a central role in recognizing ionizing radiation damage in DNA and in controlling several cell cycle checkpoints. We describe here a murine model in which a nine-nucleotide in-frame deletion has been introduced into the Atm gene by homologous recombination followed by removal of the selectable marker cassette by Cre-loxP site-specific, recombination-mediated excision. This mouse, Atm-DeltaSRI, was designed as a model of one of the most common deletion mutations (7636del9) found in A-T patients. The murine Atm deletion results in the loss of three amino acid residues (SRI; 2556-2558) but produces near full-length detectable Atm protein that lacks protein kinase activity. Radiosensitivity was observed in Atm-DeltaSRI mice, whereas the immunological profile of these mice showed greater heterogeneity of T-cell subsets than observed in Atm(-/-) mice. The life span of Atm-DeltaSRI mice was significantly longer than that of Atm(-/-) mice when maintained under nonspecific pathogen-free conditions. This can be accounted for by a lower incidence of thymic lymphomas in Atm-DeltaSRI mice up to 40 weeks, after which time the animals died of other causes. The thymic lymphomas in Atm-DeltaSRI mice were characterized by extensive apoptosis, which appears to be attributable to an increased number of cells expressing Fas ligand. A variety of other tumors including B-cell lymphomas, sarcomas, and carcinomas not seen in Atm(-/-) mice were observed in older Atm-DeltaSRI animals. Thus, expression of mutant protein in Atm-DeltaSRI knock-in mice gives rise to a discernibly different phenotype to Atm(-/-) mice, which may account for the heterogeneity seen in A-T patients with different mutations.

Activation-induced cytidine deaminase (AID) expression in human B-cell precursors is essential for central B-cell tolerance

PubMed Central

Cantaert, Tineke; Schickel, Jean-Nicolas; Bannock, Jason M.; Ng, Yen-Shing; Massad, Christopher; Oe, Tyler; Wu, Renee; Lavoie, Aubert; Walter, Jolan E.; Notarangelo, Luigi D.; Al-Herz, Waleed; Kilic, Sara Sebnem; Ochs, Hans D.; Nonoyama, Shigeaki; Durandy, Anne; Meffre, Eric

2015-01-01

SUMMARY Activation-induced cytidine deaminase (AID), the enzyme mediating class switch recombination (CSR) and somatic hypermutation (SHM) of immunoglobulin genes, is essential for the removal of developing autoreactive B cells. How AID mediates central B-cell tolerance remains unknown. We report that AID enzymes were produced in a discrete population of immature B cells that expressed recombination-activating gene 2 (RAG2), suggesting that they undergo secondary recombination to edit autoreactive antibodies. However, most AID+ immature B cells lacked anti-apoptotic MCL-1 and were deleted by apoptosis. AID inhibition using lentiviral-encoded short hairpin (sh)RNA in B cells developing in humanized mice resulted in a failure to remove autoreactive clones. Hence, B-cell intrinsic AID expression mediates central B-cell tolerance potentially through its RAG-coupled genotoxic activity in self-reactive immature B cells. PMID:26546282

A boy with partial dup(18q)/del(18p) due to a maternal pericentric inversion: Genotype-phenotype correlation and risk of recombinant chromosomes based on systematic review of the literature.

PubMed

Lustosa-Mendes, Elaine; Dos Santos, Ana Paula; Viguetti-Campos, Nilma Lúcia; Vieira, Társis Paiva; Gil-da-Silva-Lopes, Vera Lúcia

2017-01-01

We report a boy carrying a recombinant chromosome 18, with terminal deletion of 10.8 Mb from 18p11.32 to 18p11.21 and a terminal duplication of 22.8 Mb from 18q21.31 to 18q23, resulting from a maternal pericentric inversion of the chromosome 18. He presented with poor growth, developmental delay, facial dysmorphisms, surgically repaired left cleft lip and palate, a mild form of holoprosencephaly characterized by single central incisor and agenesis of the septum pellucidum, and body asymmetry. Based on the systematic review of the literature, we discuss genotype-phenotype correlation and the risk for the recombinants of pericentric inversions of chromosome 18. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Nonreplicative RNA Recombination of an Animal Plus-Strand RNA Virus in the Absence of Efficient Translation of Viral Proteins.

PubMed

Kleine Büning, Maximiliane; Meyer, Denise; Austermann-Busch, Sophia; Roman-Sosa, Gleyder; Rümenapf, Tillmann; Becher, Paul

2017-04-01

RNA recombination is a major driving force for the evolution of RNA viruses and is significantly implicated in the adaptation of viruses to new hosts, changes of virulence, as well as in the emergence of new viruses including drug-resistant and escape mutants. However, the molecular details of recombination in animal RNA viruses are only poorly understood. In order to determine whether viral RNA recombination depends on translation of viral proteins, a nonreplicative recombination system was established which is based on cotransfection of cells with synthetic bovine viral diarrhea virus (family Flaviviridae) RNA genome fragments either lacking the internal ribosome entry site required for cap-independent translation or lacking almost the complete polyprotein coding region. The emergence of a number of recombinant viruses demonstrated that IRES-mediated translation of viral proteins is dispensable for efficient recombination and suggests that RNA recombination can occur in the absence of viral proteins. Analyses of 58 independently emerged viruses led to the detection of recombinant genomes with duplications, deletions and insertions in the 5' terminal region of the open reading frame, leading to enlarged core fusion proteins detectable by Western blot analysis. This demonstrates a remarkable flexibility of the pestivirus core protein. Further experiments with capped and uncapped genome fragments containing a luciferase gene for monitoring the level of protein translation revealed that even a ∼1,000-fold enhancement of translation of viral proteins did not increase the frequency of RNA recombination. Taken together, this study highlights that nonreplicative RNA recombination does not require translation of viral proteins. © The Author(s) 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Activation of human acid sphingomyelinase through modification or deletion of C-terminal cysteine.

PubMed

Qiu, Huawei; Edmunds, Tim; Baker-Malcolm, Jennifer; Karey, Kenneth P; Estes, Scott; Schwarz, Cordula; Hughes, Heather; Van Patten, Scott M

2003-08-29

One form of Niemann-Pick disease is caused by a deficiency in the enzymatic activity of acid sphingomyelinase. During efforts to develop an enzyme replacement therapy based on a recombinant form of human acid sphingomyelinase (rhASM), purified preparations of the recombinant enzyme were found to have substantially increased specific activity if cell harvest media were stored for several weeks at -20 degrees C prior to purification. This increase in activity was found to correlate with the loss of the single free thiol on rhASM, suggesting the involvement of a cysteine residue. It was demonstrated that a variety of chemical modifications of the free cysteine on rhASM all result in substantial activation of the enzyme, and the modified cysteine responsible for this activation was shown to be the C-terminal residue (Cys629). Activation was also achieved by copper-promoted dimerization of rhASM (via cysteine) and by C-terminal truncation using carboxypeptidase Y. The role of the C-terminal cysteine in activation was confirmed by creating mutant forms of rhASM in which this residue was either deleted or replaced by a serine, with both forms having substantially higher specific activity than wild-type rhASM. These results indicate that purified rhASM can be activated in vitro by loss of the free thiol on the C-terminal cysteine via chemical modification, dimerization, or deletion of this amino acid residue. This method of activation is similar to the cysteine switch mechanism described previously for matrix metalloproteinases and could represent a means of posttranslational regulation of ASM activity in vivo.

Two different types of double-strand breaks in Saccharomyces cerevisiae are repaired by similar RAD52-independent, nonhomologous recombination events.

PubMed Central

Kramer, K M; Brock, J A; Bloom, K; Moore, J K; Haber, J E

1994-01-01

In haploid rad52 Saccharomyces cerevisiae strains unable to undergo homologous recombination, a chromosomal double-strand break (DSB) can be repaired by imprecise rejoining of the broken chromosome ends. We have used two different strategies to generate broken chromosomes: (i) a site-specific DSB generated at the MAT locus by HO endonuclease cutting or (ii) a random DSB generated by mechanical rupture during mitotic segregation of a conditionally dicentric chromosome. Broken chromosomes were repaired by deletions that were highly variable in size, all of which removed more sequences than was required either to prevent subsequent HO cleavage or to eliminate a functional centromere, respectively. The junction of the deletions frequently occurred where complementary strands from the flanking DNA could anneal to form 1 to 5 bp, although 12% (4 of 34) of the events appear to have occurred by blunt-end ligation. These types of deletions are very similar to the junctions observed in the repair of DSBs by mammalian cells (D. B. Roth and J. H. Wilson, Mol. Cell. Biol. 6:4295-4304, 1986). When a high level of HO endonuclease, expressed in all phases of the cell cycle, was used to create DSBs, we also recovered a large class of very small (2- or 3-bp) insertions in the HO cleavage site. These insertions appear to represent still another mechanism of DSB repair, apparently by annealing and filling in the overhanging 3' ends of the cleavage site. These types of events have also been well documented for vertebrate cells. PMID:8289808

Molecular analysis of recombination in a family with Duchenne muscular dystrophy and a large pericentric X chromosome inversion.

PubMed Central

Shashi, V.; Golden, W. L.; Allinson, P. S.; Blanton, S. H.; von Kap-Herr, C.; Kelly, T. E.

1996-01-01

It has been demonstrated in animal studies that, in animals heterozygous for pericentric chromosomal inversions, loop formation is greatly reduced during meiosis. This results in absence of recombination within the inverted segment, with recombination seen only outside the inversion. A recent study in yeast has shown that telomeres, rather than centromeres, lead in chromosome movement just prior to meiosis and may be involved in promoting recombination. We studied by cytogenetic analysis and DNA polymorphisms the nature of meiotic recombination in a three-generation family with a large pericentric X chromosome inversion, inv(X)(p21.1q26), in which Duchenne muscular dystrophy (DMD) was cosegregating with the inversion. On DNA analysis there was no evidence of meiotic recombination between the inverted and normal X chromosomes in the inverted segment. Recombination was seen at the telomeric regions, Xp22 and Xq27-28. No deletion or point mutation was found on analysis of the DMD gene. On the basis of the FISH results, we believe that the X inversion is the mutation responsible for DMD in this family. Our results indicate that (1) pericentric X chromosome inversions result in reduction of recombination between the normal and inverted X chromosomes; (2) meiotic X chromosome pairing in these individuals is likely initiated at the telomeres; and (3) in this family DMD is caused by the pericentric inversion. Images Figure 2 Figure 5 Figure 6 Figure 7 PMID:8651300

Ig synthesis and class switching do not require the presence of the hs4 enhancer in the 3' IgH regulatory region.

PubMed

Vincent-Fabert, Christelle; Truffinet, Véronique; Fiancette, Remi; Cogné, Nadine; Cogné, Michel; Denizot, Yves

2009-06-01

Several studies have reported that regulatory elements located 3' of the IgH locus (namely hs3a, hs1,2, hs3b, and hs4) might play a role during class switch recombination (CSR) and Ig synthesis. While individual deletion of hs3a or hs1,2 had no effect, pairwise deletion of hs3b (an inverted copy of hs3a) and hs4 markedly affected CSR and Ig expression. Among these two elements, hs4 was tentatively presented with the master role due to its unique status within the 3' regulatory region: distal position outside repeated regions, early activation in pre-B cells, strong activity throughout B cell ontogeny. To clarify its role, we generated mice with a clean deletion of the hs4 after replacement with a floxed neo(R) cassette. Surprisingly, and as for previous deletion of hs3a or hs1,2, deletion of hs4 did not affect either in vivo CSR or the secretion level of any Ig isotype. In vitro CSR and Ig secretion in response to LPS and cytokines was not affected either. The only noticeable effects of the hs4 deletion were a decrease in the number of B splenocytes and a decreased membrane IgM expression. In conclusion, while dispensable for CSR and Ig transcription in plasma cells, hs4 mostly appears to contribute to Ig transcription in resting B lymphocytes.

Mitochondrial genome maintenance: roles for nuclear nonhomologous end-joining proteins in Saccharomyces cerevisiae.

PubMed

Kalifa, Lidza; Quintana, Daniel F; Schiraldi, Laura K; Phadnis, Naina; Coles, Garry L; Sia, Rey A; Sia, Elaine A

2012-03-01

Mitochondrial DNA (mtDNA) deletions are associated with sporadic and inherited diseases and age-associated neurodegenerative disorders. Approximately 85% of mtDNA deletions identified in humans are flanked by short directly repeated sequences; however, mechanisms by which these deletions arise are unknown. A limitation in deciphering these mechanisms is the essential nature of the mitochondrial genome in most living cells. One exception is budding yeast, which are facultative anaerobes and one of the few organisms for which directed mtDNA manipulation is possible. Using this model system, we have developed a system to simultaneously monitor spontaneous direct-repeat-mediated deletions (DRMDs) in the nuclear and mitochondrial genomes. In addition, the mitochondrial DRMD reporter contains a unique KpnI restriction endonuclease recognition site that is not present in otherwise wild-type (WT) mtDNA. We have expressed KpnI fused to a mitochondrial localization signal to induce a specific mitochondrial double-strand break (mtDSB). Here we report that loss of the MRX (Mre11p, Rad50p, Xrs2p) and Ku70/80 (Ku70p, Ku80p) complexes significantly impacts the rate of spontaneous deletion events in mtDNA, and these proteins contribute to the repair of induced mtDSBs. Furthermore, our data support homologous recombination (HR) as the predominant pathway by which mtDNA deletions arise in yeast, and suggest that the MRX and Ku70/80 complexes are partially redundant in mitochondria.

Mitochondrial Genome Maintenance: Roles for Nuclear Nonhomologous End-Joining Proteins in Saccharomyces cerevisiae

PubMed Central

Kalifa, Lidza; Quintana, Daniel F.; Schiraldi, Laura K.; Phadnis, Naina; Coles, Garry L.; Sia, Rey A.; Sia, Elaine A.

2012-01-01

Mitochondrial DNA (mtDNA) deletions are associated with sporadic and inherited diseases and age-associated neurodegenerative disorders. Approximately 85% of mtDNA deletions identified in humans are flanked by short directly repeated sequences; however, mechanisms by which these deletions arise are unknown. A limitation in deciphering these mechanisms is the essential nature of the mitochondrial genome in most living cells. One exception is budding yeast, which are facultative anaerobes and one of the few organisms for which directed mtDNA manipulation is possible. Using this model system, we have developed a system to simultaneously monitor spontaneous direct-repeat–mediated deletions (DRMDs) in the nuclear and mitochondrial genomes. In addition, the mitochondrial DRMD reporter contains a unique KpnI restriction endonuclease recognition site that is not present in otherwise wild-type (WT) mtDNA. We have expressed KpnI fused to a mitochondrial localization signal to induce a specific mitochondrial double-strand break (mtDSB). Here we report that loss of the MRX (Mre11p, Rad50p, Xrs2p) and Ku70/80 (Ku70p, Ku80p) complexes significantly impacts the rate of spontaneous deletion events in mtDNA, and these proteins contribute to the repair of induced mtDSBs. Furthermore, our data support homologous recombination (HR) as the predominant pathway by which mtDNA deletions arise in yeast, and suggest that the MRX and Ku70/80 complexes are partially redundant in mitochondria. PMID:22214610

CRISPR/Cas9-Induced Double-Strand Break Repair in Arabidopsis Nonhomologous End-Joining Mutants.

PubMed

Shen, Hexi; Strunks, Gary D; Klemann, Bart J P M; Hooykaas, Paul J J; de Pater, Sylvia

2017-01-05

Double-strand breaks (DSBs) are one of the most harmful DNA lesions. Cells utilize two main pathways for DSB repair: homologous recombination (HR) and nonhomologous end-joining (NHEJ). NHEJ can be subdivided into the KU-dependent classical NHEJ (c-NHEJ) and the more error-prone KU-independent backup-NHEJ (b-NHEJ) pathways, involving the poly (ADP-ribose) polymerases (PARPs). However, in the absence of these factors, cells still seem able to adequately maintain genome integrity, suggesting the presence of other b-NHEJ repair factors or pathways independent from KU and PARPs. The outcome of DSB repair by NHEJ pathways can be investigated by using artificial sequence-specific nucleases such as CRISPR/Cas9 to induce DSBs at a target of interest. Here, we used CRISPR/Cas9 for DSB induction at the Arabidopsis cruciferin 3 (CRU3) and protoporphyrinogen oxidase (PPO) genes. DSB repair outcomes via NHEJ were analyzed using footprint analysis in wild-type plants and plants deficient in key factors of c-NHEJ (ku80), b-NHEJ (parp1 parp2), or both (ku80 parp1 parp2). We found that larger deletions of >20 bp predominated after DSB repair in ku80 and ku80 parp1 parp2 mutants, corroborating with a role of KU in preventing DSB end resection. Deletion lengths did not significantly differ between ku80 and ku80 parp1 parp2 mutants, suggesting that a KU- and PARP-independent b-NHEJ mechanism becomes active in these mutants. Furthermore, microhomologies and templated insertions were observed at the repair junctions in the wild type and all mutants. Since these characteristics are hallmarks of polymerase θ-mediated DSB repair, we suggest a possible role for this recently discovered polymerase in DSB repair in plants. Copyright © 2017 Shen et al.

Increase in Middle East Respiratory Syndrome-Coronavirus Cases in Saudi Arabia Linked to Hospital Outbreak With Continued Circulation of Recombinant Virus, July 1–August 31, 2015

PubMed Central

Assiri, Abdullah M.; Biggs, Holly M.; Abedi, Glen R.; Lu, Xiaoyan; Bin Saeed, Abdulaziz; Abdalla, Osman; Mohammed, Mutaz; Al-Abdely, Hail M.; Algarni, Homoud S.; Alhakeem, Raafat F.; Almasri, Malak M.; Alsharef, Ali A.; Nooh, Randa; Erdman, Dean D.; Gerber, Susan I.; Watson, John T.

2016-01-01

During July–August 2015, the number of cases of Middle East respiratory syndrome (MERS) reported from Saudi Arabia increased dramatically. We reviewed the 143 confirmed cases from this period and classified each based upon likely transmission source. We found that the surge in cases resulted predominantly (90%) from secondary transmission largely attributable to an outbreak at a single healthcare facility in Riyadh. Genome sequencing of MERS coronavirus from 6 cases demonstrated continued circulation of the recently described recombinant virus. A single unique frameshift deletion in open reading frame 5 was detected in the viral sequence from 1 case. PMID:27704019

Engineering the bacterial shapes for enhanced inclusion bodies accumulation.

PubMed

Jiang, Xiao-Ran; Wang, Huan; Shen, Rui; Chen, Guo-Qiang

2015-05-01

Many bacteria can accumulate inclusion bodies such as sulfur, polyphosphate, glycogen, proteins or polyhydroxyalkanoates. To exploit bacteria as factories for effective production of inclusion bodies, a larger intracellular space is needed for more inclusion body accumulation. In this study, polyhydroxybutyrate (PHB) was investigated as an inclusion bodies representative to be accumulated by Escherichia coli JM109SG. Various approaches were taken to increase the bacterial cell sizes including deletion on actin-like protein gene mreB, weak expression of mreB in mreB deletion mutant, and weak expression of mreB in mreB deletion mutant under inducible expression of SulA, the inhibitor of division ring protein FtsZ. All of the methods resulted in different levels of increases in bacterial sizes and PHB granules accumulation. Remarkably, an increase of over 100% PHB accumulation was observed in recombinant E. coli overexpressing mreB in an mreB deletion mutant under inducible expression of FtsZ inhibiting protein SulA. The molecular mechanism of enlarged bacterial size was found to be directly relate to weakened cytoskeleton which was the result of broken skeleton helix. Copyright © 2015 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Genetic Dosage Compensation in a Family with Velo-cardio-facial/DiGeorge/22q11.2 Deletion Syndrome

PubMed Central

Alkalay, Avishai A.; Guo, Tingwei; Montagna, Cristina; Digilio, M. Cristina; Marino, Bruno; Dallapiccola, Bruno; Morrow, Bernice

2014-01-01

Cytogenetic studies of a male child carrying the 22q11.2 deletion common in patients with velo-cardio-facial/DiGeorge syndrome revealed an unexpected rearrangement of the 22q11.2 region in his normal appearing mother. The mother carries a 3 Mb deletion on one copy and a reciprocal, similar sized duplication on the other copy of chromosome 22q11.2 as revealed by fluorescence in situ hybridization and array comparative genome hybridization analysis. The most parsimonious mechanism for the rearrangement is a mitotic non-allelic homologous recombination event in a cell in the early embryo soon after fertilization. The normal phenotype of the mother can be explained by the theory of genetic dosage compensation. This is the second documented case of such an event for this or any genomic disorder. This finding helps to reinforce this phenomenon in a human model, and has significant implications for genetic counseling of future children. PMID:21337693

Rag Deletion in Peripheral T Cells Blocks TCR Revision

PubMed Central

Hale, J. Scott; Ames, Kristina T.; Boursalian, Tamar E.; Fink, Pamela J.

2010-01-01

Mature CD4+Vβ5+ T cells that recognize a peripherally expressed endogenous superantigen are tolerized either by deletion or T cell receptor (TCR) revision. In Vβ5 transgenic mice, this latter tolerance pathway results in the appearance of CD4+Vβ5−TCRβ+ T cells, coinciding with Rag1, Rag2, and TdT expression and the accumulation of Vβ-DJβ recombination intermediates in peripheral CD4+ T cells. Because post-thymic RAG-dependent TCR rearrangement has remained controversial, we sought to definitively determine whether TCR revision is an extrathymic process that occurs in mature peripheral T cells. We now show that Rag deletion in post-positive selection T cells in Vβ5 transgenic mice blocks TCR revision in vivo, and that mature peripheral T cells sorted to remove cells bearing endogenous TCRβ chains can express newly generated TCRβ molecules in adoptive hosts. These findings unambiguously demonstrate post-thymic, RAG-dependent TCR rearrangement and define TCR revision as a tolerance pathway that targets mature peripheral CD4+ T cells. PMID:20435935

Cutting Edge: Rag deletion in peripheral T cells blocks TCR revision.

PubMed

Hale, J Scott; Ames, Kristina T; Boursalian, Tamar E; Fink, Pamela J

2010-06-01

Mature CD4(+)Vbeta5(+) T cells that recognize a peripherally expressed endogenous superantigen are tolerized either by deletion or TCR revision. In Vbeta5 transgenic mice, this latter tolerance pathway results in the appearance of CD4(+)Vbeta5(-)TCRbeta(+) T cells, coinciding with Rag1, Rag2, and TdT expression and the accumulation of V(beta)-DJ(beta) recombination intermediates in peripheral CD4(+) T cells. Because postthymic RAG-dependent TCR rearrangement has remained controversial, we sought to definitively determine whether TCR revision is an extrathymic process that occurs in mature peripheral T cells. We show in this study that Rag deletion in post-positive selection T cells in Vbeta5 transgenic mice blocks TCR revision in vivo and that mature peripheral T cells sorted to remove cells bearing endogenous TCRbeta-chains can express newly generated TCRbeta molecules in adoptive hosts. These findings unambiguously demonstrate postthymic, RAG-dependent TCR rearrangement and define TCR revision as a tolerance pathway that targets mature peripheral CD4(+) T cells.

Bombyx mori nucleopolyhedrovirus ORF54, a viral desmoplakin gene, is associated with the infectivity of budded virions.

PubMed

Zhang, Min-Juan; Tian, Cai-Hong; Fan, Xiao-Ying; Lou, Yi-Han; Cheng, Ruo-Lin; Zhang, Chuan-Xi

2012-07-01

Bombyx mori nucleopolyhedrovirus (BmNPV) ORF54 (Bm54), a member of the viral desmoplakin N-terminus superfamily, is homologous to Autographa californica nucleopolyhedrovirus (AcMNPV) ORF66, which is required for the efficient egress of nucleocapsids from the nucleus and occlusion body formation. In this paper, we generated a bacmid with the Bm54 gene deleted via homologous recombination in Escherichia coli and characterized the mutant virus using a transfection-infection assay and transmission electron microscopy analysis. Our results demonstrated that the cells transfected with viral DNA lacking Bm54 produced non-infectious budded viruses (BVs). Electron microscopy showed that although the deletion of Bm54 did not affect assembly and release of nucleocapsids, it severely affected polyhedron formation. In conclusion, deletion of Bm54 resulted in non-infectious BV and defective polyhedra. Although the sequences of Bm54 and Ac66 are very similar, the two genes function quite differently in the regulation of viral life cycle.

Msh1p counteracts oxidative lesion-induced instability of mtDNA and stimulates mitochondrial recombination in Saccharomyces cerevisiae.

PubMed

Kaniak, Aneta; Dzierzbicki, Piotr; Rogowska, Agata T; Malc, Ewa; Fikus, Marta; Ciesla, Zygmunt

2009-03-01

The proximity of the mitochondrial genome to the respiratory chain, a major source of ROS (radical oxygen species), makes mtDNA more vulnerable to oxidative damage than nuclear DNA. Mitochondrial BER (base excision repair) is generally considered to be the main pathway involved in the prevention of oxidative lesion-induced mutations in mtDNA. However, we previously demonstrated that the increased frequency of mitochondrial Oli(r) mutants in an ogg1Delta strain, lacking the activity of a crucial mtBER glycosylase, is reduced in the presence of plasmids encoding Msh1p, the mitochondrial homologue of the bacterial mismatch protein MutS. This finding suggested that Msh1p might be involved in the prevention of mitochondrial mutagenesis induced by oxidative stress. Here we show that a double mutant carrying the msh1-R813W allele, encoding a variant of the protein defective in the ATP hydrolysis activity, combined with deletion of SOD2, encoding the mitochondrial superoxide dismutase, displays a synergistic effect on the frequency of Oli(r) mutants, indicating that Msh1p prevents generation of oxidative lesion-induced mitochondrial mutations. We also show that double mutants carrying the msh1-R813W allele, combined with deletion of either OGG1 or APN1, the latter resulting in deficiency of the Apn1 endonuclease, exhibit a synergistic effect on the frequency of respiration-defective mutants having gross rearrangements of the mitochondrial genome. This suggests that Msh1p, Ogg1p and Apn1p play overlapping functions in maintaining the stability of mtDNA. In addition, we demonstrate, using a novel ARG8(m) recombination assay, that a surplus of Msh1p results in enhanced mitochondrial recombination. Interestingly, the mutant forms of the protein, msh1p-R813W and msh1p-G776D, fail to stimulate recombination. We postulate that the Msh1p-enhanced homologous recombination may play an important role in the prevention of oxidative lesion-induced rearrangements of the mitochondrial genome.

Recombinant Escherichia coli K5 strain with the deletion of waaR gene decreases the molecular weight of the heparosan capsular polysaccharide.

PubMed

Huang, Haichan; Liu, Xiaobo; Lv, Shencong; Zhong, Weihong; Zhang, Fuming; Linhardt, Robert J

2016-09-01

Heparosan, the capsular polysaccharide of Escherichia coli K5 having a carbohydrate backbone similar to that of heparin, has become a potential precursor for bioengineering heparin. In the heparosan biosynthesis pathway, the gene waaR encoding α-1-, 2- glycosyltransferase catalyze s the third glucosyl residues linking to the oligosaccharide chain. In the present study, a waaR deletion mutant of E. coli K5 was constructed. The mutant showed improvement of capsule polysaccharide yield. It is interesting that the heparosan molecular weight of the mutant is reduced and may become more suitable as a precursor for the production of low molecular weight heparin derived from the wild-type K5 capsular polysaccharide.

FOXF1 transcription factor is required for formation of embryonic vasculature by regulating VEGF signaling in endothelial cells.

PubMed

Ren, Xiaomeng; Ustiyan, Vladimir; Pradhan, Arun; Cai, Yuqi; Havrilak, Jamie A; Bolte, Craig S; Shannon, John M; Kalin, Tanya V; Kalinichenko, Vladimir V

2014-09-26

Inactivating mutations in the Forkhead Box transcription factor F1 (FOXF1) gene locus are frequently found in patients with alveolar capillary dysplasia with misalignment of pulmonary veins, a lethal congenital disorder, which is characterized by severe abnormalities in the respiratory, cardiovascular, and gastrointestinal systems. In mice, haploinsufficiency of the Foxf1 gene causes alveolar capillary dysplasia and developmental defects in lung, intestinal, and gall bladder morphogenesis. Although FOXF1 is expressed in multiple mesenchyme-derived cell types, cellular origins and molecular mechanisms of developmental abnormalities in FOXF1-deficient mice and patients with alveolar capillary dysplasia with misalignment of pulmonary veins remain uncharacterized because of lack of mouse models with cell-restricted inactivation of the Foxf1 gene. In the present study, the role of FOXF1 in endothelial cells was examined using a conditional knockout approach. A novel mouse line harboring Foxf1-floxed alleles was generated by homologous recombination. Tie2-Cre and Pdgfb-CreER transgenes were used to delete Foxf1 from endothelial cells. FOXF1-deficient embryos exhibited embryonic lethality, growth retardation, polyhydramnios, cardiac ventricular hypoplasia, and vascular abnormalities in the lung, placenta, yolk sac, and retina. Deletion of FOXF1 from endothelial cells reduced endothelial proliferation, increased apoptosis, inhibited vascular endothelial growth factor signaling, and decreased expression of endothelial genes critical for vascular development, including vascular endothelial growth factor receptors Flt1 and Flk1, Pdgfb, Pecam1, CD34, integrin β3, ephrin B2, Tie2, and the noncoding RNA Fendrr. Chromatin immunoprecipitation assay demonstrated that Flt1, Flk1, Pdgfb, Pecam1, and Tie2 genes are direct transcriptional targets of FOXF1. FOXF1 is required for the formation of embryonic vasculature by regulating endothelial genes critical for vascular development and vascular endothelial growth factor signaling. © 2014 American Heart Association, Inc.

Recombinant Human Acidic Fibroblast Growth Factor (aFGF) Expressed in Nicotiana benthamiana Potentially Inhibits Skin Photoaging.

PubMed

Ha, Jang-Ho; Kim, Ha-Neul; Moon, Ki-Beom; Jeon, Jae-Heung; Jung, Dai-Hyun; Kim, Su-Jung; Mason, Hugh S; Shin, Seo-Yeon; Kim, Hyun-Soon; Park, Kyung-Mok

2017-07-01

Responding to the need for recombinant acidic fibroblast growth factor in the pharmaceutical and cosmetic industries, we established a scalable expression system for recombinant human aFGF using transient and a DNA replicon vector expression in Nicotiana benthamiana . Recombinant human-acidic fibroblast growth factor was recovered following Agrobacterium infiltration of N. benthamiana . The optimal time point at which to harvest recombinant human acidic fibroblast growth factor expressing leaves was found to be 4 days post-infiltration, before necrosis was evident. Commassie-stained SDS-PAGE gels of His-tag column eluates, concentrated using a 10 000 molecular weight cut-off column, showed an intense band at the expected molecular weight for recombinant human acidic fibroblast growth factor. An immunoblot confirmed that this band was recombinant human acidic fibroblast growth factor. Up to 10 µg recombinant human-acidic fibroblast growth factor/g of fresh leaves were achieved by a simple affinity purification protocol using protein extract from the leaves of agroinfiltrated N. benthamiana . The purified recombinant human acidic fibroblast growth factor improved the survival rate of UVB-irradiated HaCaT and CCD-986sk cells approximately 89 and 81 %, respectively. N. benthamiana -derived recombinant human acidic fibroblast growth factor showed similar effects on skin cell proliferation and UVB protection compared to those of Escherichia coli -derived recombinant human acidic fibroblast growth factor. Additionally, N. benthamiana- derived recombinant human acidic fibroblast growth factor increased type 1 procollagen synthesis up to 30 % as well as reduced UVB-induced intracellular reactive oxygen species generation in fibroblast (CCD-986sk) cells.UVB is a well-known factor that causes various types of skin damage and premature aging. Therefore, the present study demonstrated that N. benthamiana -derived recombinant human acidic fibroblast growth factor effectively protects skin cell from UVB, suggesting its potential use as a cosmetic or therapeutic agent against skin photoaging. Georg Thieme Verlag KG Stuttgart · New York.

Prenatal diagnosis and molecular cytogenetic characterization of rec(10)dup(10p)inv(10)(p11.2q26.3) in a fetus associated with paternal pericentric inversion.

PubMed

Chen, Chih-Ping; Ko, Tsang-Ming; Su, Yi-Ning; Wang, Liang-Kai; Chern, Schu-Rern; Wu, Peih-Shan; Chen, Yen-Ni; Chen, Shin-Wen; Ko, Kevin; Lee, Chen-Chi; Chen, Li-Feng; Yang, Chien-Wen; Wang, Wayseen

2016-10-01

We present prenatal diagnosis and molecular cytogenetic characterization of a recombinant chromosome 10 in a fetus associated with a paternal pericentric inversion. A 35-year-old woman underwent amniocentesis at 18 weeks of gestation because of an advanced maternal age. Amniocentesis revealed a karyotype of 46,XY,der(10)del(10) (q26.3)dup(10)(p11.2p15). She underwent repeat amniocentesis at 21 weeks of gestation and array comparative genomic hybridization revealed a 31.65-Mb duplication of chromosome 10p15.3-p11.22 and a 3.07-Mb deletion of chromosome 10q26.3. Prenatal ultrasound findings were unremarkable. She was referred for genetic counseling and cytogenetic analysis revealed a karyotype of 46,XY,inv(10)(p11.2q26.3) in the father and a karyotype of 46,XX in the mother. The pregnancy was subsequently terminated, and a fetus was delivered with prominent facial dysmorphism. Postnatal cytogenetic analysis of the placenta revealed a karyotype of 46,XY, rec(10)dup(10p)inv(10)(p11.2q26.3). Fluorescence in situ hybridization analysis revealed a duplication of terminal 10p and a deletion of terminal 10q in the recombinant chromosome 10. Array comparative genomic hybridization analysis of the cord blood and umbilical cord confirmed the prenatal diagnosis. Prenatal diagnosis of a recombinant chromosome because of an advanced maternal age should alert the possibility of a paternal pericentric inversion. Copyright © 2016. Published by Elsevier B.V.

Comparative Evaluation of Vaccine Efficacy of Recombinant Marek's Disease Virus Vaccine Lacking Meq Oncogene in Commercial Chickens

USDA-ARS?s Scientific Manuscript database

Marek's disease virus oncogene meq has been identified as the gene involved in tumorigenesis in chickens. We have recently developed a Meq-null virus, rMd5delMeq, in which the oncogene Meq was deleted. Vaccine efficacy experiments conducted in ADOL 15I5 x 71 chickens vaccinated with rMd5delMeq virus...

Identification of a 3.0-kb Major Recombination Hotspot in Patients with Sotos Syndrome Who Carry a Common 1.9-Mb Microdeletion

PubMed Central

Visser, Remco; Shimokawa, Osamu; Harada, Naoki; Kinoshita, Akira; Ohta, Tohru; Niikawa, Norio; Matsumoto, Naomichi

2005-01-01

Sotos syndrome (SoS) is a congenital dysmorphic disorder characterized by overgrowth in childhood, distinctive craniofacial features, and mental retardation. Haploinsufficiency of the NSD1 gene owing to either intragenic mutations or microdeletions is known to be the major cause of SoS. The common ∼2.2-Mb microdeletion encompasses the whole NSD1 gene and neighboring genes and is flanked by low-copy repeats (LCRs). Here, we report the identification of a 3.0-kb major recombination hotspot within these LCRs, in which we mapped deletion breakpoints in 78.7% (37/47) of patients with SoS who carry the common microdeletion. The deletion size was subsequently refined to 1.9 Mb. Sequencing of breakpoint fragments from all 37 patients revealed junctions between a segment of the proximal LCR (PLCR-B) and the corresponding region of the distal LCR (DLCR-2B). PLCR-B and DLCR-2B are the only directly oriented regions, whereas the remaining regions of the PLCR and DLCR are in inverted orientation. The PLCR, with a size of 394.0 kb, and the DLCR, with a size of of 429.8 kb, showed high overall homology (∼98.5%), with an increased sequence similarity (∼99.4%) within the 3.0-kb breakpoint cluster. Several recombination-associated motifs were identified in the hotspot and/or its vicinity. Interestingly, a 10-fold average increase of a translin motif, as compared with the normal distribution within the LCRs, was recognized. Furthermore, a heterozygous inversion of the interval between the LCRs was detected in all fathers of the children carrying a deletion in the paternally derived chromosome. The functional significance of these findings remains to be elucidated. Segmental duplications of the primate genome play a major role in chromosomal evolution. Evolutionary study showed that the duplication of the SoS LCRs occurred 23.3–47.6 million years ago, before the divergence of Old World monkeys. PMID:15580547

Understanding the role of argininosuccinate lyase transcript variants in the clinical and biochemical variability of the urea cycle disorder argininosuccinic aciduria.

PubMed

Hu, Liyan; Pandey, Amit V; Eggimann, Sandra; Rüfenacht, Véronique; Möslinger, Dorothea; Nuoffer, Jean-Marc; Häberle, Johannes

2013-11-29

Argininosuccinic aciduria (ASA) is an autosomal recessive urea cycle disorder caused by deficiency of argininosuccinate lyase (ASL) with a wide clinical spectrum from asymptomatic to severe hyperammonemic neonatal onset life-threatening courses. We investigated the role of ASL transcript variants in the clinical and biochemical variability of ASA. Recombinant proteins for ASL wild type, mutant p.E189G, and the frequently occurring transcript variants with exon 2 or 7 deletions were (co-)expressed in human embryonic kidney 293T cells. We found that exon 2-deleted ASL forms a stable truncated protein with no relevant activity but a dose-dependent dominant negative effect on enzymatic activity after co-expression with wild type or mutant ASL, whereas exon 7-deleted ASL is unstable but seems to have, nevertheless, a dominant negative effect on mutant ASL. These findings were supported by structural modeling predictions for ASL heterotetramer/homotetramer formation. Illustrating the physiological relevance, the predominant occurrence of exon 7-deleted ASL was found in two patients who were both heterozygous for the ASL mutant p.E189G. Our results suggest that ASL transcripts can contribute to the highly variable phenotype in ASA patients if expressed at high levels. Especially, the exon 2-deleted ASL variant may form a heterotetramer with wild type or mutant ASL, causing markedly reduced ASL activity.

Increased volume of distribution for recombinant activated factor VII and longer plasma-derived factor VII half-life may explain their long lasting prophylactic effect.

PubMed

Mathijssen, Natascha C J; Masereeuw, Rosalinde; Holme, Pal Andre; van Kraaij, Marian G J; Laros-van Gorkom, Britta A P; Peyvandi, Flora; van Heerde, Waander L

2013-08-01

Prophylaxis with plasma-derived or recombinant activated factor VII is beneficial in severe factor VII deficiency. To understand why prophylactic treatment with both products is efficacious, we conducted a pharmacokinetic study. Ten factor VII deficient patients were treated with either recombinant activated (20 μg/kg) or plasma-derived (25 IU/kg) factor VII in a cross-over design. Pharmacokinetic parameters were analyzed through activated factor VII activity, factor VII clotting activity, and factor VII antigen levels on depicted time points. Factor VII activity half-lifes, determined by non-compartmental and one-compartmental analysis (results in brackets), were shorter for recombinant activated (1.4h; 0.7h) than for plasma-derived factor VII (6.8h; 3.2h); both recombinant activated (5.1h; 2.1h and plasma-derived factor VII (5.8h; 3.2h) resulted in longer half-lives of factor VII antigen. Activated factor VII half-lives (based on activated factor VII activity levels) were significantly higher compared to factor VII clotting activity (1.6h; 0.9h). Volumes of distribution were significantly higher for activated factor VII (236 ml/kg; 175 ml/kg, measured by activated factor VII) as compared to plasma-derived factor VII (206 ml/kg; 64 ml/kg, measured by factor FVII activity), suggesting a plasma- and extracellular fluid distribution for recombinant activated factor VII. Recombinant activated factor VII showed significantly shorter half-lifes than plasma-derived factor VII. Volumes of distribution were significantly higher for treatment with recombinant activated factor VII. The longer half-life for plasma-derived factor VII, compared to recombinant activated factor VII, and the increased volume of distribution for recombinant activated factor VII, compared to plasma-derived factor VII may further elucidate the beneficial effect of prophylactic treatment of both products. Copyright © 2013 Elsevier Ltd. All rights reserved.

Molecular characterization of the breakpoints of a 12-kb deletion in the NF1 gene in a family showing germ-line mosaicism.

PubMed Central

Lázaro, C; Gaona, A; Lynch, M; Kruyer, H; Ravella, A; Estivill, X

1995-01-01

Neurofibromatosis type 1 (NF1) is caused by deletions, insertions, translocations, and point mutations in the NF1 gene, which spans 350 kb on the long arm of human chromosome 17. Although several point mutations have been described, large molecular abnormalities have rarely been characterized in detail. We describe here the molecular breakpoints of a 12-kb deletion of the NF1 gene, which is responsible for the NF1 phenotype in a kindred with two children affected because of germline mosaicism in the unaffected father, who has the mutation in 10% of his spermatozoa. The mutation spans introns 31-39, removing 12,021 nt and inserting 30 bp, of which 19 bp are a direct repetition of a sequence located in intron 31, just 4 bp before the 5' breakpoint. The 5' and 3' breakpoints contain the sequence TATTTTA, which could be involved in the generation of the deletion. The most plausible explanation for the mechanism involved in the generation of this 12-kb deletion is homologous/nonhomologous recombination. Since sperm of the father does not contain the corresponding insertion of the 12-kb deleted sequence, this deletion could have occurred within the NF1 chromosome through loop formation. RNA from lymphocytes of one of the NF1 patients showed similar levels of the mutated and normal transcripts, suggesting that the NF1-mRNA from mutations causing frame shifts of the reading frame or stop codons in this gene is not degraded during its processing. The mutation was not detected in fresh lymphocytes from the unaffected father by PCR analysis, supporting the case for true germ-line mosaicism. Images Figure 1 Figure 3 PMID:7485153

The influence of the multi-basic cleavage site of the H5 hemagglutinin on the attenuation, immunogenicity and efficacy of a live attenuated influenza A H5N1 cold-adapted vaccine virus.

PubMed

Suguitan, Amorsolo L; Marino, Michael P; Desai, Purvi D; Chen, Li-Mei; Matsuoka, Yumiko; Donis, Ruben O; Jin, Hong; Swayne, David E; Kemble, George; Subbarao, Kanta

2009-12-20

A recombinant live attenuated influenza virus DeltaH5N1 vaccine with a modified hemagglutinin (HA) and intact neuraminidase genes from A/Vietnam/1203/04 (H5N1) and six remaining genome segments from A/Ann Arbor/6/60 (H2N2) cold-adapted (AA ca) virus was previously shown to be attenuated in chickens, mice and ferrets. Evaluation of the recombinant H5N1 viruses in mice indicated that three independent factors contributed to the attenuation of the DeltaH5N1 vaccine: the attenuating mutations specified by the AA ca loci had the greatest influence, followed by the deletion of the H5 HA multi-basic cleavage site (MBS), and the constellation effects of the AA genes acting in concert with the H5N1 glycoproteins. Restoring the MBS in the H5 HA of the vaccine virus improved its immunogenicity and efficacy, likely as a consequence of increased virus replication, indicating that removal of the MBS had a deleterious effect on the immunogenicity and efficacy of the DeltaH5N1 vaccine in mice.

The influence of the multi-basic cleavage site of the H5 hemagglutinin on the attenuation, immunogenicity and efficacy of a live attenuated influenza A H5N1 cold-adapted vaccine virus

PubMed Central

Suguitan, Amorsolo L.; Marino, Michael P.; Desai, Purvi D.; Chen, Li-Mei; Matsuoka, Yumiko; Donis, Ruben O.; Jin, Hong; Swayne, David E.; Kemble, George; Subbarao, Kanta

2009-01-01

A recombinant live attenuated influenza virus ΔH5N1 vaccine with a modified hemagglutinin (HA) and intact neuraminidase genes from A/Vietnam/1203/04 (H5N1) and six remaining genome segments from A/Ann Arbor/6/60 (H2N2) cold-adapted (AA ca) virus was previously shown to be attenuated in chickens, mice and ferrets. Evaluation of the recombinant H5N1 viruses in mice indicated that three independent factors contributed to the attenuation of the ΔH5N1 vaccine: the attenuating mutations specified by the AA ca loci had the greatest influence, followed by the deletion of the H5 HA multi-basic cleavage site (MBS), and the constellation effects of the AA genes acting in concert with the H5N1 glycoproteins. Restoring the MBS in the H5 HA of the vaccine virus improved its immunogenicity and efficacy, likely as a consequence of increased virus replication, indicating that removal of the MBS had a deleterious effect on the immunogenicity and efficacy of the ΔH5N1 vaccine in mice. PMID:19833372

Factor IX[sub Madrid 2]: A deletion/insertion in Facotr IX gene which abolishes the sequence of the donor junction at the exon IV-intron d splice site

DOE Office of Scientific and Technical Information (OSTI.GOV)

Solera, J.; Magallon, M.; Martin-Villar, J.

1992-02-01

DNA from a patient with severe hemophilia B was evaluated by RFLP analysis, producing results which suggested the existence of a partial deletion within the factor IX gene. The deletion was further localized and characterized by PCR amplification and sequencing. The altered allele has a 4,442-bp deletion which removes both the donor splice site located at the 5[prime] end of intron d and the two last coding nucleotides located at the 3[prime] end of exon IV in the normal factor IX gene; this fragment has been inserted in inverted orientation. Two homologous sequences have been discovered at the ends ofmore » the deleted DNA fragment.« less

Runx2 Expression in Smooth Muscle Cells Is Required for Arterial Medial Calcification in Mice

PubMed Central

Lin, Mu-En; Chen, Theodore; Leaf, Elizabeth M.; Speer, Mei Y.; Giachelli, Cecilia M.

2016-01-01

Arterial medial calcification (AMC) is a hallmark of aging, diabetes, and chronic kidney disease. Smooth muscle cell (SMC) transition to an osteogenic phenotype is a common feature of AMC, and is preceded by expression of runt-related transcription factor 2 (Runx2), a master regulator of bone development. Whether SMC-specific Runx2 expression is required for osteogenic phenotype change and AMC remains unknown. We therefore created an improved targeting construct to generate mice with floxed Runx2 alleles (Runx2f/f) that do not produce truncated Runx2 proteins after Cre recombination, thereby preventing potential off-target effects. SMC-specific deletion using SM22–recombinase transgenic allele mice (Runx2ΔSM) led to viable mice with normal bone and arterial morphology. After vitamin D overload, arterial SMCs in Runx2f/f mice expressed Runx2, underwent osteogenic phenotype change, and developed severe AMC. In contrast, vitamin D–treated Runx2ΔSM mice had no Runx2 in blood vessels, maintained SMC phenotype, and did not develop AMC. Runx2 deletion did not affect serum calcium, phosphate, fibroblast growth factor-23, or alkaline phosphatase levels. In vitro, Runx2f/f SMCs calcified to a much greater extent than those derived from Runx2ΔSM mice. These data indicate a critical role of Runx2 in SMC osteogenic phenotype change and mineral deposition in a mouse model of AMC, suggesting that Runx2 and downstream osteogenic pathways in SMCs may be useful therapeutic targets for treating or preventing AMC in high-risk patients. PMID:25987250

Attenuation and efficacy of live-attenuated Rift Valley fever virus vaccine candidates in non-human primates.

PubMed

Smith, Darci R; Johnston, Sara C; Piper, Ashley; Botto, Miriam; Donnelly, Ginger; Shamblin, Joshua; Albariño, César G; Hensley, Lisa E; Schmaljohn, Connie; Nichol, Stuart T; Bird, Brian H

2018-05-09

Rift Valley fever virus (RVFV) is an important mosquito-borne veterinary and human pathogen that has caused large outbreaks of severe disease throughout Africa and the Arabian Peninsula. Currently, no licensed vaccine or therapeutics exists to treat this potentially deadly disease. The explosive nature of RVFV outbreaks and the severe consequences of its accidental or intentional introduction into RVFV-free areas provide the impetus for the development of novel vaccine candidates for use in both livestock and humans. Rationally designed vaccine candidates using reverse genetics have been used to develop deletion mutants of two known RVFV virulence factors, the NSs and NSm genes. These recombinant viruses were demonstrated to be protective and immunogenic in rats, mice, and sheep, without producing clinical illness in these animals. Here, we expand upon those findings and evaluate the single deletion mutant (ΔNSs rRVFV) and double deletion mutant (ΔNSs-ΔNSm rRVFV) vaccine candidates in the common marmoset (Callithrix jacchus), a non-human primate (NHP) model resembling severe human RVF disease. We demonstrate that both the ΔNSs and ΔNSs-ΔNSm rRVFV vaccine candidates were found to be safe and immunogenic in the current study. The vaccinated animals received a single dose of vaccine that led to the development of a robust antibody response. No vaccine-induced adverse reactions, signs of clinical illness or infectious virus were detected in the vaccinated marmosets. All vaccinated animals that were subsequently challenged with RVFV were protected against viremia and liver disease. In summary, our results provide the basis for further development of the ΔNSs and ΔNSs-ΔNSm rRVFV as safe and effective human RVFV vaccines for this significant public health threat.

Osteocytes, not Osteoblasts or Lining Cells, are the Main Source of the RANKL Required for Osteoclast Formation in Remodeling Bone

PubMed Central

Xiong, Jinhu; Piemontese, Marilina; Onal, Melda; Campbell, Josh; Goellner, Joseph J.; Dusevich, Vladimir; Bonewald, Lynda; Manolagas, Stavros C.; O’Brien, Charles A.

2015-01-01

The cytokine receptor activator of nuclear factor kappa B ligand (RANKL), encoded by the Tnfsf11 gene, is essential for osteoclastogenesis and previous studies have shown that deletion of the Tnfsf11 gene using a Dmp1-Cre transgene reduces osteoclast formation in cancellous bone by more than 70%. However, the Dmp1-Cre transgene used in those studies leads to recombination in osteocytes, osteoblasts, and lining cells making it unclear whether one or more of these cell types produce the RANKL required for osteoclast formation in cancellous bone. Because osteoblasts, osteocytes, and lining cells have distinct locations and functions, distinguishing which of these cell types are sources of RANKL is essential for understanding the orchestration of bone remodeling. To distinguish between these possibilities, we have now created transgenic mice expressing the Cre recombinase under the control of regulatory elements of the Sost gene, which is expressed in osteocytes but not osteoblasts or lining cells in murine bone. Activity of the Sost-Cre transgene in osteocytes, but not osteoblast or lining cells, was confirmed by crossing Sost-Cre transgenic mice with tdTomato and R26R Cre-reporter mice, which express tdTomato fluorescent protein or LacZ, respectively, only in cells expressing the Cre recombinase or their descendants. Deletion of the Tnfsf11 gene in Sost-Cre mice led to a threefold decrease in osteoclast number in cancellous bone and increased cancellous bone mass, mimicking the skeletal phenotype of mice in which the Tnfsf11 gene was deleted using the Dmp1-Cre transgene. These results demonstrate that osteocytes, not osteoblasts or lining cells, are the main source of the RANKL required for osteoclast formation in remodeling cancellous bone. PMID:26393791

Signature-tagged mutagenesis screening revealed a novel smooth-to-rough transition determinant of Salmonella enterica serovar Enteritidis.

PubMed

Jiao, Yang; Guo, Rongxian; Tang, Peipei; Kang, Xilong; Yin, Junlei; Wu, Kaiyue; Geng, Shizhong; Li, Qiuchun; Sun, Jun; Xu, Xiulong; Zhou, Xiaohui; Gan, Junji; Jiao, Xinan; Liu, Xiufan; Pan, Zhiming

2017-03-03

Salmonella enterica serovar Enteritidis (S. Enteritidis) has emerged as one of the most important food-borne pathogens for humans. Lipopolysaccharide (LPS), as a component of the outer membrane, is responsible for the virulence and smooth-to-rough transition in S. Enteritidis. In this study, we screened S. Enteritidis signature-tagged transposon mutant library using monoclonal antibody against somatic O 9 antigen (O 9 MAb) and O 9 factor rabbit antiserum to identify novel genes that are involved in smooth-to-rough transition. A total of 480 mutants were screened and one mutant with transposon insertion in rfbG gene had smooth-to-rough transition phenotype. In order to verify the role of rfbG gene, an rfbG insertion or deletion mutant was constructed using λ-Red recombination system. Phenotypic and biological analysis revealed that rfbG insertion or deletion mutants were similar to the wild-type strain in growth rate and biochemical properties, but the swimming motility was reduced. SE Slide Agglutination test and ELISA test showed that rfbG mutants do not stimulate animals to produce agglutinating antibody. In addition, the half-lethal dose (LD 50 ) of the rfbG deletion mutant strain was 10 6.6 -fold higher than that of the parent strain in a mouse model when injected intraperitoneally. These data indicate that the rfbG gene is involved in smooth-to-rough transition, swimming motility and virulence of S. Enteritidis. Furthermore, somatic O-antigen antibody-based approach to screen signature-tagged transposon mutants is feasible to clarify LPS biosynthesis and to find suitable markers in DIVA-vaccine research.

Different levels of immunogenicity of two strains of Fowlpox virus as recombinant vaccine vectors eliciting T-cell responses in heterologous prime-boost vaccination strategies.

PubMed

Cottingham, Matthew G; van Maurik, Andre; Zago, Manola; Newton, Angela T; Anderson, Richard J; Howard, M Keith; Schneider, Jörg; Skinner, Michael A

2006-07-01

The FP9 strain of F has been described as a more immunogenic recombinant vaccine vector than the Webster FPV-M (FPW) strain (R. J. Anderson et al., J. Immunol. 172:3094-3100, 2004). This study expands the comparison to include two separate recombinant antigens and multiple, rather than single, independent viral clones derived from the two strains. Dual-poxvirus heterologous prime-boost vaccination regimens using individual clones of recombinant FP9 or FPW in combination with recombinant modified V Ankara expressing the same antigen were evaluated for their ability to elicit T-cell responses against recombinant antigens from Plasmodium berghei (circumsporozoite protein) or human immunodeficiency virus type 1 (a Gag-Pol-Nef fusion protein). Gamma interferon enzyme-linked immunospot assay and fluorescence-activated cell sorting assays of the responses to specific epitopes confirmed the approximately twofold-greater cellular immunogenicity of FP9 compared to FPW, when given as the priming or boosting immunization. Equality of transgene expression in mouse cells infected with the two strains in vitro was verified by Western blotting. Directed partial sequence analysis and PCR analysis of FPW and comparison to available whole-genome sequences revealed that many loci that are mutated in the highly attenuated and culture-adapted FP9 strain are wild type in FPW, including the seven multikilobase deletions. These "passage-specific" alterations are hypothesized to be involved in determining the immunogenicity of fowlpox virus as a recombinant vaccine vector.

Construction of an infectious clone of canine herpesvirus genome as a bacterial artificial chromosome.

PubMed

Arii, Jun; Hushur, Orkash; Kato, Kentaro; Kawaguchi, Yasushi; Tohya, Yukinobu; Akashi, Hiroomi

2006-04-01

Canine herpesvirus (CHV) is an attractive candidate not only for use as a recombinant vaccine to protect dogs from a variety of canine pathogens but also as a viral vector for gene therapy in domestic animals. However, developments in this area have been impeded by the complicated techniques used for eukaryotic homologous recombination. To overcome these problems, we used bacterial artificial chromosomes (BACs) to generate infectious BACs. Our findings may be summarized as follows: (i) the CHV genome (pCHV/BAC), in which a BAC flanked by loxP sites was inserted into the thymidine kinase gene, was maintained in Escherichia coli; (ii) transfection of pCHV/BAC into A-72 cells resulted in the production of infectious virus; (iii) the BAC vector sequence was almost perfectly excisable from the genome of the reconstituted virus CHV/BAC by co-infection with CHV/BAC and a recombinant adenovirus that expressed the Cre recombinase; and (iv) a recombinant virus in which the glycoprotein C gene was deleted was generated by lambda recombination followed by Flp recombination, which resulted in a reduction in viral titer compared with that of the wild-type virus. The infectious clone pCHV/BAC is useful for the modification of the CHV genome using bacterial genetics, and CHV/BAC should have multiple applications in the rapid generation of genetically engineered CHV recombinants and the development of CHV vectors for vaccination and gene therapy in domestic animals.

Din7 and Mhr1 expression levels regulate double-strand-break-induced replication and recombination of mtDNA at ori5 in yeast.

PubMed

Ling, Feng; Hori, Akiko; Yoshitani, Ayako; Niu, Rong; Yoshida, Minoru; Shibata, Takehiko

2013-06-01

The Ntg1 and Mhr1 proteins initiate rolling-circle mitochondrial (mt) DNA replication to achieve homoplasmy, and they also induce homologous recombination to maintain mitochondrial genome integrity. Although replication and recombination profoundly influence mitochondrial inheritance, the regulatory mechanisms that determine the choice between these pathways remain unknown. In Saccharomyces cerevisiae, double-strand breaks (DSBs) introduced by Ntg1 at the mitochondrial replication origin ori5 induce homologous DNA pairing by Mhr1, and reactive oxygen species (ROS) enhance production of DSBs. Here, we show that a mitochondrial nuclease encoded by the nuclear gene DIN7 (DNA damage inducible gene) has 5'-exodeoxyribonuclease activity. Using a small ρ(-) mtDNA bearing ori5 (hypersuppressive; HS) as a model mtDNA, we revealed that DIN7 is required for ROS-enhanced mtDNA replication and recombination that are both induced at ori5. Din7 overproduction enhanced Mhr1-dependent mtDNA replication and increased the number of residual DSBs at ori5 in HS-ρ(-) cells and increased deletion mutagenesis at the ori5 region in ρ(+) cells. However, simultaneous overproduction of Mhr1 suppressed all of these phenotypes and enhanced homologous recombination. Our results suggest that after homologous pairing, the relative activity levels of Din7 and Mhr1 modulate the preference for replication versus homologous recombination to repair DSBs at ori5.

Intestinal cell targeting of a stable recombinant Cu-Zn SOD from Cucumis melo fused to a gliadin peptide.

PubMed

Intes, Laurent; Bahut, Muriel; Nicole, Pascal; Couvineau, Alain; Guette, Catherine; Calenda, Alphonse

2012-05-31

The mRNA encoding full length chloroplastic Cu-Zn SOD (superoxide dismutase) of Cucumis melo (Cantaloupe melon) was cloned. This sequence was then used to generate a mature recombinant SOD by deleting the first 64 codons expected to encode a chloroplastic peptide signal. A second hybrid SOD was created by inserting ten codons to encode a gliadin peptide at the N-terminal end of the mature SOD. Taking account of codon bias, both recombinant proteins were successfully expressed and produced in Escherichia coli. Both recombinant SODs display an enzymatic activity of ~5000U mg(-1) and were shown to be stable for at least 4h at 37°C in biological fluids mimicking the conditions of intestinal transit. These recombinant proteins were capable in vitro, albeit at different levels, of reducing ROS-induced-apoptosis of human epithelial cells. They also stimulated production and release in a time-dependent manner of an autologous SOD activity from cells located into jejunum biopsies. Nevertheless, the fused gliadin peptide enable the recombinant Cu-Zn SOD to maintain a sufficiently sustained interaction with the intestinal cells membrane in vivo rather than being eliminated with the flow. According to these observations, the new hybrid Cu-Zn SOD should show promise in applications for managing inflammatory bowel diseases. Copyright © 2012 Elsevier B.V. All rights reserved.

The structural basis for the functional comparability of factor VIII and the long-acting variant recombinant factor VIII Fc fusion protein

DOE PAGES

Leksa, N. C.; Chiu, P. -L.; Bou-Assaf, G. M.; ...

2017-05-03

Fusion of the human IgG 1 Fc domain to the C-terminal C2 domain of B-domain-deleted (BDD) factor VIII (FVIII) results in the recombinant FVIII Fc (rFVIIIFc) fusion protein, which has a 1.5-fold longer half-life in humans. To assess the structural properties of rFVIIIFc by comparing its constituent FVIII and Fc elements with their respective isolated components, and evaluating their structural independence within rFVIIIFc. rFVIIIFc and its isolated FVIII and Fc components were compared by the use of hydrogen–deuterium exchange mass spectrometry (HDX-MS). The structure of rFVIIIFc was also evaluated by the use of X-ray crystallography, small-angle X-ray scattering (SAXS), andmore » electron microscopy (EM). The degree of steric interference by the appended Fc domain was assessed by EM and surface plasmon resonance (SPR). HDX-MS analysis of rFVIIIFc revealed that fusion caused no structural perturbations in FVIII or Fc. The rFVIIIFc crystal structure showed that the FVIII component is indistinguishable from published BDD FVIII structures. The Fc domain was not observed, indicating high mobility. SAXS analysis was consistent with an ensemble of rigid-body models in which the Fc domain exists in a largely extended orientation relative to FVIII. Binding of Fab fragments of anti-C2 domain antibodies to BDD FVIII was visualized by EM, and the affinities of the corresponding intact antibodies for BDD FVIII and rFVIIIFc were comparable by SPR analysis. Thus, the FVIII and Fc components of rFVIIIFc are structurally indistinguishable from their isolated constituents, and show a high degree of structural independence, consistent with the functional comparability of rFVIIIFc and unmodified FVIII.« less

The structural basis for the functional comparability of factor VIII and the long-acting variant recombinant factor VIII Fc fusion protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Leksa, N. C.; Chiu, P. -L.; Bou-Assaf, G. M.

Fusion of the human IgG 1 Fc domain to the C-terminal C2 domain of B-domain-deleted (BDD) factor VIII (FVIII) results in the recombinant FVIII Fc (rFVIIIFc) fusion protein, which has a 1.5-fold longer half-life in humans. To assess the structural properties of rFVIIIFc by comparing its constituent FVIII and Fc elements with their respective isolated components, and evaluating their structural independence within rFVIIIFc. rFVIIIFc and its isolated FVIII and Fc components were compared by the use of hydrogen–deuterium exchange mass spectrometry (HDX-MS). The structure of rFVIIIFc was also evaluated by the use of X-ray crystallography, small-angle X-ray scattering (SAXS), andmore » electron microscopy (EM). The degree of steric interference by the appended Fc domain was assessed by EM and surface plasmon resonance (SPR). HDX-MS analysis of rFVIIIFc revealed that fusion caused no structural perturbations in FVIII or Fc. The rFVIIIFc crystal structure showed that the FVIII component is indistinguishable from published BDD FVIII structures. The Fc domain was not observed, indicating high mobility. SAXS analysis was consistent with an ensemble of rigid-body models in which the Fc domain exists in a largely extended orientation relative to FVIII. Binding of Fab fragments of anti-C2 domain antibodies to BDD FVIII was visualized by EM, and the affinities of the corresponding intact antibodies for BDD FVIII and rFVIIIFc were comparable by SPR analysis. Thus, the FVIII and Fc components of rFVIIIFc are structurally indistinguishable from their isolated constituents, and show a high degree of structural independence, consistent with the functional comparability of rFVIIIFc and unmodified FVIII.« less

Recombinant activated factor VII in cardiac surgery: single-center experience.

PubMed

Singh, Sarvesh Pal; Chauhan, Sandeep; Choudhury, Minati; Malik, Vishwas; Choudhary, Shiv Kumar

2014-02-01

The widespread off-label use of recombinant activated factor VII for the control of refractory postoperative hemorrhage continues despite a warning from the Food and Drug Administration. Although effective in reducing the need for transfusion of blood and blood products, safety concerns still prevail. To compare the dosing and efficacy of recombinant activated factor VII between pediatric and adult patients, and in the operating room and intensive care unit. The records of 69 patients (33 children and 36 adults) who underwent cardiovascular surgery and received recombinant activated factor VII were reviewed retrospectively. The dose of recombinant activated factor VII, mediastinal drainage, use of blood and blood products, incidence of thrombosis, and 28-day mortality were studied. the efficacy of recombinant activated factor VII was comparable in adults and children, despite the lower dose in adults. Prophylactic use of recombinant activated factor VII decreased the incidence of mediastinal exploration and the duration of intensive care unit stay. A 4.3% incidence of thrombotic complications was observed in this study. The efficacious dose of recombinant activated factor VII is much less in adults compared to children. Prophylactic use of recombinant activated factor VII decreases the dose required, the incidence of mediastinal exploration, and intensive care unit stay, with no survival benefit.

The generation of oxidative stress-induced rearrangements in Saccharomyces cerevisiae mtDNA is dependent on the Nuc1 (EndoG/ExoG) nuclease and is enhanced by inactivation of the MRX complex.

PubMed

Dzierzbicki, Piotr; Kaniak-Golik, Aneta; Malc, Ewa; Mieczkowski, Piotr; Ciesla, Zygmunt

2012-12-01

Oxidative stress is known to enhance the frequency of two major types of alterations in the mitochondrial genome of Saccharomyces cerevisiae: point mutations and large deletions resulting in the generation of respiration-deficient petite rhō mutants. We investigated the effect of antimycin A, a well-known agent inducing oxidative stress, on the stability of mtDNA. We show that antimycin enhances exclusively the generation of respiration-deficient petite mutants and this is accompanied by a significant increase in the level of reactive oxygen species (ROS) and in a marked drop of cellular ATP. Whole mitochondrial genome sequencing revealed that mtDNAs of antimycin-induced petite mutants are deleted for most of the wild-type sequence and usually contain one of the active origins of mtDNA replication: ori1, ori2 ori3 or ori5. We show that the frequency of antimycin-induced rhō mutants is significantly elevated in mutants deleted either for the RAD50 or XRS2 gene, both encoding the components of the MRX complex, which is known to be involved in the repair of double strand breaks (DSBs) in DNA. Furthermore, enhanced frequency of rhō mutants in cultures of antimycin-treated cells lacking Rad50 was further increased by the simultaneous absence of the Ogg1 glycosylase, an important enzyme functioning in mtBER. We demonstrate also that rad50Δ and xrs2Δ deletion mutants display a considerable reduction in the frequency of allelic mitochondrial recombination, suggesting that it is the deficiency in homologous recombination which is responsible for enhanced rearrangements of mtDNA in antimycin-treated cells of these mutants. Finally, we show that the generation of large-scale mtDNA deletions induced by antimycin is markedly decreased in a nuc1Δ mutant lacking the activity of the Nuc1 nuclease, an ortholog of the mammalian mitochondrial nucleases EndoG and ExoG. This result indicates that the nuclease plays an important role in processing of oxidative stress-induced lesions in the mitochondrial genome. Copyright © 2012 Elsevier B.V. All rights reserved.

Human structural variation: mechanisms of chromosome rearrangements

PubMed Central

Weckselblatt, Brooke; Rudd, M. Katharine

2015-01-01

Chromosome structural variation (SV) is a normal part of variation in the human genome, but some classes of SV can cause neurodevelopmental disorders. Analysis of the DNA sequence at SV breakpoints can reveal mutational mechanisms and risk factors for chromosome rearrangement. Large-scale SV breakpoint studies have become possible recently owing to advances in next-generation sequencing (NGS) including whole-genome sequencing (WGS). These findings have shed light on complex forms of SV such as triplications, inverted duplications, insertional translocations, and chromothripsis. Sequence-level breakpoint data resolve SV structure and determine how genes are disrupted, fused, and/or misregulated by breakpoints. Recent improvements in breakpoint sequencing have also revealed non-allelic homologous recombination (NAHR) between paralogous long interspersed nuclear element (LINE) or human endogenous retrovirus (HERV) repeats as a cause of deletions, duplications, and translocations. This review covers the genomic organization of simple and complex constitutional SVs, as well as the molecular mechanisms of their formation. PMID:26209074

Deficiency of Interleukin-1 Receptor Antagonist (DIRA): Report of the First Indian Patient and a Novel Deletion Affecting IL1RN.

PubMed

Mendonca, Leonardo O; Malle, Louise; Donovan, Frank X; Chandrasekharappa, Settara C; Montealegre Sanchez, Gina A; Garg, Megha; Tedgard, Ulf; Castells, Mariana; Saini, Shiv S; Dutta, Sourabh; Goldbach-Mansky, Raphaela; Suri, Deepti; Jesus, Adriana A

2017-07-01

Deficiency of interleukin-1 receptor antagonist (DIRA) is a rare life-threatening autoinflammatory disease caused by autosomal recessive mutations in IL1RN. DIRA presents clinically with early onset generalized pustulosis, multifocal osteomyelitis, and elevation of acute phase reactants. We evaluated and treated an antibiotic-unresponsive patient with presumed DIRA with recombinant IL-1Ra (anakinra). The patient developed anaphylaxis to anakinra and was subsequently desensitized. Genetic analysis of IL1RN was undertaken and treatment with anakinra was initiated. A 5-month-old Indian girl born to healthy non-consanguineous parents presented at the third week of life with irritability, sterile multifocal osteomyelitis including ribs and clavicles, a mild pustular rash, and elevated acute phase reactants. SNP array of the patient's genomic DNA revealed a previously unrecognized homozygous deletion of approximately 22.5 Kb. PCR and Sanger sequencing of the borders of the deleted area allowed identification of the breakpoints of the deletion, thus confirming a homozygous 22,216 bp deletion that spans the first four exons of IL1RN. Due to a clinical suspicion of DIRA, anakinra was initiated which resulted in an anaphylactic reaction that triggered desensitization with subsequent marked and sustained clinical and laboratory improvement. We report a novel DIRA-causing homozygous deletion affecting IL1RN in an Indian patient. The mutation likely is a founder mutation; the design of breakpoint-specific primers will enable genetic screening in Indian patients suspected of DIRA. The patient developed anaphylaxis to anakinra, was desensitized, and is in clinical remission on continued treatment.

High frequency of TTTY2-like gene-related deletions in patients with idiopathic oligozoospermia and azoospermia.

PubMed

Yapijakis, C; Serefoglou, Z; Papadimitriou, K; Makrinou, E

2015-06-01

Genes located on Y chromosome and expressed in testis are likely to be involved in spermatogenesis. TTTY2 is a Y-linked multicopy gene family of unknown function that includes TTTY2L2A and TTTY2L12A at Yq11 and Yp11 loci respectively. Using PCR amplification, we screened for TTTY2L2A- and TTTY2L12A-associated deletions, in 94 Greek men with fertility problems. Patients were divided into three groups as following: group A (n = 28) included men with idiopathic moderate oligozoospermia, group B (n = 34) with idiopathic severe oligozoospermia and azoospermia, and group C (n = 32) with oligo- and azoospermia of various known etiologies. No deletions were detected in group C patients and 50 fertile controls. However, two patients from group A had deletions in TTTY2L2A (7.1%) and six in TTTY2L12A (21.4%), whereas from group B, four patients had deletions in TTTY2L2A (11.8%) and 10 in TTTY2L12A (29.4%). In addition, five patients from both groups A and B (8%) appeared to have deletions in both studied TTTY2 genes, although these are located very far apart. These results indicate that the TTTY2 gene family may play a significant role in spermatogenesis and suggest a possible mechanism of nonhomologous recombinational events that may cause genomic instability and ultimately lead to male infertility. © 2014 Blackwell Verlag GmbH.

Functional role of a distal (3'-phosphate) group of CoA in the recombinant human liver medium-chain acyl-CoA dehydrogenase-catalysed reaction.

PubMed Central

Peterson, K L; Srivastava, D K

1997-01-01

The X-ray crystallographic structure of medium-chain acyl-CoA dehydrogenase (MCAD)-octenoyl-CoA complex reveals that the 3'-phosphate group of CoA is confined to the exterior of the protein structure [approx. 15 A (1.5 nm) away from the enzyme active site], and is fully exposed to the outside solvent environment. To ascertain whether such a distal (3'-phosphate) fragment of CoA plays any significant role in the enzyme catalysis, we investigated the recombinant human liver MCAD (HMCAD)-catalysed reaction by using normal (phospho) and 3'-phosphate-truncated (dephospho) forms of octanoyl-CoA and butyryl-CoA substrates. The steady-state kinetic data revealed that deletion of the 3'-phosphate group from octanoyl-CoA substrate increased the turnover rate of the enzyme to about one-quarter, whereas that from butyryl-CoA substrate decreased the turnover rate of the enzyme to about one-fifth; the Km values of both these substrates were increased by 5-10-fold on deletion of the 3'-phosphate group from the corresponding acyl-CoA substrates. The transient kinetics for the reductive half-reaction, oxidative half-reaction and the dissociation 'off-rate' (of the reaction product from the oxidized enzyme site) were all found to be affected by deletions of the 3'-phosphate group from octanoyl-CoA and butyryl-CoA substrates. A cumulative account of these results reveals that, although the 3'-phosphate group of acyl-CoA substrates might seem 'useless' on the basis of the structural data, it has an essential functional role during HMCAD catalysis. PMID:9271097

Synthesis and accumulation of aromatic aldehydes in an engineered strain of Escherichia coli.

PubMed

Kunjapur, Aditya M; Tarasova, Yekaterina; Prather, Kristala L J

2014-08-20

Aromatic aldehydes are useful in numerous applications, especially as flavors, fragrances, and pharmaceutical precursors. However, microbial synthesis of aldehydes is hindered by rapid, endogenous, and redundant conversion of aldehydes to their corresponding alcohols. We report the construction of an Escherichia coli K-12 MG1655 strain with reduced aromatic aldehyde reduction (RARE) that serves as a platform for aromatic aldehyde biosynthesis. Six genes with reported activity on the model substrate benzaldehyde were rationally targeted for deletion: three genes that encode aldo-keto reductases and three genes that encode alcohol dehydrogenases. Upon expression of a recombinant carboxylic acid reductase in the RARE strain and addition of benzoate during growth, benzaldehyde remained in the culture after 24 h, with less than 12% conversion of benzaldehyde to benzyl alcohol. Although individual overexpression results demonstrated that all six genes could contribute to benzaldehyde reduction in vivo, additional experiments featuring subset deletion strains revealed that two of the gene deletions were dispensable under the conditions tested. The engineered strain was next investigated for the production of vanillin from vanillate and succeeded in preventing formation of the byproduct vanillyl alcohol. A pathway for the biosynthesis of vanillin directly from glucose was introduced and resulted in a 55-fold improvement in vanillin titer when using the RARE strain versus the wild-type strain. Finally, synthesis of the chiral pharmaceutical intermediate L-phenylacetylcarbinol (L-PAC) was demonstrated from benzaldehyde and glucose upon expression of a recombinant mutant pyruvate decarboxylase in the RARE strain. Beyond allowing accumulation of aromatic aldehydes as end products in E. coli, the RARE strain expands the classes of chemicals that can be produced microbially via aldehyde intermediates.

A Functional Element Necessary for Fetal Hemoglobin Silencing

PubMed Central

Sankaran, Vijay G.; Xu, Jian; Byron, Rachel; Greisman, Harvey A.; Fisher, Chris; Weatherall, David J.; Sabath, Daniel E.; Groudine, Mark; Orkin, Stuart H.; Premawardhena, Anuja; Bender, M.A.

2011-01-01

BACKGROUND An improved understanding of the regulation of the fetal hemoglobin genes holds promise for the development of targeted therapeutic approaches for fetal hemoglobin induction in the β-hemoglobinopathies. Although recent studies have uncovered trans-acting factors necessary for this regulation, limited insight has been gained into the cis-regulatory elements involved. METHODS We identified three families with unusual patterns of hemoglobin expression, suggestive of deletions in the locus of the β-globin gene (β-globin locus). We performed array comparative genomic hybridization to map these deletions and confirmed breakpoints by means of polymerase-chain-reaction assays and DNA sequencing. We compared these deletions, along with previously mapped deletions, and studied the trans-acting factors binding to these sites in the β-globin locus by using chromatin immunoprecipitation. RESULTS We found a new (δβ)0-thalassemia deletion and a rare hereditary persistence of fetal hemoglobin deletion with identical downstream breakpoints. Comparison of the two deletions resulted in the identification of a small intergenic region required for γ-globin (fetal hemoglobin) gene silencing. We mapped a Kurdish β0-thalassemia deletion, which retains the required intergenic region, deletes other surrounding sequences, and maintains fetal hemoglobin silencing. By comparing these deletions and other previously mapped deletions, we elucidated a 3.5-kb intergenic region near the 5′ end of the δ-globin gene that is necessary for γ-globin silencing. We found that a critical fetal hemoglobin silencing factor, BCL11A, and its partners bind within this region in the chromatin of adult erythroid cells. CONCLUSIONS By studying three families with unusual deletions in the β-globin locus, we identified an intergenic region near the δ-globin gene that is necessary for fetal hemoglobin silencing. (Funded by the National Institutes of Health and others.) PMID:21879898

Evolution and Emergence of Enteroviruses through Intra- and Inter-species Recombination: Plasticity and Phenotypic Impact of Modular Genetic Exchanges in the 5' Untranslated Region.

PubMed

Muslin, Claire; Joffret, Marie-Line; Pelletier, Isabelle; Blondel, Bruno; Delpeyroux, Francis

2015-01-01

Genetic recombination shapes the diversity of RNA viruses, including enteroviruses (EVs), which frequently have mosaic genomes. Pathogenic circulating vaccine-derived poliovirus (cVDPV) genomes consist of mutated vaccine poliovirus (PV) sequences encoding capsid proteins, and sequences encoding nonstructural proteins derived from other species' C EVs, including certain coxsackieviruses A (CV-A) in particular. Many cVDPV genomes also have an exogenous 5' untranslated region (5' UTR). This region is involved in virulence and includes the cloverleaf (CL) and the internal ribosomal entry site, which play major roles in replication and the initiation of translation, respectively. We investigated the plasticity of the PV genome in terms of recombination in the 5' UTR, by developing an experimental model involving the rescue of a bipartite PV/CV-A cVDPV genome rendered defective by mutations in the CL, following the co-transfection of cells with 5' UTR RNAs from each of the four human EV species (EV-A to -D). The defective cVDPV was rescued by recombination with 5' UTR sequences from the four EV species. Homologous and nonhomologous recombinants with large deletions or insertions in three hotspots were isolated, revealing a striking plasticity of the 5' UTR. By contrast to the recombination of the cVDPV with the 5' UTR of group II (EV-A and -B), which can decrease viral replication and virulence, recombination with the 5' UTRs of group I (EV-C and -D) appeared to be evolutionarily neutral or associated with a gain in fitness. This study illustrates how the genomes of positive-strand RNA viruses can evolve into mosaic recombinant genomes through intra- or inter-species modular genetic exchanges, favoring the emergence of new recombinant lineages.

Long G2 accumulates recombination intermediates and disturbs chromosome segregation at dysfunction telomere in Schizosaccharomyces pombe

DOE Office of Scientific and Technical Information (OSTI.GOV)

Habib, Ahmed G.K.; Masuda, Kenta; Yukawa, Masashi

Protection of telomere (Pot1) is a single-stranded telomere binding protein which is essential for chromosome ends protection. Fission yeast Rqh1 is a member of RecQ helicases family which has essential roles in the maintenance of genomic stability and regulation of homologous recombination. Double mutant between fission yeast pot1Δ and rqh1 helicase dead (rqh1-hd) maintains telomere by homologous recombination. In pot1Δ rqh1-hd double mutant, recombination intermediates accumulate near telomere which disturb chromosome segregation and make cells sensitive to microtubule inhibitors thiabendazole (TBZ). Deletion of chk1{sup +} or mutation of its kinase domain shortens the G2 of pot1Δ rqh1-hd double mutant andmore » suppresses both the accumulation of recombination intermediates and the TBZ sensitivity of that double mutant. In this study, we asked whether the long G2 is the reason for the TBZ sensitivity of pot1Δ rqh1-hd double mutant. We found that shortening the G2 of pot1Δ rqh1-hd double mutant by additional mutations of wee1 and mik1 or gain of function mutation of Cdc2 suppresses both the accumulation of recombination intermediates and the TBZ sensitivity of pot1Δ rqh1-hd double mutant. Our results suggest that long G2 of pot1Δ rqh1-hd double mutant may allow time for the accumulation of recombination intermediates which disturb chromosome segregation and make cells sensitive to TBZ. - Ηighlights: • We show link between long G2 and accumulation of toxic recombination intermediates. • Accumulation of recombination intermediates at telomere results in TBZ sensitivity. • Activation of DNA damage checkpoint worsens cells' viability in presence of TBZ.« less

Genetic algorithms as global random search methods

NASA Technical Reports Server (NTRS)

Peck, Charles C.; Dhawan, Atam P.

1995-01-01

Genetic algorithm behavior is described in terms of the construction and evolution of the sampling distributions over the space of candidate solutions. This novel perspective is motivated by analysis indicating that the schema theory is inadequate for completely and properly explaining genetic algorithm behavior. Based on the proposed theory, it is argued that the similarities of candidate solutions should be exploited directly, rather than encoding candidate solutions and then exploiting their similarities. Proportional selection is characterized as a global search operator, and recombination is characterized as the search process that exploits similarities. Sequential algorithms and many deletion methods are also analyzed. It is shown that by properly constraining the search breadth of recombination operators, convergence of genetic algorithms to a global optimum can be ensured.

Genome Editing in Escherichia coli with Cas9 and synthetic CRISPRs

DOE Office of Scientific and Technical Information (OSTI.GOV)

Peng, Ze; Richardson, Sarah; Robinson, David

Recently, the Cas9-CRISPR system has proven to be a useful tool for genome editing in eukaryotes, which repair the double stranded breaks made by Cas9 with non-homologous end joining or homologous recombination. Escherichia coli lacks non-homologous end joining and has a very low homologous recombination rate, effectively rendering targeted Cas9 activity lethal. We have developed a heat curable, serializable, plasmid based system for selectionless Cas9 editing in arbitrary E. coli strains that uses synthetic CRISPRs for targeting and -red to effect repairs of double stranded breaks. We have demonstrated insertions, substitutions, and multi-target deletions with our system, which we havemore » tested in several strains.« less

Genetic algorithms as global random search methods

NASA Technical Reports Server (NTRS)

Peck, Charles C.; Dhawan, Atam P.

1995-01-01

Genetic algorithm behavior is described in terms of the construction and evolution of the sampling distributions over the space of candidate solutions. This novel perspective is motivated by analysis indicating that that schema theory is inadequate for completely and properly explaining genetic algorithm behavior. Based on the proposed theory, it is argued that the similarities of candidate solutions should be exploited directly, rather than encoding candidate solution and then exploiting their similarities. Proportional selection is characterized as a global search operator, and recombination is characterized as the search process that exploits similarities. Sequential algorithms and many deletion methods are also analyzed. It is shown that by properly constraining the search breadth of recombination operators, convergence of genetic algorithms to a global optimum can be ensured.

Deletion of the Lymantria dispar multicapsid nucleopolyhedrovirus ecdysteroid UDP-glucosyl transferase gene enhances viral killing speed in the last instar of the gypsy moth

Treesearch

James M. Slavicek; Holly J.R. Popham; C.I. Riegel

1999-01-01

The Lymantria dispar multicapsid nucleopolyhedrovirus (LdMNPV) is used on a limited basis as a gypsy moth (L. dispar) control agent. In an effort to improve the efficacy (i.e., killing speed) of the LdMNPV, we generated a recombinant viral strain (vEGT-) that does not produce the enzyme ecdysteroid UDP-glucosyltransferase (EGT). We...

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dai, Ziyu; Aryal, Uma K.; Shukla, Anil

ALG3 is a Family 58 glycosyltransferase enzyme involved in early N-linked glycan synthesis. Here, we investigated the effect of the alg3 gene disruption on growth, development, metabolism, and protein secretion in Aspergillus niger. The alg3 gene deletion resulted in a significant reduction of growth on complete (CM) and potato dextrose agar (PDA) media and a substantial reduction of spore production on CM. It also delayed spore germination in the liquid cultures of both CM and PDA media, but led to a significant accumulation of red pigment on both CM and liquid modified minimal medium (MM) supplemented with yeast extract. Themore » relative abundance of 55 proteins of the total 190 proteins identified in the secretome was significantly different as a result of alg3 gene deletion. Comparison of a Trichoderma reesei cellobiohydrolase (Cel7A) heterologously expressed in A. niger parental and Δalg3 strains showed that the recombinant Cel7A expressed in the mutant background was smaller in size than that from the parental strains. This study suggests that ALG3 is critical for growth and development, pigment production, and protein secretion in A. niger. Functional analysis of recombinant Cel7A with aberrant glycosylation demonstrates the feasibility of this alternative approach to evaluate the role of N-linked glycosylation in glycoprotein secretion and function.« less

Mutational analysis of the reverse transcriptase and ribonuclease H domains of the human foamy virus.

PubMed Central

Kögel, D; Aboud, M; Flügel, R M

1995-01-01

Human foamy or spuma virus (HFV) codes for a distinct set of pol gen products. To determine the minimal requirements for the HFV enzymatic activities, defined residues of the reverse transcriptase (RT) and ribo-nuclease H (RNase H) domain of the HFV pol gene were mutated by site-specific PCR mutagenesis. The mutant gene products were bacterially expressed, purified by Ni2+ chelate affinity chromatography and characterised by Western blotting. The enzymatic activities of the individual recombinant HFV pol mutant proteins were characterised by the situ RT, RNase H and RNase H assays. Two substitution mutants reached RT activity levels higher than that of the intact recombinant HFV RT-RH-His. When the catalytically essential D508 was substituted by A508, 5% of RNase H activity was retained while DNA polymerase activity increased 2-fold. A deletion of 11 amino acid residues in the hinge region completely abolished DNA polymerase while RNase H activity decreased 2-fold. A deletion mutant in the C-terminal RH domain showed no RNase H but retained RNase H activity indicating that the activities are genetically separable. The combined data reveal that the HFV DNA polymerase and RNase H activities are interdependent. Images PMID:7544460

Resistance of herpes simplex virus type 2 to neomycin maps to the N-terminal portion of glycoprotein C.

PubMed Central

Oyan, A M; Dolter, K E; Langeland, N; Goins, W F; Glorioso, J C; Haarr, L; Crumpacker, C S

1993-01-01

Entry of herpes simplex virus (HSV) into cells is believed to be mediated by specific binding of envelope proteins to a cellular receptor. Neomycin specifically blocks this initial step in infection by HSV-1 but not HSV-2. Resistance of HSV-2 to this compound maps to a region of the genome encoding glycoprotein C (gC-2). We have studied the function of gC-2 in the initial interaction of the virus with the host cell, using HSV-2 mutants deleted for gC-2 and gC-2-rescued recombinants. Resistance to neomycin was directly linked to the presence of gC-2 within the viral genome. In addition, deletion of the gC-2 gene caused a marked delay in adsorption to cells relative to the wild-type virus. HSV-1 recombinants containing chimeric gC genes composed of HSV-1 and HSV-2 sequences were used to localize neomycin resistance within the N-terminal 223 amino acids of gC-2. This region of the glycoprotein comprises an important domain responsible for binding of HSV-2 to cell receptors in the presence of neomycin. A gC-2-negative mutant is still infectious, indicating that HSV-2 also has an alternative pathway of adsorption. Images PMID:8386261

The yeast Saccharomyces cerevisiae DNA polymerase IV: possible involvement in double strand break DNA repair.

PubMed

Leem, S H; Ropp, P A; Sugino, A

1994-08-11

We identified and purified a new DNA polymerase (DNA polymerase IV), which is similar to mammalian DNA polymerase beta, from Saccharomyces cerevisiae and suggested that it is encoded by YCR14C (POLX) on chromosome III. Here, we provided a direct evidence that the purified DNA polymerase IV is indeed encoded by POLX. Strains harboring a pol4 deletion mutation exhibit neither mitotic growth defect nor a meiosis defect, suggesting that DNA polymerase IV participates in nonessential functions in DNA metabolism. The deletion strains did not exhibit UV-sensitivity. However, they did show weak sensitivity to MMS-treatment and exhibited a hyper-recombination phenotype when intragenic recombination was measured during meiosis. Furthermore, MAT alpha pol4 delta segregants had a higher frequency of illegitimate mating with a MAT alpha tester strain than that of wild-type cells. These results suggest that DNA polymerase IV participates in a double-strand break repair pathway. A 3.2kb of the POL4 transcript was weakly expressed in mitotically growing cells. During meiosis, a 2.2 kb POL4 transcript was greatly induced, while the 3.2 kb transcript stayed at constant levels. This induction was delayed in a swi4 delta strain during meiosis, while no effect was observed in a swi6 delta strain.

Different domains of the murine RNA polymerase I-specific termination factor mTTF-I serve distinct functions in transcription termination.

PubMed

Evers, R; Smid, A; Rudloff, U; Lottspeich, F; Grummt, I

1995-03-15

Termination of mouse ribosomal gene transcription by RNA polymerase I (Pol I) requires the specific interaction of a DNA binding protein, mTTF-I, with an 18 bp sequence element located downstream of the rRNA coding region. Here we describe the molecular cloning and functional characterization of the cDNA encoding this transcription termination factor. Recombinant mTTF-I binds specifically to the murine terminator elements and terminates Pol I transcription in a reconstituted in vitro system. Deletion analysis has defined a modular structure of mTTF-I comprising a dispensable N-terminal half, a large C-terminal DNA binding region and an internal domain which is required for transcription termination. Significantly, the C-terminal region of mTTF-I reveals striking homology to the DNA binding domains of the proto-oncogene c-Myb and the yeast transcription factor Reb1p. Site-directed mutagenesis of one of the tryptophan residues that is conserved in the homology region of c-Myb, Reb1p and mTTF-I abolishes specific DNA binding, a finding which underscores the functional relevance of these residues in DNA-protein interactions.

Different domains of the murine RNA polymerase I-specific termination factor mTTF-I serve distinct functions in transcription termination.

PubMed Central

Evers, R; Smid, A; Rudloff, U; Lottspeich, F; Grummt, I

1995-01-01

Termination of mouse ribosomal gene transcription by RNA polymerase I (Pol I) requires the specific interaction of a DNA binding protein, mTTF-I, with an 18 bp sequence element located downstream of the rRNA coding region. Here we describe the molecular cloning and functional characterization of the cDNA encoding this transcription termination factor. Recombinant mTTF-I binds specifically to the murine terminator elements and terminates Pol I transcription in a reconstituted in vitro system. Deletion analysis has defined a modular structure of mTTF-I comprising a dispensable N-terminal half, a large C-terminal DNA binding region and an internal domain which is required for transcription termination. Significantly, the C-terminal region of mTTF-I reveals striking homology to the DNA binding domains of the proto-oncogene c-Myb and the yeast transcription factor Reb1p. Site-directed mutagenesis of one of the tryptophan residues that is conserved in the homology region of c-Myb, Reb1p and mTTF-I abolishes specific DNA binding, a finding which underscores the functional relevance of these residues in DNA-protein interactions. Images PMID:7720715

Mutant POLG2 Disrupts DNA Polymerase γ Subunits and Causes Progressive External Ophthalmoplegia

PubMed Central

Longley, Matthew J.; Clark, Susanna; Yu Wai Man, Cynthia; Hudson, Gavin; Durham, Steve E.; Taylor, Robert W.; Nightingale, Simon; Turnbull, Douglass M.; Copeland, William C.; Chinnery, Patrick F.

2006-01-01

DNA polymerase γ (pol γ) is required to maintain the genetic integrity of the 16,569-bp human mitochondrial genome (mtDNA). Mutation of the nuclear gene for the catalytic subunit of pol γ (POLG) has been linked to a wide range of mitochondrial diseases involving mutation, deletion, and depletion of mtDNA. We describe a heterozygous dominant mutation (c.1352G→A/p.G451E) in POLG2, the gene encoding the p55 accessory subunit of pol γ, that causes progressive external ophthalmoplegia with multiple mtDNA deletions and cytochrome c oxidase (COX)–deficient muscle fibers. Biochemical characterization of purified, recombinant G451E-substituted p55 protein in vitro revealed incomplete stimulation of the catalytic subunit due to compromised subunit interaction. Although G451E p55 retains a wild-type ability to bind DNA, it fails to enhance the DNA-binding strength of the p140-p55 complex. In vivo, the disease most likely arises through haplotype insufficiency or heterodimerization of the mutated and wild-type proteins, which promote mtDNA deletions by stalling the DNA replication fork. The progressive accumulation of mtDNA deletions causes COX deficiency in muscle fibers and results in the clinical phenotype. PMID:16685652

An integer programming formulation of the parsimonious loss of heterozygosity problem.

PubMed

Catanzaro, Daniele; Labbé, Martine; Halldórsson, Bjarni V

2013-01-01

A loss of heterozygosity (LOH) event occurs when, by the laws of Mendelian inheritance, an individual should be heterozygote at a given site but, due to a deletion polymorphism, is not. Deletions play an important role in human disease and their detection could provide fundamental insights for the development of new diagnostics and treatments. In this paper, we investigate the parsimonious loss of heterozygosity problem (PLOHP), i.e., the problem of partitioning suspected polymorphisms from a set of individuals into a minimum number of deletion areas. Specifically, we generalize Halldórsson et al.'s work by providing a more general formulation of the PLOHP and by showing how one can incorporate different recombination rates and prior knowledge about the locations of deletions. Moreover, we show that the PLOHP can be formulated as a specific version of the clique partition problem in a particular class of graphs called undirected catch-point interval graphs and we prove its general $({\\cal NP})$-hardness. Finally, we provide a state-of-the-art integer programming (IP) formulation and strengthening valid inequalities to exactly solve real instances of the PLOHP containing up to 9,000 individuals and 3,000 SNPs. Our results give perspectives on the mathematics of the PLOHP and suggest new directions on the development of future efficient exact solution approaches.

A 1.5 million–base pair inversion polymorphism in families with Williams-Beuren syndrome

PubMed Central

Osborne, Lucy R.; Li, Martin; Pober, Barbara; Chitayat, David; Bodurtha, Joann; Mandel, Ariane; Costa, Teresa; Grebe, Theresa; Cox, Sarah; Tsui, Lap-Chee; Scherer, Stephen W.

2010-01-01

Williams-Beuren syndrome (WBS) is most often caused by hemizygous deletion of a 1.5-Mb interval encompassing at least 17 genes at 7q11.23 (refs. 1, 2). As with many other haploinsufficiency diseases, the mechanism underlying the WBS deletion is thought to be unequal meiotic recombination, probably mediated by the highly homologous DNA that flanks the commonly deleted region3. Here, we report the use of interphase fluorescence in situ hybridization (FISH) and pulsed-field gel electrophoresis (PFGE) to identify a genomic polymorphism in families with WBS, consisting of an inversion of the WBS region. We have observed that the inversion is hemizygous in 3 of 11 (27%) atypical affected individuals who show a subset of the WBS phenotypic spectrum but do not carry the typical WBS microdeletion. Two of these individuals also have a parent who carries the inversion. In addition, in 4 of 12 (33%) families with a proband carrying the WBS deletion, we observed the inversion exclusively in the parent transmitting the disease-related chromosome. These results suggest the presence of a newly identified genomic variant within the population that may be associated with the disease. It may result in predisposition to primarily WBS-causing microdeletions, but may also cause translocations and inversions. PMID:11685205

Ig heavy chain class switch recombination: mechanism and regulation

PubMed Central

Stavnezer, Janet; Schrader, Carol E.

2014-01-01

Ig heavy chain class switching occurs rapidly after activation of mature naïve B cells, resulting in a switch from expressing IgM and IgD to expression of IgG, IgE, or IgA; this switch improves the ability of antibodies to remove the pathogen that induces the humoral immune response. Class switching occurs by a deletional recombination between two different switch (S) regions, each of which is associated with a heavy chain constant (CH) region gene. Class switch recombination (CSR) is instigated by activation-induced cytidine deaminase (AID), which converts cytosines in S regions to uracils. The uracils are subsequently removed by two DNA repair pathways, resulting in mutations, single-strand DNA breaks, and the double-strand breaks required for CSR. We discuss several aspects of CSR, including how CSR is induced, CSR in B-cell progenitors, the roles for transcription and chromosomal looping in CSR, and the roles of certain DNA repair enzymes in CSR. PMID:25411432

Infectious mutants of cassava latent virus generated in vivo from intact recombinant DNA clones containing single copies of the genome.

PubMed Central

Stanley, J; Townsend, R

1986-01-01

Intact recombinant DNAs containing single copies of either component of the cassava latent virus genome can elicit infection when mechanically inoculated to host plants in the presence of the appropriate second component. Characterisation of infectious mutant progeny viruses, by analysis of virus-specific supercoiled DNA intermediates, indicates that most if not all of the cloning vector has been deleted, achieved at least in some cases by intermolecular recombination in vivo between DNAs 1 and 2. Significant rearrangements within the intergenic region of DNA 2, predominantly external to the common region, can be tolerated without loss of infectivity suggesting a somewhat passive role in virus multiplication for the sequences in question. Although packaging constraints might impose limits on the amount of DNA within geminate particles, isolation of an infectious coat protein mutant defective in virion production suggests that packaging is not essential for systemic spread of the viral DNA. Images PMID:2875435

Transient dominant host-range selection using Chinese hamster ovary cells to generate marker-free recombinant viral vectors from vaccinia virus.

PubMed

Liu, Liang; Cooper, Tamara; Eldi, Preethi; Garcia-Valtanen, Pablo; Diener, Kerrilyn R; Howley, Paul M; Hayball, John D

2017-04-01

Recombinant vaccinia viruses (rVACVs) are promising antigen-delivery systems for vaccine development that are also useful as research tools. Two common methods for selection during construction of rVACV clones are (i) co-insertion of drug resistance or reporter protein genes, which requires the use of additional selection drugs or detection methods, and (ii) dominant host-range selection. The latter uses VACV variants rendered replication-incompetent in host cell lines by the deletion of host-range genes. Replicative ability is restored by co-insertion of the host-range genes, providing for dominant selection of the recombinant viruses. Here, we describe a new method for the construction of rVACVs using the cowpox CP77 protein and unmodified VACV as the starting material. Our selection system will expand the range of tools available for positive selection of rVACV during vector construction, and it is substantially more high-fidelity than approaches based on selection for drug resistance.

[Understanding mitochondrial genome fragmentation in parasitic lice (Insecta: Phthiraptera)].

PubMed

Dong, Wen-Ge; Guo, Xian-Guo; Jin, Dao-Chao; Xue, Shi-Peng; Qin, Feng; Simon, Song; Stephen, C Barker; Renfu, Shao

2013-07-01

Lice are obligate ectoparasites of mammals and birds. Extensive fragmentation of mitochondrial genomes has been found in some louse species in the families Pediculidae, Pthiridae, Philopteridae and Trichodectidae. For example, the mt genomes of human body louse (Pediculus humanus), head louse (Pediculus capitis), and public louse (Pthirus pubis) have 20, 20 and 14 mini-chromosomes, respectively. These mini-chromosomes might be the results of deletion and recombination of mt genes. The factors and mechanisms of mitochondrial genome fragmentation are currently unknown. The fragmentation might be the results of evolutionary selection or random genetic drift or it is probably related to the lack of mtSSB (mitochondrial single-strand DNA binding protein). Understanding the fragmentation of mitochondrial genomes is of significance for understanding the origin and evolution of mitochondria. This paper reviews the recent advances in the studies of mito-chondrial genome fragmentation in lice, including the phenomena of mitochondrial genome fragmentation, characteristics of fragmented mitochondrial genomes, and some factors and mechanisms possibly leading to the mitochondrial genome fragmentation of lice. Perspectives for future studies on fragmented mt genomes are also discussed.

Structure and polymorphism of the mouse prion protein gene.

PubMed Central

Westaway, D; Cooper, C; Turner, S; Da Costa, M; Carlson, G A; Prusiner, S B

1994-01-01

Missense mutations in the prion protein (PrP) gene, overexpression of the cellular isoform of PrP (PrPC), and infection with prions containing the scrapie isoform of PrP (PrPSc) all cause neurodegenerative disease. To understand better the physiology and expression of PrPC, we retrieved mouse PrP gene (Prn-p) yeast artificial chromosome (YAC), cosmid, phage, and cDNA clones. Physical mapping positions Prn-p approximately 300 kb from ecotropic virus integration site number 4 (Evi-4), compatible with failure to detect recombination between Prn-p and Evi-4 in genetic crosses. The Prn-pa allele encompasses three exons, with exons 1 and 2 encoding the mRNA 5' untranslated region. Exon 2 has no equivalent in the Syrian hamster and human PrP genes. The Prn-pb gene shares this intron/exon structure but harbors an approximately 6-kb deletion within intron 2. While the Prn-pb open reading frame encodes two amino acid substitutions linked to prolonged scrapie incubation periods, a deletion of intron 2 sequences also characterizes inbred strains such as RIII/S and MOLF/Ei with shorter incubation periods, making a relationship between intron 2 size and scrapie pathogenesis unlikely. The promoter regions of a and b Prn-p alleles include consensus Sp1 and AP-1 sites, as well as other conserved motifs which may represent binding sites for as yet unidentified transcription factors. Images PMID:7912827

Polygalacturonase gene pgxB in Aspergillus niger is a virulence factor in apple fruit.

PubMed

Liu, Cheng-Qian; Hu, Kang-Di; Li, Ting-Ting; Yang, Ying; Yang, Feng; Li, Yan-Hong; Liu, He-Ping; Chen, Xiao-Yan; Zhang, Hua

2017-01-01

Aspergillus niger, a saprophytic fungus, is widely distributed in soil, air and cereals, and can cause postharvest diseases in fruit. Polygalacturonase (PG) is one of the main enzymes in fungal pathogens to degrade plant cell wall. To evaluate whether the deletion of an exo-polygalacturonase gene pgxB would influence fungal pathogenicity to fruit, pgxB gene was deleted in Aspergillus niger MA 70.15 (wild type) via homologous recombination. The ΔpgxB mutant showed similar growth behavior compared with the wild type. Pectin medium induced significant higher expression of all pectinase genes in both wild type and ΔpgxB in comparison to potato dextrose agar medium. However, the ΔpgxB mutant was less virulent on apple fruits as the necrosis diameter caused by ΔpgxB mutant was significantly smaller than that of wild type. Results of quantitive-PCR showed that, in the process of infection in apple fruit, gene expressions of polygalacturonase genes pgaI, pgaII, pgaA, pgaC, pgaD and pgaE were enhanced in ΔpgxB mutant in comparison to wild type. These results prove that, despite the increased gene expression of other polygalacturonase genes in ΔpgxB mutant, the lack of pgxB gene significantly reduced the virulence of A. niger on apple fruit, suggesting that pgxB plays an important role in the infection process on the apple fruit.

B cell IFN-γ receptor signaling promotes autoimmune germinal centers via cell-intrinsic induction of BCL-6

PubMed Central

Jackson, Shaun W.; Jacobs, Holly M.; Arkatkar, Tanvi; Dam, Elizabeth M.; Scharping, Nicole E.; Kolhatkar, Nikita S.; Hou, Baidong; Buckner, Jane H.

2016-01-01

Dysregulated germinal center (GC) responses are implicated in the pathogenesis of human autoimmune diseases, including systemic lupus erythematosus (SLE). Although both type 1 and type 2 interferons (IFNs) are involved in lupus pathogenesis, their respective impacts on the establishment of autoimmune GCs has not been addressed. In this study, using a chimeric model of B cell-driven autoimmunity, we demonstrate that B cell type 1 IFN receptor signals accelerate, but are not required for, lupus development. In contrast, B cells functioning as antigen-presenting cells initiate CD4+ T cell activation and IFN-γ production, and strikingly, B cell–intrinsic deletion of the IFN-γ receptor (IFN-γR) abrogates autoimmune GCs, class-switched autoantibodies (auto-Abs), and systemic autoimmunity. Mechanistically, although IFN-γR signals increase B cell T-bet expression, B cell–intrinsic deletion of T-bet exerts an isolated impact on class-switch recombination to pathogenic auto-Ab subclasses without impacting GC development. Rather, in both mouse and human B cells, IFN-γ synergized with B cell receptor, toll-like receptor, and/or CD40 activation signals to promote cell-intrinsic expression of the GC master transcription factor, B cell lymphoma 6 protein. Our combined findings identify a novel B cell–intrinsic mechanism whereby IFN signals promote lupus pathogenesis, implicating this pathway as a potential therapeutic target in SLE. PMID:27069113

The Zinc-finger protein ASCIZ regulates B cell development via DYNLL1 and Bim

PubMed Central

Jurado, Sabine; Gleeson, Kimberly; O’Donnell, Kristy; Izon, David J.; Walkley, Carl R.; Strasser, Andreas; Tarlinton, David M.

2012-01-01

Developing B lymphocytes expressing defective or autoreactive pre-B or B cell receptors (BCRs) are eliminated by programmed cell death, but how the balance between death and survival signals is regulated to prevent immunodeficiency and autoimmunity remains incompletely understood. In this study, we show that absence of the essential ATM (ataxia telangiectasia mutated) substrate Chk2-interacting Zn2+-finger protein (ASCIZ; also known as ATMIN/ZNF822), a protein with dual functions in the DNA damage response and as a transcription factor, leads to progressive cell loss from the pre-B stage onwards and severely diminished splenic B cell numbers in mice. This lymphopenia cannot be suppressed by deletion of p53 or complementation with a prearranged BCR, indicating that it is not caused by impaired DNA damage responses or defective V(D)J recombination. Instead, ASCIZ-deficient B cell precursors contain highly reduced levels of DYNLL1 (dynein light chain 1; LC8), a recently identified transcriptional target of ASCIZ, and normal B cell development can be restored by ectopic Dynll1 expression. Remarkably, the B cell lymphopenia in the absence of ASCIZ can also be fully suppressed by deletion of the proapoptotic DYNLL1 target Bim. Our findings demonstrate a key role for ASCIZ in regulating the survival of developing B cells by activating DYNLL1 expression, which may then modulate Bim-dependent apoptosis. PMID:22891272

The relationship between BIM deletion polymorphism and clinical significance of epidermal growth factor receptor-mutated non-small cell lung cancer patients with epidermal growth factor receptor-tyrosine kinase inhibitor therapy: a meta-analysis.

PubMed

Zou, Qian; Zhan, Ping; Lv, Tangfeng; Song, Yong

2015-12-01

BIM deletion polymorphism is a germline that might lead to little or no BH3 expression, which affects epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) related apoptosis. Recent studies show that BIM deletion polymorphism might be a critical factor leading to the resistance of EGFR-TKIs in EGFR mutation-positive non-small cell lung cancer (NSCLC) patients. Thus, a meta-analysis was conducted by combing seven original eligible studies including 778 NSCLC patients to investigate a steady and reliable conclusion. Our study indicated that BIM deletion polymorphism was significantly associated with the poor objective response rate (ORR) of EGFR-TKIs in EGFR-mutated NSCLC patients [odds ratios (OR) =0.55, 95% confidence interval (CI), 0.33-0.92]. And disease control rate (DCR) in EGFR-mutate NSCLC patients treated with EGFR-TKIs was significantly decreased in patients with BIM deletion polymorphism (OR=0.55, 95% CI, 0.27-1.12). Moreover, the progression-free survival (PFS) of patients with BIM deletion polymorphism is shorter. These findings suggested that BIM deletion polymorphism might be a genetic cause of intrinsic resistance to TKI therapy and it could be emerged as an independent predictor to identify patients who would benefit from TKI targeted therapy in EGFR-mutated NSCLC.

Clinical significance of BIM deletion polymorphism in non-small-cell lung cancer with epidermal growth factor receptor mutation.

PubMed

Isobe, Kazutoshi; Hata, Yoshinobu; Tochigi, Naobumi; Kaburaki, Kyohei; Kobayashi, Hiroshi; Makino, Takashi; Otsuka, Hajime; Sato, Fumitomo; Ishida, Fumiaki; Kikuchi, Naoshi; Hirota, Nao; Sato, Keita; Sano, Go; Sugino, Keishi; Sakamoto, Susumu; Takai, Yujiro; Shibuya, Kazutoshi; Iyoda, Akira; Homma, Sakae

2014-04-01

Germline alterations in the proapoptotic protein Bcl-2-like 11 (BIM) can have a crucial role in tumor response to treatment. To determine the clinical utility of detecting BIM deletion polymorphism in non-small-cell lung cancer positive for epidermal growth factor receptor (EGFR) mutation, we examined outcomes of patients with and without BIM alterations. We studied 70 patients with EGFR mutation-positive non-small-cell lung cancer who were treated with an EGFR tyrosine kinase inhibitor between January 2008 and January 2013. BIM deletion was analyzed by polymerase chain reaction in 58 samples of peripheral blood and 24 formalin-fixed paraffin-embedded slides of surgical specimens (20 of lung tissue and four of brain tissue); both blood and tissue specimens were available for 12 patients. We retrospectively analyzed clinical characteristics, response rate, toxicity, and outcomes among patients with and without BIM deletion. BIM deletion was present in 13 of 70 patients (18.6%). There were no significant differences between patients with and without BIM deletion in clinical characteristics, rate of response to EGFR tyrosine kinase inhibitor, or incidence of adverse events. Patients with BIM deletion had significantly shorter progression-free survival (PFS) than those without BIM deletion (median, 227 versus 533 days; p < 0.001). Multivariate Cox regression analysis showed that BIM deletion was an independent indicator of shorter PFS (hazard ratio, 3.99; 95% confidence interval, 1.864-8.547; p < 0.001). Polymerase chain reaction successfully detected BIM deletion in samples of peripheral blood and formalin-fixed paraffin-embedded slides of surgical specimens. BIM deletion was the most important independent prognostic factor in shorter PFS.

The effects of upaB deletion and the double/triple deletion of upaB, aatA, and aatB genes on pathogenicity of avian pathogenic Escherichia coli.

PubMed

Zhu-Ge, Xiang-Kai; Pan, Zi-Hao; Tang, Fang; Mao, Xiang; Hu, Lin; Wang, Shao-Hui; Xu, Bin; Lu, Cheng-Ping; Fan, Hong-Jie; Dai, Jian-Jun

2015-12-01

Autotransporters (ATs) are associated with pathogenesis of Avian Pathogenic Escherichia coli (APEC). The molecular characterization of APEC ATs can provide insights about their relevance to APEC pathogenesis. Here, we characterized a conventional autotransporter UpaB in APEC DE205B genome. The upaB existed in 41.9 % of 236 APEC isolates and was predominantly associated with ECOR B2 and D. Our studies showed that UpaB mediates the DE205B adhesion in DF-1 cells, and enhances autoaggregation and biofilm formation of fimbria-negative E. coli AAEC189 (MG1655Δfim) in vitro. Deletion of upaB of DE205B attenuates the virulence in duck model and early colonization in the duck lungs during APEC systemic infection. Furthermore, double and triple deletion of upaB, aatA, and aatB genes cumulatively attenuated DE205B adhesion in DF-1 cells, accompanying with decreased 50 % lethal dose (LD50) in duck model and the early colonization in the duck lungs. However, DE205BΔupaB/ΔaatA/ΔaatB might "compensate" the influence of gene deletion by upregulating the expression of fimbrial adhesin genes yqiL, yadN, and vacuolating autotransporter vat during early colonization of APEC. Finally, we demonstrated that vaccination with recombinant UpaB, AatA, and AatB proteins conferred protection against colisepticemia caused by DE205B infection in duck model.

Inversion of the Williams syndrome region is a common polymorphism found more frequently in parents of children with Williams syndrome.

PubMed

Hobart, Holly H; Morris, Colleen A; Mervis, Carolyn B; Pani, Ariel M; Kistler, Doris J; Rios, Cecilia M; Kimberley, Kendra W; Gregg, Ronald G; Bray-Ward, Patricia

2010-05-15

Williams syndrome (WS) is a multisystem disorder caused by deletion of about 1.55 Mb of DNA (including 26 genes) on chromosome 7q11.23, a region predisposed to recombination due to its genomic structure. Deletion of the Williams syndrome chromosome region (WSCR) occurs sporadically. To better define chance for familial recurrence and to investigate the prevalence of genomic rearrangements of the region, 257 children with WS and their parents were studied. We determined deletion size in probands by metaphase FISH, parent-of-origin of the deleted chromosome by molecular genetic methods, and inversion status of the WSCR in both parents by interphase FISH. The frequency of WSCR inversion in the transmitting parent group was 24.9%. In contrast, the rate of inversion in the non-transmitting parent group (a reasonable estimate of the rate in the general population) was 5.8%. There were no significant gender differences with respect to parent-of-origin for the deleted chromosome or the incidence of the inversion polymorphism. There was no difference in the rate of spontaneous abortion for mothers heterozygous for the WSCR inversion relative to mothers without the inversion. We calculate that for a parent heterozygous for a WSCR inversion, the chance to have a child with WS is about 1 in 1,750, in contrast to the 1 in 9,500 chance for a parent without an inversion.

Paracentric inversions in humans: A review of 446 paracentric inversions with presentation of 120 new cases

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pettenati, M.J.; Rao, P.N.; Grss, F.

1995-01-16

We present a large review of 446 cases of paracentric inversions (PAI), including 120 new cases, to assess their incidence, distribution, inheritance, modes of ascertainment, interchromosomal effects, viable recombinant offspring, and clinical relevance. All 23 autosomes and sex chromosomes had inversions. However, none were identified in chromosome arms 18p, 19q, 20q, and Yp. PAI were most commonly reported in chromosomes 4, 16, 17, 18, 19, 20, 21, 22, and Y. Inversions were most common in chromosome arms 6p, 7q, 11q, and 14q and observed least in chromosome arms 2p, 2q, 3q, 4q, and 6q. Frequently encountered breakpoints included 3(p13p25), 6(p12p23),more » 6(p12p25), 7(q11q22), and 11(q21q23). Ascertainment was primarily incidental (54.5%), mental retardation and/or congenital anomalies (22.2%), spontaneous abortions (11.4%), associations with syndromes (3.0%), and infertility (2.0%) accounted for the remainder. Ascertainment was neither related to the length of the inverted segment nor to specific inversions except for PAI of Xq which often presented with manifestations of Ullrich-Turner syndrome. Sixty-six percent of PAI were inherited while 8.5% were de novo. Recombination was observed in 17 cases, 15 of which resulted in a monocentric chromosomal deletion or duplication. No common factors were identified that suggested a tendency toward recombination. The incidence of viable recombinants was estimated to be 3.8%. This review documents that PAI are perhaps more commonly identified than suggested in previous reviews. Despite the possible bias of ascertainment in some cases, there may be associated risks with PAI that require further examination. Our data suggest that PAI carriers do not appear to be free of risks of abnormalities or abnormal progeny and caution is recommended when counseling. 162 refs., 4 figs., 7 tabs.« less

Using Recombinant Lactococci as an Approach to Dissect the Immunomodulating Capacity of Surface Piliation in Probiotic Lactobacillus rhamnosus GG

PubMed Central

Nummenmaa, Elina; Mäkinen, Veli-Matti; Reunanen, Justus; Satokari, Reetta; de Vos, Willem M.; Palva, Ilkka; Palva, Airi

2013-01-01

Primarily arising from their well understood beneficial health effects, many lactobacilli strains are considered good candidates for use as probiotics in humans and animals. Lactobacillar probiosis can itself be best typified by the Lactobacillus rhamnosus GG strain, which, with its well-documented clinical benefits, has emerged as one of the most widely used probiotics in the food and health-supplement industries. Even so, many facets of its molecular mechanisms and limitations as a beneficial commensal bacterium still remain to be thoroughly explored and dissected. Because L. rhamnosus GG is one of only a few such strains exhibiting surface piliation (called SpaCBA), we sought to examine whether this particular type of cell-surface appendage has a discernible immunomodulating capacity and is able to trigger targeted responses in human immune-related cells. Thus, presented herein for this study, we recombinantly engineered Lactococcus lactis to produce native (and pilin-deleted) SpaCBA pili that were assembled in a structurally authentic form and anchored to the cell surface, and which had retained mucus-binding functionality. By using these recombinant lactococcal constructs, we were able to demonstrate that the SpaCBA pilus can be a contributory factor in the activation of Toll-like receptor 2-dependent signaling in HEK cells as well as in the modulation of pro- and anti-inflammatory cytokine (TNF-α, IL-6, IL-10, and IL-12) production in human monocyte-derived dendritic cells. From these data, we suggest that the recombinant-expressed and surface-anchored SpaCBA pilus, given its projected functioning in the gut environment, might be viewed as a new microbe-associated molecular pattern (MAMP)-like modulator of innate immunity. Accordingly, our study has brought some new insight to the molecular immunogenicity of the SpaCBA pilus, thus opening the way to a better understanding of its possible role in the multifaceted nature of L. rhamnosus GG probiosis within the human gut. PMID:23691212

Imperatorin inhibits HIV-1 replication through an Sp1-dependent pathway.

PubMed

Sancho, Rocío; Márquez, Nieves; Gómez-Gonzalo, Marta; Calzado, Marco A; Bettoni, Giorgio; Coiras, Maria Teresa; Alcamí, José; López-Cabrera, Manuel; Appendino, Giovanni; Muñoz, Eduardo

2004-09-03

Coumarins and structurally related compounds have been recently shown to present anti-human immunodeficiency virus, type 1 (HIV-1) activity. Among them, the dietary furanocoumarin imperatorin is present in citrus fruits, in culinary herbs, and in some medicinal plants. In this study we report that imperatorin inhibits either vesicular stomatitis virus-pseudotyped or gp160-enveloped recombinant HIV-1 infection in several T cell lines and in HeLa cells. These recombinant viruses express luciferase as a marker of viral replication. Imperatorin did not inhibit the reverse transcription nor the integration steps in the viral cell cycle. Using several 5' long terminal repeat-HIV-1 constructs where critical response elements were either deleted or mutated, we found that the transcription factor Sp1 is critical for the inhibitory activity of imperatorin induced by both phorbol 12-myristate 13-acetate and HIV-1 Tat. Moreover in transient transfections imperatorin specifically inhibited phorbol 12-myristate 13-acetate-induced transcriptional activity of the Gal4-Sp1 fusion protein. Since Sp1 is also implicated in cell cycle progression we further studied the effect of imperatorin on cyclin D1 gene transcription and protein expression and in HeLa cell cycle progression. We found that imperatorin strongly inhibited cyclin D1 expression and arrested the cells at the G(1) phase of the cell cycle. These results highlight the potential of Sp1 transcription factor as a target for natural anti-HIV-1 compounds such as furanocoumarins that might have a potential therapeutic role in the management of AIDS.

Identification of novel extracellular protein for PCB/biphenyl metabolism in Rhodococcus jostii RHA1.

PubMed

Atago, Yuki; Shimodaira, Jun; Araki, Naoto; Bin Othman, Nor'azizi; Zakaria, Zuriati; Fukuda, Masao; Futami, Junichiro; Hara, Hirofumi

2016-05-01

Rhodococcus jostii RHA1 (RHA1) degrades polychlorinated biphenyl (PCB) via co-metabolism with biphenyl. To identify the novel open reading frames (ORFs) that contribute to PCB/biphenyl metabolism in RHA1, we compared chromatin immunoprecipitation chip and transcriptomic data. Six novel ORFs involved in PCB/biphenyl metabolism were identified. Gene deletion mutants of these 6 ORFs were made and were tested for their ability to grow on biphenyl. Interestingly, only the ro10225 deletion mutant showed deficient growth on biphenyl. Analysis of Ro10225 protein function showed that growth of the ro10225 deletion mutant on biphenyl was recovered when exogenous recombinant Ro10225 protein was added to the culture medium. Although Ro10225 protein has no putative secretion signal sequence, partially degraded Ro10225 protein was detected in conditioned medium from wild-type RHA1 grown on biphenyl. This Ro10225 fragment appeared to form a complex with another PCB/biphenyl oxidation enzyme. These results indicated that Ro10225 protein is essential for the formation of the PCB/biphenyl dioxygenase complex in RHA1.

STS map of genes and anonymous DNA fragments on human chromosome 18 using a panel of somatic cell hybrids

DOE Office of Scientific and Technical Information (OSTI.GOV)

Overhauser, J.; Mewar, R.; Rojas, K.

1993-02-01

Somatic cell hybrids containing different deleted regions of chromosome 18 derived form patients with balanced translocations or terminal deletions were used to create a deletion mapping panel. Twenty-four sequence-tagged sites (STSs) for 17 genes and 7 anonymous polymorphic DNA fragments were identified. These STSs were used to map the 24 loci to 18 defined regions of chromosome 18. Both ERV1, previously mapped to 18q22-q23, and YES1, previously mapped to 18q21.3, were found to map to 18p11.21-pter. Several genes previously mapped to 18q21 were found to be in the order cen-SSAV1-DCC-FECH-GRP-BCL2-PLANH2-tel. The precise mapping of genes to chromosome 18 should helpmore » in determining whether these genes may be involved in the etiology of specific chromosomal syndromes associated with chromosome 18. The mapping of the poloymorphic loci will assist in the integration of the physical map with the recombination map of chromosome 18. 43 refs., 2 figs., 1 tab.« less

Deletion of the thymidine kinase gene induces complete attenuation of the Georgia isolate of African swine fever virus.

PubMed

Sanford, B; Holinka, L G; O'Donnell, V; Krug, P W; Carlson, J; Alfano, M; Carrillo, C; Wu, Ping; Lowe, Andre; Risatti, G R; Gladue, D P; Borca, M V

2016-02-02

African swine fever virus (ASFV) is the etiological agent of a contagious and often lethal viral disease of domestic pigs. There are no vaccines to control Africa swine fever (ASF). Experimental vaccines have been developed using genetically modified live attenuated ASFVs obtained by specifically deleting virus genes involved in virulence, including the thymidine kinase (TK) gene. TK has been shown to be involved in the virulence of several viruses, including ASFV. Here we report the construction of a recombinant virus (ASFV-G/V-ΔTK) obtained by deleting the TK gene in a virulent strain of ASFV Georgia adapted to replicate in Vero cells (ASFV-G/VP30). ASFV-G/P-ΔTK demonstrated decreased replication both in primary swine macrophage cell cultures and in Vero cells compared with ASFV-G/VP30. In vivo, intramuscular administration of up to 10(6) TCID50 of ASFV-G/V-ΔTK does not result in ASF disease. However, these animals are not protected when challenged with the virulent parental Georgia strain. Published by Elsevier B.V.

Interchromosomal recombination in Zea mays.

PubMed Central

Hu, W; Timmermans, M C; Messing, J

1998-01-01

A new allele of the 27-kD zein locus in maize has been generated by interchromosomal recombination between chromosomes of two different inbred lines. A continuous patch of at least 11,817 bp of inbred W64A, containing the previously characterized Ra allele of the 27-kD zein gene, has been inserted into the genome of A188 by a single crossover. While both junction sequences are conserved, sequences of the two homologs between these junctions differ considerably. W64A contains the 7313-bp-long retrotransposon, Zeon-1. A188 contains a second copy of the 27-kD zein gene and a 2-kb repetitive element. Therefore, recombination results in a 7.3-kb insertion and a 14-kb deletion compared to the original S+A188 allele. If nonpairing sequences are looped out, 206 single base changes, frequently clustered, are present. The structure of this allele may explain how a recently discovered example of somatic recombination occurred in an A188/W64A hybrid. This would indicate that despite these sequence differences, pairing between these alleles could occur early during plant development. Therefore, such a somatically derived chimeric chromosome can also be heritable and give rise to new alleles. PMID:9799274

Recombination Can Cause Telomere Elongations as Well as Truncations Deep within Telomeres in Wild-Type Kluyveromyces lactis Cells ▿

PubMed Central

Bechard, Laura H.; Jamieson, Nathan; McEachern, Michael J.

2011-01-01

In this study, we examined the role of recombination at the telomeres of the yeast Kluyveromyces lactis. We demonstrated that an abnormally long and mutationally tagged telomere was subject to high rates of telomere rapid deletion (TRD) that preferentially truncated the telomere to near-wild-type size. Unlike the case in Saccharomyces cerevisiae, however, there was not a great increase in TRD in meiosis. About half of mitotic TRD events were associated with deep turnover of telomeric repeats, suggesting that telomeres were often cleaved to well below normal length prior to being reextended by telomerase. Despite its high rate of TRD, the long telomere showed no increase in the rate of subtelomeric gene conversion, a highly sensitive test of telomere dysfunction. We also showed that the long telomere was subject to appreciable rates of becoming elongated substantially further through a recombinational mechanism that added additional tagged repeats. Finally, we showed that the deep turnover that occurs within normal-length telomeres was diminished in the absence of RAD52. Taken together, our results suggest that homologous recombination is a significant process acting on both abnormally long and normally sized telomeres in K. lactis. PMID:21148753

The Bxb1 recombination system demonstrates heritable transmission of site-specific excision in Arabidopsis

PubMed Central

2012-01-01

Background The mycobacteriophage large serine recombinase Bxb1 catalyzes site-specific recombination between its corresponding attP and attB recognition sites. Previously, we and others have shown that Bxb1 has catalytic activity in various eukaryotic species including Nicotiana tabacum, Schizosaccharomyces pombe, insects and mammalian cells. Results In this work, the Bxb1 recombinase gene was transformed and constitutively expressed in Arabidopsis thaliana plants harboring a chromosomally integrated attP and attB-flanked target sequence. The Bxb1 recombinase successfully excised the target sequence in a conservative manner and the resulting recombination event was heritably transmitted to subsequent generations in the absence of the recombinase transgene. In addition, we also show that Bxb1 recombinase expressing plants can be manually crossed with att-flanked target transgenic plants to generate excised progeny. Conclusion The Bxb1 large serine recombinase performs site-specific recombination in Arabidopsis thaliana germinal tissue, producing stable lines free of unwanted DNA. The precise site-specific deletion produced by Bxb1 in planta demonstrates that this enzyme can be a useful tool for the genetic engineering of plants without selectable marker transgenes or other undesirable exogenous sequences. PMID:22436504

Specialized Genetic Recombination Systems in Bacteria: Their Involvement in Gene Expression and Evolution,

DTIC Science & Technology

1980-01-01

genetics (Hayes 1968). This marvelous process is important in providing us with the breadth of phenotypic diversity that one sees within a single plant or...separate overall pro- cesses, but may share common components of DNA metabolism, such as winding/unwinding enzymes, ligase, polymerases , various nucle...incorpuoted DNA segmnent are re- paired by DNA polymerase and ligase. Any diffoernces (base mispairing’S, nil- cleotide additions or deletions) between

Homologous and heterologous recombination between adenovirus vector DNA and chromosomal DNA.

PubMed

Stephen, Sam Laurel; Sivanandam, Vijayshankar Ganesh; Kochanek, Stefan

2008-11-01

Adenovirus vector DNA is perceived to remain as episome following gene transfer. We quantitatively and qualitatively analysed recombination between high capacity adenoviral vector (HC-AdV) and chromosomal DNA following gene transfer in vitro. We studied homologous and heterologous recombination with a single HC-AdV carrying (i) a large genomic HPRT fragment with the HPRT CHICAGO mutation causing translational stop upon homologous recombination with the HPRT locus and (ii) a selection marker to allow for clonal selection in the event of heterologous recombination. We analysed the sequences at the junctions between vector and chromosomal DNA. In primary cells and in cell lines, the frequency of homologous recombination ranged from 2 x 10(-5) to 1.6 x 10(-6). Heterologous recombination occurred at rates between 5.5 x 10(-3) and 1.1 x 10(-4). HC-AdV DNA integrated via the termini mostly as intact molecules. Analysis of the junction sequences indicated vector integration in a relatively random manner without an obvious preference for particular chromosomal regions, but with a preference for integration into genes. Integration into protooncogenes or tumor suppressor genes was not observed. Patchy homologies between vector termini and chromosomal DNA were found at the site of integration. Although the majority of integrations had occurred without causing mutations in the chromosomal DNA, cases of nucleotide substitutions and insertions were observed. In several cases, deletions of even relative large chromosomal regions were likely. These results extend previous information on the integration patterns of adenovirus vector DNA and contribute to a risk-benefit assessment of adenovirus-mediated gene transfer.

The SMC-5/6 Complex and the HIM-6 (BLM) Helicase Synergistically Promote Meiotic Recombination Intermediate Processing and Chromosome Maturation during Caenorhabditis elegans Meiosis

PubMed Central

Hong, Ye; Sonneville, Remi; Agostinho, Ana; Meier, Bettina; Wang, Bin; Blow, J. Julian; Gartner, Anton

2016-01-01

Meiotic recombination is essential for the repair of programmed double strand breaks (DSBs) to generate crossovers (COs) during meiosis. The efficient processing of meiotic recombination intermediates not only needs various resolvases but also requires proper meiotic chromosome structure. The Smc5/6 complex belongs to the structural maintenance of chromosome (SMC) family and is closely related to cohesin and condensin. Although the Smc5/6 complex has been implicated in the processing of recombination intermediates during meiosis, it is not known how Smc5/6 controls meiotic DSB repair. Here, using Caenorhabditis elegans we show that the SMC-5/6 complex acts synergistically with HIM-6, an ortholog of the human Bloom syndrome helicase (BLM) during meiotic recombination. The concerted action of the SMC-5/6 complex and HIM-6 is important for processing recombination intermediates, CO regulation and bivalent maturation. Careful examination of meiotic chromosomal morphology reveals an accumulation of inter-chromosomal bridges in smc-5; him-6 double mutants, leading to compromised chromosome segregation during meiotic cell divisions. Interestingly, we found that the lethality of smc-5; him-6 can be rescued by loss of the conserved BRCA1 ortholog BRC-1. Furthermore, the combined deletion of smc-5 and him-6 leads to an irregular distribution of condensin and to chromosome decondensation defects reminiscent of condensin depletion. Lethality conferred by condensin depletion can also be rescued by BRC-1 depletion. Our results suggest that SMC-5/6 and HIM-6 can synergistically regulate recombination intermediate metabolism and suppress ectopic recombination by controlling chromosome architecture during meiosis. PMID:27010650

The SMC-5/6 Complex and the HIM-6 (BLM) Helicase Synergistically Promote Meiotic Recombination Intermediate Processing and Chromosome Maturation during Caenorhabditis elegans Meiosis.

PubMed

Hong, Ye; Sonneville, Remi; Agostinho, Ana; Meier, Bettina; Wang, Bin; Blow, J Julian; Gartner, Anton

2016-03-01

Meiotic recombination is essential for the repair of programmed double strand breaks (DSBs) to generate crossovers (COs) during meiosis. The efficient processing of meiotic recombination intermediates not only needs various resolvases but also requires proper meiotic chromosome structure. The Smc5/6 complex belongs to the structural maintenance of chromosome (SMC) family and is closely related to cohesin and condensin. Although the Smc5/6 complex has been implicated in the processing of recombination intermediates during meiosis, it is not known how Smc5/6 controls meiotic DSB repair. Here, using Caenorhabditis elegans we show that the SMC-5/6 complex acts synergistically with HIM-6, an ortholog of the human Bloom syndrome helicase (BLM) during meiotic recombination. The concerted action of the SMC-5/6 complex and HIM-6 is important for processing recombination intermediates, CO regulation and bivalent maturation. Careful examination of meiotic chromosomal morphology reveals an accumulation of inter-chromosomal bridges in smc-5; him-6 double mutants, leading to compromised chromosome segregation during meiotic cell divisions. Interestingly, we found that the lethality of smc-5; him-6 can be rescued by loss of the conserved BRCA1 ortholog BRC-1. Furthermore, the combined deletion of smc-5 and him-6 leads to an irregular distribution of condensin and to chromosome decondensation defects reminiscent of condensin depletion. Lethality conferred by condensin depletion can also be rescued by BRC-1 depletion. Our results suggest that SMC-5/6 and HIM-6 can synergistically regulate recombination intermediate metabolism and suppress ectopic recombination by controlling chromosome architecture during meiosis.

[Asymmetric effects of addition versus deletion on change detection task: the role of feeling of something strange].

PubMed

Uchino, Yashio; Hakoda, Yuji; Shibata, Mariko

2005-06-01

Two experiments were conducted to examine the asymmetric effect of alterations (i.e., addition versus deletion) on recognition memory. In Experiment 1, a scale for measuring the FSS (Feeling of Something Strange) was developed (n=50) using added or deleted pictures from previous research (e.g., Uchino, Hakoda, & Yamada, 2000). Result showed that altered pictures were evaluated by "pleasant" and "odd" factors. In Experiment 2, 80 participants observed 20 pictures, and then they answered whether each test picture was altered or not. Test pictures varied in significance of the objects added or deleted on a scene. Additions were detected more easily than deletions only when added object was unexpected or unusual, while deleted object was essential to a scene (TD: typicality-disrupted condition). Then, 60 participants rated the FSS scale for test pictures. Ratings of odd factor for added pictures were higher than deleted pictures presented in the TD condition. These results suggest that superiority of addition over deletion might be due to their different effect on FSS.

Myotubular Myopathy in a girl with a deletion of Xq27-q28 and unbalanced X inactivation assigns the MTM1 gene to a 600-kb region

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dahl, N.; Mandel, J.L.; Chery, M.

1995-05-01

A young girl with a clinically moderate form of myotubular myopathy was found to carry a cytogenetically detectable deletion in Xq27-q28. The deletion had occurred de novo on the paternal X chromosome. It encompasses the fragile X (FRAXA) and Hunter syndrome (IDS) loci, and the DXS304 and DXS455 markers, in Xq27.3 and proximal Xq28. Other loci from the proximal half of Xq28 (DXS49, DXS256, DXS258, DXS305, and DXS497) were found intact. As the X-linked myotubular myopathy locus (MTM1) was previously mapped to Xq28 by linkage analysis, the present observation suggested that MTM1 is included in the deletion. However, a significantmore » clinical phenotype is unexpected in a female MTM1 carrier. Analysis of inactive X-specific methylation at the androgen receptor gene showed that the deleted X chromosome was active in {approximately}80% of leukocytes. Such unbalanced inactivation may account for the moderate MTM1 phenotype and for the mental retardation that later developed in the patient. This observation is discussed in relation to the hypothesis that a locus modulating X inactivation may lie in the region. Comparison of this deletion with that carried by a male patient with a severe Hunter syndrome phenotype but no myotubular myopathy, in light of recent linkage data on recombinant MTM1 families, led to a considerable refinement of the position of the MTM1 locus, to a region of {approximately}600 kb, between DXS304 and DXS497. 46 refs., 4 figs.« less

DNA Sequences Proximal to Human Mitochondrial DNA Deletion Breakpoints Prevalent in Human Disease Form G-quadruplexes, a Class of DNA Structures Inefficiently Unwound by the Mitochondrial Replicative Twinkle Helicase*

PubMed Central

Bharti, Sanjay Kumar; Sommers, Joshua A.; Zhou, Jun; Kaplan, Daniel L.; Spelbrink, Johannes N.; Mergny, Jean-Louis; Brosh, Robert M.

2014-01-01

Mitochondrial DNA deletions are prominent in human genetic disorders, cancer, and aging. It is thought that stalling of the mitochondrial replication machinery during DNA synthesis is a prominent source of mitochondrial genome instability; however, the precise molecular determinants of defective mitochondrial replication are not well understood. In this work, we performed a computational analysis of the human mitochondrial genome using the “Pattern Finder” G-quadruplex (G4) predictor algorithm to assess whether G4-forming sequences reside in close proximity (within 20 base pairs) to known mitochondrial DNA deletion breakpoints. We then used this information to map G4P sequences with deletions characteristic of representative mitochondrial genetic disorders and also those identified in various cancers and aging. Circular dichroism and UV spectral analysis demonstrated that mitochondrial G-rich sequences near deletion breakpoints prevalent in human disease form G-quadruplex DNA structures. A biochemical analysis of purified recombinant human Twinkle protein (gene product of c10orf2) showed that the mitochondrial replicative helicase inefficiently unwinds well characterized intermolecular and intramolecular G-quadruplex DNA substrates, as well as a unimolecular G4 substrate derived from a mitochondrial sequence that nests a deletion breakpoint described in human renal cell carcinoma. Although G4 has been implicated in the initiation of mitochondrial DNA replication, our current findings suggest that mitochondrial G-quadruplexes are also likely to be a source of instability for the mitochondrial genome by perturbing the normal progression of the mitochondrial replication machinery, including DNA unwinding by Twinkle helicase. PMID:25193669

Deletion of alpha-synuclein decreases impulsivity in mice.

PubMed

Peña-Oliver, Y; Buchman, V L; Dalley, J W; Robbins, T W; Schumann, G; Ripley, T L; King, S L; Stephens, D N

2012-03-01

The presynaptic protein alpha-synuclein, associated with Parkinson's Disease (PD), plays a role in dopaminergic neurotransmission and is implicated in impulse control disorders (ICDs) such as drug addiction. In this study we investigated a potential causal relationship between alpha-synuclein and impulsivity, by evaluating differences in motor impulsivity in the 5-choice serial reaction time task (5-CSRTT) in strains of mice that differ in the expression of the alpha-synuclein gene. C57BL/6JOlaHsd mice differ from their C57BL/6J ancestors in possessing a chromosomal deletion resulting in the loss of two genes, snca, encoding alpha-synuclein, and mmrn1, encoding multimerin-1. C57BL/6J mice displayed higher impulsivity (more premature responding) than C57BL/6JOlaHsd mice when the pre-stimulus waiting interval was increased in the 5-CSRTT. In order to ensure that the reduced impulsivity was indeed related to snca, and not adjacent gene deletion, wild type (WT) and mice with targeted deletion of alpha-synuclein (KO) were tested in the 5-CSRTT. Similarly, WT mice were more impulsive than mice with targeted deletion of alpha-synuclein. Interrogation of our ongoing analysis of impulsivity in BXD recombinant inbred mouse lines revealed an association of impulsive responding with levels of alpha-synuclein expression in hippocampus. Expression of beta- and gamma-synuclein, members of the synuclein family that may substitute for alpha-synuclein following its deletion, revealed no differential compensations among the mouse strains. These findings suggest that alpha-synuclein may contribute to impulsivity and potentially, to ICDs which arise in some PD patients treated with dopaminergic medication. © 2011 The Authors. Genes, Brain and Behavior © 2011 Blackwell Publishing Ltd and International Behavioural and Neural Genetics Society.

Paracentric inversion of chromosome 2 associated with cryptic duplication of 2q14 and deletion of 2q37 in a patient with autism.

PubMed

Devillard, Françoise; Guinchat, Vincent; Moreno-De-Luca, Daniel; Tabet, Anne-Claude; Gruchy, Nicolas; Guillem, Pascale; Nguyen Morel, Marie-Ange; Leporrier, Nathalie; Leboyer, Marion; Jouk, Pierre-Simon; Lespinasse, James; Betancur, Catalina

2010-09-01

We describe a patient with autism and a paracentric inversion of chromosome 2q14.2q37.3, with a concurrent duplication of the proximal breakpoint at 2q14.1q14.2 and a deletion of the distal breakpoint at 2q37.3. The abnormality was derived from his mother with a balanced paracentric inversion. The inversion in the child appeared to be cytogenetically balanced but subtelomere FISH revealed a cryptic deletion at the 2q37.3 breakpoint. High-resolution single nucleotide polymorphism array confirmed the presence of a 3.5 Mb deletion that extended to the telomere, and showed a 4.2 Mb duplication at 2q14.1q14.2. FISH studies using a 2q14.2 probe showed that the duplicated segment was located at the telomeric end of chromosome 2q. This recombinant probably resulted from breakage of a dicentric chromosome. The child had autism, mental retardation, speech and language delay, hyperactivity, growth retardation with growth hormone deficiency, insulin-dependent diabetes, and mild facial dysmorphism. Most of these features have been previously described in individuals with simple terminal deletion of 2q37. Pure duplications of the proximal chromosome 2q are rare and no specific syndrome has been defined yet, so the contribution of the 2q14.1q14.2 duplication to the phenotype of the patient is unknown. These findings underscore the need to explore apparently balanced chromosomal rearrangements inherited from a phenotypically normal parent in subjects with autism and/or developmental delay. In addition, they provide further evidence indicating that chromosome 2q terminal deletions are among the most frequently reported cytogenetic abnormalities in individuals with autism.

Recurrent Distal 7q11.23 Deletion Including HIP1 and YWHAG Identified in Patients with Intellectual Disabilities, Epilepsy, and Neurobehavioral Problems

PubMed Central

Ramocki, Melissa B.; Bartnik, Magdalena; Szafranski, Przemyslaw; Kołodziejska, Katarzyna E.; Xia, Zhilian; Bravo, Jaclyn; Miller, G. Steve; Rodriguez, Diana L.; Williams, Charles A.; Bader, Patricia I.; Szczepanik, Elżbieta; Mazurczak, Tomasz; Antczak-Marach, Dorota; Coldwell, James G.; Akman, Cigdem I.; McAlmon, Karen; Cohen, Melinda P.; McGrath, James; Roeder, Elizabeth; Mueller, Jennifer; Kang, Sung-Hae L.; Bacino, Carlos A.; Patel, Ankita; Bocian, Ewa; Shaw, Chad A.; Cheung, Sau Wai; Mazurczak, Tadeusz; Stankiewicz, Paweł

2010-01-01

We report 26 individuals from ten unrelated families who exhibit variable expression and/or incomplete penetrance of epilepsy, learning difficulties, intellectual disabilities, and/or neurobehavioral abnormalities as a result of a heterozygous microdeletion distally adjacent to the Williams-Beuren syndrome region on chromosome 7q11.23. In six families with a common recurrent ∼1.2 Mb deletion that includes the Huntingtin-interacting protein 1 (HIP1) and tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein gamma (YWHAG) genes and that is flanked by large complex low-copy repeats, we identified sites for nonallelic homologous recombination in two patients. There were no cases of this ∼1.2 Mb distal 7q11.23 deletion copy number variant identified in over 20,000 control samples surveyed. Three individuals with smaller, nonrecurrent deletions (∼180–500 kb) that include HIP1 but not YWHAG suggest that deletion of HIP1 is sufficient to cause neurological disease. Mice with targeted mutation in the Hip1 gene (Hip1−/−) develop a neurological phenotype characterized by failure to thrive, tremor, and gait ataxia. Overall, our data characterize a neurodevelopmental and epilepsy syndrome that is likely caused by recurrent and nonrecurrent deletions, including HIP1. These data do not exclude the possibility that YWHAG loss of function is also sufficient to cause neurological phenotypes. Based on the current knowledge of Hip1 protein function and its proposed role in AMPA and NMDA ionotropic glutamate receptor trafficking, we believe that HIP1 haploinsufficiency in humans will be amenable to rational drug design for improved seizure control and cognitive and behavioral function. PMID:21109226

Recurrent distal 7q11.23 deletion including HIP1 and YWHAG identified in patients with intellectual disabilities, epilepsy, and neurobehavioral problems.

PubMed

Ramocki, Melissa B; Bartnik, Magdalena; Szafranski, Przemyslaw; Kołodziejska, Katarzyna E; Xia, Zhilian; Bravo, Jaclyn; Miller, G Steve; Rodriguez, Diana L; Williams, Charles A; Bader, Patricia I; Szczepanik, Elżbieta; Mazurczak, Tomasz; Antczak-Marach, Dorota; Coldwell, James G; Akman, Cigdem I; McAlmon, Karen; Cohen, Melinda P; McGrath, James; Roeder, Elizabeth; Mueller, Jennifer; Kang, Sung-Hae L; Bacino, Carlos A; Patel, Ankita; Bocian, Ewa; Shaw, Chad A; Cheung, Sau Wai; Mazurczak, Tadeusz; Stankiewicz, Paweł

2010-12-10

We report 26 individuals from ten unrelated families who exhibit variable expression and/or incomplete penetrance of epilepsy, learning difficulties, intellectual disabilities, and/or neurobehavioral abnormalities as a result of a heterozygous microdeletion distally adjacent to the Williams-Beuren syndrome region on chromosome 7q11.23. In six families with a common recurrent ∼1.2 Mb deletion that includes the Huntingtin-interacting protein 1 (HIP1) and tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein gamma (YWHAG) genes and that is flanked by large complex low-copy repeats, we identified sites for nonallelic homologous recombination in two patients. There were no cases of this ∼1.2 Mb distal 7q11.23 deletion copy number variant identified in over 20,000 control samples surveyed. Three individuals with smaller, nonrecurrent deletions (∼180-500 kb) that include HIP1 but not YWHAG suggest that deletion of HIP1 is sufficient to cause neurological disease. Mice with targeted mutation in the Hip1 gene (Hip1⁻(/)⁻) develop a neurological phenotype characterized by failure to thrive, tremor, and gait ataxia. Overall, our data characterize a neurodevelopmental and epilepsy syndrome that is likely caused by recurrent and nonrecurrent deletions, including HIP1. These data do not exclude the possibility that YWHAG loss of function is also sufficient to cause neurological phenotypes. Based on the current knowledge of Hip1 protein function and its proposed role in AMPA and NMDA ionotropic glutamate receptor trafficking, we believe that HIP1 haploinsufficiency in humans will be amenable to rational drug design for improved seizure control and cognitive and behavioral function. Copyright © 2010 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

Protective Immunity and Safety of a Genetically Modified Influenza Virus Vaccine

PubMed Central

Garcia, Cristiana Couto; Filho, Bruno Galvão; Gonçalves, Ana Paula de Faria; Lima, Braulio Henrique Freire; Lopes, Gabriel Augusto Oliveira; Rachid, Milene Alvarenga; Peixoto, Andiara Cristina Cardoso; de Oliveira, Danilo Bretas; Ataíde, Marco Antônio; Zirke, Carla Aparecida; Cotrim, Tatiane Marques; Costa, Érica Azevedo; Almeida, Gabriel Magno de Freitas; Russo, Remo Castro; Gazzinelli, Ricardo Tostes; Machado, Alexandre de Magalhães Vieira

2014-01-01

Recombinant influenza viruses are promising viral platforms to be used as antigen delivery vectors. To this aim, one of the most promising approaches consists of generating recombinant viruses harboring partially truncated neuraminidase (NA) segments. To date, all studies have pointed to safety and usefulness of this viral platform. However, some aspects of the inflammatory and immune responses triggered by those recombinant viruses and their safety to immunocompromised hosts remained to be elucidated. In the present study, we generated a recombinant influenza virus harboring a truncated NA segment (vNA-Δ) and evaluated the innate and inflammatory responses and the safety of this recombinant virus in wild type or knock-out (KO) mice with impaired innate (Myd88 -/-) or acquired (RAG -/-) immune responses. Infection using truncated neuraminidase influenza virus was harmless regarding lung and systemic inflammatory response in wild type mice and was highly attenuated in KO mice. We also demonstrated that vNA-Δ infection does not induce unbalanced cytokine production that strongly contributes to lung damage in infected mice. In addition, the recombinant influenza virus was able to trigger both local and systemic virus-specific humoral and CD8+ T cellular immune responses which protected immunized mice against the challenge with a lethal dose of homologous A/PR8/34 influenza virus. Taken together, our findings suggest and reinforce the safety of using NA deleted influenza viruses as antigen delivery vectors against human or veterinary pathogens. PMID:24927156

Din7 and Mhr1 expression levels regulate double-strand-break–induced replication and recombination of mtDNA at ori5 in yeast

PubMed Central

Ling, Feng; Hori, Akiko; Yoshitani, Ayako; Niu, Rong; Yoshida, Minoru; Shibata, Takehiko

2013-01-01

The Ntg1 and Mhr1 proteins initiate rolling-circle mitochondrial (mt) DNA replication to achieve homoplasmy, and they also induce homologous recombination to maintain mitochondrial genome integrity. Although replication and recombination profoundly influence mitochondrial inheritance, the regulatory mechanisms that determine the choice between these pathways remain unknown. In Saccharomyces cerevisiae, double-strand breaks (DSBs) introduced by Ntg1 at the mitochondrial replication origin ori5 induce homologous DNA pairing by Mhr1, and reactive oxygen species (ROS) enhance production of DSBs. Here, we show that a mitochondrial nuclease encoded by the nuclear gene DIN7 (DNA damage inducible gene) has 5′-exodeoxyribonuclease activity. Using a small ρ− mtDNA bearing ori5 (hypersuppressive; HS) as a model mtDNA, we revealed that DIN7 is required for ROS-enhanced mtDNA replication and recombination that are both induced at ori5. Din7 overproduction enhanced Mhr1-dependent mtDNA replication and increased the number of residual DSBs at ori5 in HS-ρ− cells and increased deletion mutagenesis at the ori5 region in ρ+ cells. However, simultaneous overproduction of Mhr1 suppressed all of these phenotypes and enhanced homologous recombination. Our results suggest that after homologous pairing, the relative activity levels of Din7 and Mhr1 modulate the preference for replication versus homologous recombination to repair DSBs at ori5. PMID:23598996

Major psychological factors affecting acceptance of gene-recombination technology.

PubMed

Tanaka, Yutaka

2004-12-01

The purpose of this study was to verify the validity of a causal model that was made to predict the acceptance of gene-recombination technology. A structural equation model was used as a causal model. First of all, based on preceding studies, the factors of perceived risk, perceived benefit, and trust were set up as important psychological factors determining acceptance of gene-recombination technology in the structural equation model. An additional factor, "sense of bioethics," which I consider to be important for acceptance of biotechnology, was added to the model. Based on previous studies, trust was set up to have an indirect influence on the acceptance of gene-recombination technology through perceived risk and perceived benefit in the model. Participants were 231 undergraduate students in Japan who answered a questionnaire with a 5-point bipolar scale. The results indicated that the proposed model fits the data well, and showed that acceptance of gene-recombination technology is explained largely by four factors, that is, perceived risk, perceived benefit, trust, and sense of bioethics, whether the technology is applied to plants, animals, or human beings. However, the relative importance of the four factors was found to vary depending on whether the gene-recombination technology was applied to plants, animals, or human beings. Specifically, the factor of sense of bioethics is the most important factor in acceptance of plant gene-recombination technology and animal gene-recombination technology, and the factors of trust and perceived risk are the most important factors in acceptance of human being gene-recombination technology.

MNF, an ankyrin repeat protein of myxoma virus, is part of a native cellular SCF complex during viral infection

PubMed Central

2010-01-01

Myxoma virus (MYXV), a member of the Poxviridae family, is the agent responsible for myxomatosis, a fatal disease in the European rabbit (Oryctolagus cuniculus). Like all poxviruses, MYXV is known for encoding multiple proteins that regulate cellular signaling pathways. Among them, four proteins share the same ANK/PRANC structure: M148R, M149R, MNF (Myxoma Nuclear factor) and M-T5, all of them described as virulence factors. This family of poxvirus proteins, recently identified, has drawn considerable attention for its potential role in modulating the host ubiquitin-proteasome system during viral infection. To date, many members of this novel protein family have been shown to interact with SCF components, in vitro. Here, we focus on MNF gene, which has been shown to express a nuclear protein presenting nine ANK repeats, one of which has been identified as a nuclear localization signal. In transfection, MNF has been shown to colocalise with the transcription factor NF-κB in the nucleus of TNFα-stimulated cells. Functionally, MNF is a critical virulence factor since its deletion generates an almost apathogenic virus. In this study, to pursue the investigation of proteins interacting with MNF and of its mechanism of action, we engineered a recombinant MYXV expressing a GFP-linked MNF under the control of MNF native promoter. Infection of rabbits with MYXV-GFPMNF recombinant virus provided the evidence that the GFP fusion does not disturb the main function of MNF. Hence, cells were infected with MYXV-GFPMNF and immunoprecipitation of the GFPMNF fusion protein was performed to identify MNF's partners. For the first time, endogenous components of SCF (Cullin-1 and Skp1) were co-precipitated with an ANK myxoma virus protein, expressed in an infectious context, and without over-expression of any protein. PMID:20211013

A recombinant lentiviral PDGF-driven mouse model of proneural glioblastoma.

PubMed

Rahme, Gilbert J; Luikart, Bryan W; Cheng, Chao; Israel, Mark A

2018-02-19

Mouse models of glioblastoma (GBM), the most aggressive primary brain tumor, are critical for understanding GBM pathology and can contribute to the preclinical evaluation of therapeutic agents. Platelet-derived growth factor (PDGF) signaling has been implicated in the development and pathogenesis of GBM, specifically the proneural subtype. Although multiple mouse models of PDGF-driven glioma have been described, they require transgenic mice engineered to activate PDGF signaling and/or impair tumor suppressor genes and typically represent lower-grade glioma. We designed recombinant lentiviruses expressing both PDGFB and a short hairpin RNA targeting Cdkn2a to induce gliomagenesis following stereotactic injection into the dentate gyrus of adult immunocompetent mice. We engineered these viruses to coexpress CreERT2 with PDGFB, allowing for deletion of floxed genes specifically in transduced cells, and designed another version of this recombinant lentivirus in which enhanced green fluorescent protein was coexpressed with PDGFB and CreERT2 to visualize transduced cells. The dentate gyrus of injected mice showed hypercellularity one week post-injection and subsequently developed bona fide tumors with the pathologic hallmarks of GBM leading to a median survival of 77 days post-injection. Transcriptomic analysis of these tumors revealed a proneural gene expression signature. Informed by the genetic alterations observed in human GBM, we engineered a novel mouse model of proneural GBM. While reflecting many of the advantages of transgenic mice, this model allows for the facile in vivo testing of gene function in tumor cells and makes possible the rapid production of large numbers of immunocompetent tumor-bearing mice for preclinical testing of therapeutics. © The Author(s) 2017. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com

Transcription of a recombinant bunyavirus RNA template by transiently expressed bunyavirus proteins.

PubMed

Dunn, E F; Pritlove, D C; Jin, H; Elliott, R M

1995-08-01

We describe a convenient system for analyzing bunyavirus transcription using a recombinant RNA template derived from the plasmid pBUNSCAT, which comprises a negative-sense reporter gene (chloramphenicol acetyltransferase or CAT) flanked by the exact 5' and 3' untranslated regions of the Bunyamwera virus (BUN) S RNA segment. When cells which expressed bunyavirus proteins (either by recombinant vaccinia viruses or by the vaccinia virus-T7 system) were transfected with BUNSCAT RNA, CAT activity could be measured, indicating transcription of the negative-sense reporter RNA into mRNA. The system permits investigation of both the protein and RNA sequence requirements for transcription. Extensions of 2 bases at the 5' end or 11 or 35 bases at the 3' end of BUNSCAT RNA allowed transcription but a lower level than the wild-type template. Deletion of the 5 nucleotides at the 3' end of BUNSCAT RNA reduced CAT activity by > 99%. Investigation of the viral protein requirements of the system showed that only the bunyavirus L and N proteins were needed for CAT activity. The BUN L protein was also able to transcribe the reporter RNA in concert with the N proteins of closely related bunyaviruses such as Batai, Cache Valley, Maguari, Main Drain, and Northway, but only inefficiently with those of Kairi, Guaroa, or Lumbo viruses. When BUN L proteins containing specific mutations were expressed CAT activity was only observed using those mutated L proteins previously reported to be active in a nucleocapsid transfection assay (H. Jin and R. M. Elliott, 1992, J. Gen. Virol. 73, 2235-2244). These results illustrate the utility of this system for a detailed genetic analysis of the factors involved in bunyavirus transcription.

Depletion of ε-COP in the COPI Vesicular Coat Reduces Cleistothecium Production in Aspergillus nidulans.

PubMed

Kang, Eun-Hye; Song, Eun-Jung; Kook, Jun Ho; Lee, Hwan-Hee; Jeong, Bo-Ri; Park, Hee-Moon

2015-03-01

We have previously isolated ε-COP, the α-COP interactor in COPI of Aspergillus nidulans, by yeast two-hybrid screening. To understand the function of ε-COP, the aneA (+) gene for ε-COP/AneA was deleted by homologous recombination using a gene-specific disruption cassette. Deletion of the ε-COP gene showed no detectable changes in vegetative growth or asexual development, but resulted in decrease in the production of the fruiting body, cleistothecium, under conditions favorable for sexual development. Unlike in the budding yeast Saccharomyces cerevisiae, in A. nidulans, over-expression of ε-COP did not rescue the thermo-sensitive growth defect of the α-COP mutant at 42℃. Together, these data show that ε-COP is not essential for viability, but it plays a role in fruiting body formation in A. nidulans.

Delimitation of essential genes of cassava latent virus DNA 2.

PubMed Central

Etessami, P; Callis, R; Ellwood, S; Stanley, J

1988-01-01

Insertion and deletion mutagenesis of both extended open reading frames (ORFs) of cassava latent virus DNA 2 destroys infectivity. Infectivity is restored by coinoculating constructs that contain single mutations within different ORFs. Although frequent intermolecular recombination produces dominant parental-type virus, mutants can be retained within the virus population indicating that they are competent for replication and suggesting that rescue can occur by complementation of trans acting gene products. By cloning specific fragments into DNA 1 coat protein deletion vectors we have delimited the DNA 2 coding regions and provide substantive evidence that both are essential for virus infection. Although a DNA 2 component is unique to whitefly-transmitted geminiviruses, the results demonstrate that neither coding region is involved solely in insect transmission. The requirement for a bipartite genome for whitefly-transmitted geminiviruses is discussed. Images PMID:3387209

Agrobacterium tumefaciens-mediated transformation of oleaginous yeast Lipomyces species.

PubMed

Dai, Ziyu; Deng, Shuang; Culley, David E; Bruno, Kenneth S; Magnuson, Jon K

2017-08-01

Interest in using renewable sources of carbon, especially lignocellulosic biomass, for the production of hydrocarbon fuels and chemicals has fueled interest in exploring various organisms capable of producing hydrocarbon biofuels and chemicals or their precursors. The oleaginous (oil-producing) yeast Lipomyces starkeyi is the subject of active research regarding the production of triacylglycerides as hydrocarbon fuel precursors using a variety of carbohydrate and nutrient sources. The genome of L. starkeyi has been published, which opens the door to production strain improvements through the development and use of the tools of synthetic biology for this oleaginous species. The first step in establishment of synthetic biology tools for an organism is the development of effective and reliable transformation methods with suitable selectable marker genes and demonstration of the utility of the genetic elements needed for expression of introduced genes or deletion of endogenous genes. Chemical-based methods of transformation have been published but suffer from low efficiency. To address these problems, Agrobacterium-mediated transformation was investigated as an alternative method for L. starkeyi and other Lipomyces species. In this study, Agrobacterium-mediated transformation was demonstrated to be effective in the transformation of both L. starkeyi and other Lipomyces species. The deletion of the peroxisomal biogenesis factor 10 gene was also demonstrated in L. starkeyi. In addition to the bacterial antibiotic selection marker gene hygromycin B phosphotransferase, the bacterial β-glucuronidase reporter gene under the control of L. starkeyi translation elongation factor 1α promoter was also stably expressed in six different Lipomyces species. The results from this study demonstrate that Agrobacterium-mediated transformation is a reliable and effective genetic tool for homologous recombination and expression of heterologous genes in L. starkeyi and other Lipomyces species.

African Swine Fever Virus Georgia Isolate Harboring Deletions of MGF360 and MGF505 Genes Is Attenuated in Swine and Confers Protection against Challenge with Virulent Parental Virus

PubMed Central

O'Donnell, Vivian; Holinka, Lauren G.; Gladue, Douglas P.; Sanford, Brenton; Krug, Peter W.; Lu, Xiqiang; Arzt, Jonathan; Reese, Bo; Carrillo, Consuelo; Risatti, Guillermo R.

2015-01-01

ABSTRACT African swine fever virus (ASFV) is the etiological agent of a contagious and often lethal disease of domestic pigs that has significant economic consequences for the swine industry. The control of African swine fever (ASF) has been hampered by the unavailability of vaccines. Experimental vaccines have been developed using genetically modified live attenuated ASFVs where viral genes involved in virus virulence were removed from the genome. Multigene family 360 (MGF360) and MGF505 represent a group of genes sharing partial sequence and structural identities that have been connected with ASFV host range specificity, blocking of the host innate response, and virus virulence. Here we report the construction of a recombinant virus (ASFV-G-ΔMGF) derived from the highly virulent ASFV Georgia 2007 isolate (ASFV-G) by specifically deleting six genes belonging to MGF360 or MGF505: MGF505-1R, MGF360-12L, MGF360-13L, MGF360-14L, MGF505-2R, and MGF505-3R. ASFV-G-ΔMGF replicates as efficiently in primary swine macrophage cell cultures as the parental virus. In vivo, ASFV-G-ΔMGF is completely attenuated in swine, since pigs inoculated intramuscularly (i.m.) with either 102 or 104 50% hemadsorbing doses (HAD50) remained healthy, without signs of the disease. Importantly, when these animals were subsequently exposed to highly virulent parental ASFV-G, no signs of the disease were observed, although a proportion of these animals harbored the challenge virus. This is the first report demonstrating the role of MGF genes acting as independent determinants of ASFV virulence. Additionally, ASFV-G-ΔMGF is the first experimental vaccine reported to induce protection in pigs challenged with highly virulent and epidemiologically relevant ASFV-G. IMPORTANCE The main problem for controlling ASF is the lack of vaccines. Studies focusing on understanding ASFV virulence led to the production of genetically modified recombinant viruses that, while attenuated, are able to confer protection in pigs challenged with homologous viruses. Here we have produced an attenuated recombinant ASFV derived from highly virulent ASFV strain Georgia (ASFV-G) lacking only six of the multigene family 360 (MGF360) and MGF505 genes (ASFV-G-ΔMGF). It is demonstrated, by first time, that deleting specific MGF genes alone can completely attenuate a highly virulent field ASFV isolate. Recombinant virus ASFV-G-ΔMGF effectively confers protection in pigs against challenge with ASFV-G when delivered once via the intramuscular (i.m.) route. The protection against ASFV-G is highly effective by 28 days postvaccination. This is the first report of an experimental vaccine that induces solid protection against virulent ASFV-G. PMID:25810553

Impact of the Bim Deletion Polymorphism on Survival Among Patients With Completely Resected Non-Small-Cell Lung Carcinoma.

PubMed

Atsumi, Jun; Shimizu, Kimihiro; Ohtaki, Yoichi; Kaira, Kyoichi; Kakegawa, Seiichi; Nagashima, Toshiteru; Enokida, Yasuaki; Nakazawa, Seshiru; Obayashi, Kai; Takase, Yoshiaki; Kawashima, Osamu; Kamiyoshihara, Mitsuhiro; Sugano, Masayuki; Ibe, Takashi; Igai, Hitoshi; Takeyoshi, Izumi

2016-02-01

A deletion polymorphism of the Bim gene has been reported to be a prognostic factor for patients with non-small-cell lung cancer (NSCLC) treated with epidermal growth factor receptor-tyrosine kinase inhibitors in the Asian population. We investigated the impact of the Bim deletion polymorphism on survival among patients with completely resected NSCLC. The Bim polymorphism was detected by polymerase chain reaction analysis. We measured overall survival (OS) and recurrence-free survival rates in 411 patients and postrecurrence survival (PRS) in 94 patients who experienced recurrence and received additional anticancer therapy. The Bim deletion polymorphism was detected in 61 patients (14.8%). OS rates were significantly lower for patients with the Bim deletion polymorphism than for those with the wild-type sequence. On multivariable analysis, the Bim deletion polymorphism was identified as an independent prognostic factor for OS (hazard ratio, 1.98; 95% CI, 1.17 to 3.36; P = .011). Among the 94 patients who experienced recurrence and were treated with anticancer therapy, patients with the Bim deletion polymorphism showed significantly poorer PRS than those with the wild-type sequence (median, 9.8 months v 26.9 months, respectively; P < .001). Multivariable analysis revealed that the Bim deletion polymorphism was an independent predictor of PRS (hazard ratio, 3.36; 95% CI, 1.75 to 6.47; P < .001). This trend remained apparent in subgroup analyses stratified by EGFR status, histology, and therapeutic modality. The Bim deletion polymorphism is a novel indicator of shortened PRS among patients with recurrent NSCLC treated with anticancer therapy in the Asian population.

Analysis of genotoxic activity of ketamine and rocuronium bromide using the somatic mutation and recombination test in Drosophila melanogaster.

PubMed

Koksal, Pakize Muge; Gürbüzel, Mehmet

2015-03-01

The present study evaluated the mutagenic and recombinogenic effects of two commonly used anesthetic agents, ketamine and rocuronium bromide, in medicine using the wing somatic mutation and recombination test (SMART) in Drosophila. The standard (ST) cross and the high-bioactivation (HB) cross with high sensitivity to procarcinogens and promutagens were used. The SMART test is based on the loss of heterozygosity, which occurs via various mechanisms, such as chromosome loss and deletion, half-translocation, mitotic recombination, mutation, and non-disjunction. Genetic alterations occurring in the somatic cells of the wing's imaginal discs result in mutant clones in the wing blade. Three-day-old trans-heterozygous larvae with two recessive markers, multiple wing hairs (mwh) and flare (flr(3)), were treated with ketamine and rocuronium bromide. Analysis of the ST cross indicated that ketamine exhibited genotoxicity activity and that this activity was particularly dependent on homologous mitotic recombination at concentrations of 250 μg/ml and above. Rocuronium bromide did not exert mutagenic and/or recombinogenic effects. In the HB cross, ketamine at a concentration of 1000 μg/ml and rocuronium bromide at all concentrations, with the exception of 250 μg/ml (inconclusive), exerted genotoxic effects, which could also be associated with the increase in mitotic recombination. Copyright © 2015 Elsevier B.V. All rights reserved.

Virulence of a chimeric recombinant infectious haematopoietic necrosis virus expressing the spring viraemia of carp virus glycoprotein in salmonid and cyprinid fish

USGS Publications Warehouse

Emmenegger, Eveline; Biacchesi, Stéphane; Mérour, Emilie; Glenn, Jolene. A; Palmer, Alexander D.; Brémont, Michel; Kurath, Gael

2018-01-01

Infectious haematopoietic necrosis virus (IHNV) and spring viraemia of carp virus (SVCV) are both rhabdoviruses of fish, listed as notifiable disease agents by the World Organization for Animal Health. Recombinant rhabdoviruses with heterologous gene substitutions have been engineered to study genetic determinants and assess the potential of these recombinant viruses for vaccine development. A recombinant IHNV (rIHNV), containing the full-length genome of a European IHNV strain, was modified by deleting the glycoprotein (G) gene and replacing it with a European SVCV G-gene to make the rIHNV-Gsvcv. The chimeric rIHNV-Gsvcv level of virulence in rainbow trout, common carp and koi was assessed, and its ability to induce a protective immune response in surviving koi against wild-type SVCV infection was tested. The rIHNV-Gsvcv infection of trout led to high mortality, ranging from 78% to 92.5%, after immersion. In contrast, no deaths occurred in juvenile common carp after infection with rIHNV-Gsvcv by either immersion or intraperitoneal (IP) injection. Similarly, koi infected with rIHNV-Gsvcv via IP injection had little to no mortality (≤9%). Koi that survived initial infection with a high dose of recombinant virus rIHNV-Gsvcv were protected against a virulent SVCV challenge resulting in a high relative per cent survival of 82.5%.

Development of a novel engineered E. coli host cell line platform with improved column capacity performance for ion-exchange chromatography.

PubMed

Mukherjee, Rudra Palash; Fruchtl, McKinzie S; Beitle, Robert R; Brune, Ellen M

2018-02-01

This article reports on the analysis of an engineered Escherichia coli designed to reduce the host cell protein (HCP) burden on recombinant protein purification by column chromatography. Since downstream purification accounts for a major portion of production costs when using a recombinant platform, minimization of HCPs that are initially captured or otherwise interfere during chromatography will positively impact the entire purification process. Such a strategy, of course, would also require the cell line to grow, and express recombinant proteins, at levels comparable to, or better than, its parent strain. An E. coli strain with a small number of strategic deletions (LTSF06) was transformed to produce three different recombinant biologics to examine growth and expression, and with another model protein to assess growth and the effect of selectively reduced HCPs on target product capture on DEAE ion exchange medium. Cell growth levels were maintained or increased for all constructs, and a significant reduction in HCP adsorption was realized. Indeed, a breakthrough analysis indicated that as a result of reducing adsorption of particular HCPs, a 37% increase in target protein capture was observed. This increase in product capture efficiency was achieved by focusing not on HCPs that co-elute with the recombinant target, but rather on those possessing particular column adsorption and elution characteristics. Copyright © 2017 Elsevier Inc. All rights reserved.

Recombinant factor VIIa (eptacog alfa): a pharmacoeconomic review of its use in haemophilia in patients with inhibitors to clotting factors VIII or IX.

PubMed

Lyseng-Williamson, Katherine A; Plosker, Greg L

2007-01-01

Recombinant factor VIIa (NovoSeven; also known as recombinant activated factor VII or eptacog alfa) is indicated as an intravenous haemostatic agent in haemophilia patients with inhibitors to clotting factors VIII or IX. In noncomparative trials in haemophilia patients with inhibitors, on-demand home treatment with recombinant factor VIIa was effective in controlling episodes of mild to moderate bleeding and well tolerated, with early treatment being associated with a greater rate of success and the need for fewer doses than delayed treatment. Prophylactic treatment with recombinant factor VIIa was also effective in maintaining haemostasis in patients with this indication undergoing surgery. Relative to prior treatment with plasma-derived agents, treatment with recombinant factor VIIa was associated with improvements in health-related quality of life in a cost-utility study in haemophilia patients with inhibitors in Australia. In well designed decision-model cost analyses conducted from a healthcare payer perspective in several countries, on-demand treatment with recombinant factor VIIa to control mild to moderate bleeding episodes in this patient population was predicted to be cost saving or cost neutral relative to on-demand treatment with intravenous activated prothrombin complex concentrate (aPCC). Although the acquisition cost of recombinant factor VIIa was greater than that of aPCC in some studies, the greater initial efficacy of recombinant factor VIIa than aPCC resulted in lower predicted total medical costs. Results were generally robust to plausible changes in key parameters. Orthopaedic surgery with recombinant factor VIIa to maintain haemostasis in haemophilia patients with inhibitors was generally predicted to be cost saving, relative to not having surgery, over the medium to long term in modelled cost analyses from a healthcare payer perspective in the UK and US. The initial cost of surgery was high, but the difference in costs between patients undergoing or not undergoing surgery was predicted to decline over time, as savings were realised from the decrease in the number of bleeding episodes requiring treatment in patients who underwent surgery. In haemophilia patients with inhibitors, recombinant factor VIIa is clinically effective in controlling mild to moderate bleeds, and in maintaining haemostasis in patients undergoing orthopaedic surgery. Available pharmacoeconomic data from several countries, despite inherent limitations, support the use of recombinant factor VIIa as a treatment option that is at least cost neutral relative to aPCC in treating mild to moderate bleeds in this patient population. In addition, orthopaedic surgery with recombinant factor VIIa to maintain haemostasis in haemophilia patients with inhibitors is generally cost saving relative to not having surgery over the medium to long term, as the acquisition costs of recombinant factor VIIa are offset by cost savings resulting from the decrease in the number of joint-related bleeds.

Physiology of deletion mutants in the anaerobic β-myrcene degradation pathway in Castellaniella defragrans

PubMed Central

2012-01-01

Background Monoterpenes present a large and versatile group of unsaturated hydrocarbons of plant origin with widespread use in the fragrance as well as food industry. The anaerobic β-myrcene degradation pathway in Castellaniella defragrans strain 65Phen differs from well known aerobic, monooxygenase-containing pathways. The initial enzyme linalool dehydratase-isomerase ldi/LDI catalyzes the hydration of β-myrcene to (S)-(+)-linalool and its isomerization to geraniol. A high-affinity geraniol dehydrogenase geoA/GeDH and a geranial dehydrogenase geoB/GaDH contribute to the formation of geranic acid. A genetic system was for the first time applied for the betaproteobacterium to prove in vivo the relevance of the linalool dehydratase-isomerase and the geraniol dehydrogenase. In-frame deletion cassettes were introduced by conjugation and two homologous recombination events. Results Polar effects were absent in the in-frame deletion mutants C. defragrans Δldi and C. defragrans ΔgeoA. The physiological characterization of the strains demonstrated a requirement of the linalool dehydratase-isomerase for growth on acyclic monoterpenes, but not on cyclic monoterpenes. The deletion of geoA resulted in a phenotype with hampered growth rate on monoterpenes as sole carbon and energy source as well as reduced biomass yields. Enzyme assays revealed the presence of a second geraniol dehydrogenase. The deletion mutants were in trans complemented with the broad-host range expression vector pBBR1MCS-4ldi and pBBR1MCS-2geoA, restoring in both cases the wild type phenotype. Conclusions In-frame deletion mutants of genes in the anaerobic β-myrcene degradation revealed novel insights in the in vivo function. The deletion of a high-affinity geraniol dehydrogenase hampered, but did not preclude growth on monoterpenes. A second geraniol dehydrogenase activity was present that contributes to the β-myrcene degradation pathway. Growth on cyclic monoterpenes independent of the initial enzyme LDI suggests the presence of a second enzyme system activating unsaturated hydrocarbons. PMID:22947208

Characterization of Novel Hepatitis B Virus PreS/S-Gene Mutations in a Patient with Occult Hepatitis B Virus Infection

PubMed Central

Xu, Zhihui; Chen, Rongjuan; Si, Lanlan; Lu, Shanshan; Li, Xiaodong; Wang, Shuai; Zhang, Kai; Li, Jin; Han, Juqiang; Xu, Dongping

2016-01-01

Objective The impact of hepatitis B virus (HBV) preS/S-gene mutations on occult HBV infection (OBI) is not fully understood. This study characterized multiple novel HBV preS/S-gene mutants obtained from an OBI patient. Methods PreS/S-gene mutants were analyzed by clonal sequencing. Viral replication and expression were analyzed by transfecting HBV genomic recombinants into HepG2 cells. Results Twenty-one preS/S-gene mutants were cloned from four sequential serum samples, including 13 mutants that were not previously documented: (1) sI/T126V+sG145R; (2) preS1 nt 3014−3198 deletion; (3) preS1 nt 3046−3177 deletion; (4) preS1 nt 3046−3177 deletion+s115−116 “INGTST” insertion; (5) preS1 nt 3046−3177 deletion+s115−116 “INGTST” insertion+sG145R; (6) preS1 nt 3115−3123 deletion+sQ129N; (7) preS1 nt 3115−3123 deletion+s126−127 “RPCMNCTI” insertion; (8) s115−116 “INGTST” insertion; (9) s115−116 “INGTST” insertion+sG145R; (10) s126−127 “RPCMNCTI” insertion; (11) preS1 nt 2848−2862 deletion+preS2 initiation codon M→I; (12) s122−123 “KSTGLCK” insertion+sQ129N; and (13) preS2 initiation codon M→I+s131−133TSM→NST. The proportion of preS1 nt 3046−3177 deletion and preS2 initiation codon M→I+s131−133TSM→NST mutants increased in the viral pool with prolonged disease. The 13 novel OBI-related mutants showed a 51.2−99.9% decrease in HBsAg levels compared with that of the wild type. Additional N-glycosylation-associated mutations, sQ129N and s131−133TSM→NST, but not s126−127 “RPCMNCTI,” greatly attenuated anti-HBs binding to HBsAg. Compared with the wild type, replication and surface antigen promoter II activity of the preS1 nt 3046−3177 deletion mutant decreased by 43.3% and 97.0%, respectively. Conclusion PreS/S-gene mutations may play coordinated roles in the presentation of OBI and might be associated with disease progression. This has implications for HBV diagnosis and vaccine improvement. PMID:27182775

The importance of the specific Z-DNA structure and polyamines in carcinogenesis: fact or fiction.

PubMed

Juranic, Z; Kidric, M; Tomin, R; Juranić, I; Spuzić, I; Petrović, J

1991-08-01

In this work some aspects of carcinogenesis are given. The importance of the emergence of Z or H DNA structure in the gene, or in the flanking gene sequences for the gene deletion and unusual gene recombination, is discussed. Some considerations on the role of selective pressure (of polyamines, of Mg2+, of the various levels of topoisomerase II, and of ATP) in the process of oncogene amplification, are given too.

Generation and Characterization of a Double Recombinant Monkeypox Virus for use in Animal Model Development and Therapeutic Evaluation

DTIC Science & Technology

2012-09-27

time patients could reach a temperature near 103°F. The fever was typically 5     accompanied by headache, backache, vomiting , and prostration. A...were co-housed with prairie dogs . Infected prairie dogs were sold and distributed across multiple states including Wisconsin, Illinois, Indiana...deletion of C3L from the Congo Basin clade virus reduced morbidity and mortality in prairie dogs infected intranasally (29). Since 1986, passive

A new and efficient approach for construction of uridine/uracil auxotrophic mutants in the filamentous fungus Aspergillus oryzae using Agrobacterium tumefaciens-mediated transformation.

PubMed

Nguyen, Khuyen Thi; Ho, Quynh Ngoc; Do, Loc Thi Binh Xuan; Mai, Linh Thi Dam; Pham, Duc-Ngoc; Tran, Huyen Thi Thanh; Le, Diep Hong; Nguyen, Huy Quang; Tran, Van-Tuan

2017-06-01

Aspergillus oryzae is a filamentous fungus widely used in food industry and as a microbial cell factory for recombinant protein production. Due to the inherent resistance of A. oryzae to common antifungal compounds, genetic transformation of this mold usually requires auxotrophic mutants. In this study, we show that Agrobacterium tumefaciens-mediated transformation (ATMT) method is very efficient for deletion of the pyrG gene in different Aspergillus oryzae wild-type strains to generate uridine/uracil auxotrophic mutants. Our data indicated that all the obtained uridine/uracil auxotrophic transformants, which are 5- fluoroorotic acid (5-FOA) resistant, exist as the pyrG deletion mutants. Using these auxotrophic mutants and the pyrG selectable marker for genetic transformation via A. tumefaciens, we could get about 1060 transformants per 10 6 fungal spores. In addition, these A. oryzae mutants were also used successfully for expression of the DsRed fluorescent reporter gene under control of the A. oryzae amyB promoter by the ATMT method, which resulted in obvious red transformants on agar plates. Our work provides a new and effective approach for constructing the uridine/uracil auxotrophic mutants in the importantly industrial fungus A. oryzae. This strategy appears to be applicable to other filamentous fungi to develop similar genetic transformation systems based on auxotrophic/nutritional markers for food-grade recombinant applications.

Cis-acting regulatory sequences promote high-frequency gene conversion between repeated sequences in mammalian cells.

PubMed

Raynard, Steven J; Baker, Mark D

2004-01-01

In mammalian cells, little is known about the nature of recombination-prone regions of the genome. Previously, we reported that the immunoglobulin heavy chain (IgH) mu locus behaved as a hotspot for mitotic, intrachromosomal gene conversion (GC) between repeated mu constant (Cmu) regions in mouse hybridoma cells. To investigate whether elements within the mu gene regulatory region were required for hotspot activity, gene targeting was used to delete a 9.1 kb segment encompassing the mu gene promoter (Pmu), enhancer (Emu) and switch region (Smu) from the locus. In these cell lines, GC between the Cmu repeats was significantly reduced, indicating that this 'recombination-enhancing sequence' (RES) is necessary for GC hotspot activity at the IgH locus. Importantly, the RES fragment stimulated GC when appended to the same Cmu repeats integrated at ectopic genomic sites. We also show that deletion of Emu and flanking matrix attachment regions (MARs) from the RES abolishes GC hotspot activity at the IgH locus. However, no stimulation of ectopic GC was observed with the Emu/MARs fragment alone. Finally, we provide evidence that no correlation exists between the level of transcription and GC promoted by the RES. We suggest a model whereby Emu/MARS enhances mitotic GC at the endogenous IgH mu locus by effecting chromatin modifications in adjacent DNA.

Robust inducible Cre recombinase activity in the human malaria parasite Plasmodium falciparum enables efficient gene deletion within a single asexual erythrocytic growth cycle.

PubMed

Collins, Christine R; Das, Sujaan; Wong, Eleanor H; Andenmatten, Nicole; Stallmach, Robert; Hackett, Fiona; Herman, Jean-Paul; Müller, Sylke; Meissner, Markus; Blackman, Michael J

2013-05-01

Asexual blood stages of the malaria parasite, which cause all the pathology associated with malaria, can readily be genetically modified by homologous recombination, enabling the functional study of parasite genes that are not essential in this part of the life cycle. However, no widely applicable method for conditional mutagenesis of essential asexual blood-stage malarial genes is available, hindering their functional analysis. We report the application of the DiCre conditional recombinase system to Plasmodium falciparum, the causative agent of the most dangerous form of malaria. We show that DiCre can be used to obtain rapid, highly regulated site-specific recombination in P. falciparum, capable of excising loxP-flanked sequences from a genomic locus with close to 100% efficiency within the time-span of a single erythrocytic growth cycle. DiCre-mediated deletion of the SERA5 3' UTR failed to reduce expression of the gene due to the existence of alternative cryptic polyadenylation sites within the modified locus. However, we successfully used the system to recycle the most widely used drug resistance marker for P. falciparum, human dihydrofolate reductase, in the process producing constitutively DiCre-expressing P. falciparum clones that have broad utility for the functional analysis of essential asexual blood-stage parasite genes. © 2013 John Wiley & Sons Ltd.

Method for producing labeled single-stranded nucleic acid probes

DOEpatents

Dunn, John J.; Quesada, Mark A.; Randesi, Matthew

1999-10-19

Disclosed is a method for the introduction of unidirectional deletions in a cloned DNA segment. More specifically, the method comprises providing a recombinant DNA construct comprising a DNA segment of interest inserted in a cloning vector, the cloning vector having an f1 endonuclease recognition sequence adjacent to the insertion site of the DNA segment of interest. The recombinant DNA construct is then contacted with the protein pII encoded by gene II of phage f1 thereby generating a single-stranded nick. The nicked DNA is then contacted with E. coli Exonuclease III thereby expanding the single-stranded nick into a single-stranded gap. The single-stranded gapped DNA is then contacted with a single-strand-specific endonuclease thereby producing a linearized DNA molecule containing a double-stranded deletion corresponding in size to the single-stranded gap. The DNA treated in this manner is then incubated with DNA ligase under conditions appropriate for ligation. Also disclosed is a method for producing single-stranded DNA probes. In this embodiment, single-stranded gapped DNA, produced as described above, is contacted with a DNA polymerase in the presence of labeled nucleotides to fill in the gap. This DNA is then linearized by digestion with a restriction enzyme which cuts outside the DNA segment of interest. The product of this digestion is then denatured to produce a labeled single-stranded nucleic acid probe.

Development and Characterization of a High Throughput Screen to investigate the delayed Effects of Radiations Commonly Encountered in Space

NASA Astrophysics Data System (ADS)

Morgan, W. F.

Astronauts based on the space station or on long-term space missions will be exposed to high Z radiations in the cosmic environment In order to evaluate the potentially deleterious effects of exposure to radiations commonly encountered in space we have developed and characterized a high throughput assay to detect mutation deletion events and or hyperrecombination in the progeny of exposed cells This assay is based on a plasmid vector containing a green fluorescence protein reporter construct We have shown that after stable transfection of the vector into human or hamster cells this construct can identify mutations specifically base changes and deletions as well as recombination events e g gene conversion or homologous recombination occurring as a result of exposure to ionizing radiation Our focus has been on those events occurring in the progeny of an irradiated cell that are potentially associated with radiation induced genomic instability rather than the more conventional assays that evaluate the direct immediate effects of radiation exposure Considerable time has been spent automating analysis of surviving colonies as a function of time after irradiation in order to determine when delayed instability is induced and the consequences of this delayed instability The assay is now automated permitting the evaluation of potentially rare events associated with low dose low dose rate radiations commonly encountered in space

Bypass of lethality with mosaic mice generated by Cre-loxP-mediated recombination.

PubMed

Betz, U A; Vosshenrich, C A; Rajewsky, K; Müller, W

1996-10-01

The analysis of gene function based on the generation of mutant mice by homologous recombination in embryonic stem cells is limited if gene disruption results in embryonic lethality. Mosaic mice, which contain a certain proportion of mutant cells in all organs, allow lethality to be circumvented and the potential of mutant cells to contribute to different cell lineages to be analyzed. To generate mosaic animals, we used the bacteriophage P1-derived Cre-loxP recombination system, which allows gene alteration by Cre-mediated deletion of loxP-flanked gene segments. We generated nestin-cre transgenic mouse lines, which expressed the Cre recombinase under the control of the rat nestin promoter and its second intron enhancer. In crosses to animals carrying a loxP-flanked target gene, partial deletion of the loxP-flanked allele occurred before day 10.5 post coitum and was detectable in all adult organs examined, including germ-line cells. Using this approach, we generated mosaic mice containing cells deficient in the gamma-chain of the interleukin-2 receptor (IL-2R gamma); in these animals, the IL-2R gamma-deficient cells were underrepresented in the thymus and spleen. Because mice deficient in DNA polymerase beta die perinatally, we studied the effects of DNA polymerase beta deficiency in mosaic animals. We found that some of the mosaic polymerase beta-deficient animals were viable, but were often reduced in size and weight. The fraction of DNA polymerase beta-deficient cells in mosaic embryos decreased during embryonic development, presumably because wild-type cells had a competitive advantage. The nestin-cre transgenic mice can be used to generate mosaic animals in which target genes are mutated by Cre-mediated recombination of loxP-flanked target genes. By using mosaic animals, embryonic lethality can be bypassed and cell lineages for whose development a given target gene is critical can be identified. In the case of DNA polymerase beta, deficient cells are already selected against during embryonic development, demonstrating the general importance of this protein in multiple cell types.

Deletion endpoint allele-specificity in the developmentally regulated elimination of an internal sequence (IES) in Paramecium.

PubMed Central

Dubrana, K; Le Mouël, A; Amar, L

1997-01-01

Ciliated protozoa undergo thousands of site-specific DNA deletion events during the programmed development of micronuclear genomes to macronuclear genomes. Two deletion elements, W1 and W2, were identified in the Paramecium primaurelia wild-type 156 strain. Here, we report the characterization of both elements in wild-type strain 168 and show that they display variant deletion patterns when compared with those of strain 156. The W1 ( 168 ) element is defective for deletion. The W2 ( 168 ) element is excised utilizing two alternative boundaries on one side, both are different from the boundary utilized to excise the W2156 element. By crossing the 156 and 168 strains, we demonstrate that the definition of all deletion endpoints are each controlled by cis -acting determinant(s) rather than by strain-specific trans-acting factor(s). Sequence comparison of all deleted DNA segments indicates that the 5'-TA-3'terminal sequence is strictly required at their ends. Furthermore the identity of the first eight base pairs of these ends to a previously established consensus sequence correlates with the frequency of the corresponding deletion events. Our data implies the existence of an adaptive convergent evolution of these Paramecium deleted DNA segment end sequences. PMID:9171098

The HDAC inhibitor SB939 overcomes resistance to BCR-ABL kinase Inhibitors conferred by the BIM deletion polymorphism in chronic myeloid leukemia.

PubMed

Rauzan, Muhammad; Chuah, Charles T H; Ko, Tun Kiat; Ong, S Tiong

2017-01-01

Chronic myeloid leukemia (CML) treatment has been improved by tyrosine kinase inhibitors (TKIs) such as imatinib mesylate (IM) but various factors can cause TKI resistance in patients with CML. One factor which contributes to TKI resistance is a germline intronic deletion polymorphism in the BCL2-like 11 (BIM) gene which impairs the expression of pro-apoptotic splice isoforms of BIM. SB939 (pracinostat) is a hydroxamic acid based HDAC inhibitor with favorable pharmacokinetic, physicochemical and pharmaceutical properties, and we investigated if this drug could overcome BIM deletion polymorphism-induced TKI resistance. We found that SB939 corrects BIM pre-mRNA splicing in CML cells with the BIM deletion polymorphism, and induces apoptotic cell death in CML cell lines and primary cells with the BIM deletion polymorphism. More importantly, SB939 both decreases the viability of CML cell lines and primary CML progenitors with the BIM deletion and restores TKI-sensitivity. Our results demonstrate that SB939 overcomes BIM deletion polymorphism-induced TKI resistance, and suggest that SB939 may be useful in treating CML patients with BIM deletion-associated TKI resistance.

Generation of Tandem Direct Duplications by Reversed-Ends Transposition of Maize Ac Elements

PubMed Central

Peterson, Thomas

2013-01-01

Tandem direct duplications are a common feature of the genomes of eukaryotes ranging from yeast to human, where they comprise a significant fraction of copy number variations. The prevailing model for the formation of tandem direct duplications is non-allelic homologous recombination (NAHR). Here we report the isolation of a series of duplications and reciprocal deletions isolated de novo from a maize allele containing two Class II Ac/Ds transposons. The duplication/deletion structures suggest that they were generated by alternative transposition reactions involving the termini of two nearby transposable elements. The deletion/duplication breakpoint junctions contain 8 bp target site duplications characteristic of Ac/Ds transposition events, confirming their formation directly by an alternative transposition mechanism. Tandem direct duplications and reciprocal deletions were generated at a relatively high frequency (∼0.5 to 1%) in the materials examined here in which transposons are positioned nearby each other in appropriate orientation; frequencies would likely be much lower in other genotypes. To test whether this mechanism may have contributed to maize genome evolution, we analyzed sequences flanking Ac/Ds and other hAT family transposons and identified three small tandem direct duplications with the structural features predicted by the alternative transposition mechanism. Together these results show that some class II transposons are capable of directly inducing tandem sequence duplications, and that this activity has contributed to the evolution of the maize genome. PMID:23966872

Techniques for Large-Scale Bacterial Genome Manipulation and Characterization of the Mutants with Respect to In Silico Metabolic Reconstructions.

PubMed

diCenzo, George C; Finan, Turlough M

2018-01-01

The rate at which all genes within a bacterial genome can be identified far exceeds the ability to characterize these genes. To assist in associating genes with cellular functions, a large-scale bacterial genome deletion approach can be employed to rapidly screen tens to thousands of genes for desired phenotypes. Here, we provide a detailed protocol for the generation of deletions of large segments of bacterial genomes that relies on the activity of a site-specific recombinase. In this procedure, two recombinase recognition target sequences are introduced into known positions of a bacterial genome through single cross-over plasmid integration. Subsequent expression of the site-specific recombinase mediates recombination between the two target sequences, resulting in the excision of the intervening region and its loss from the genome. We further illustrate how this deletion system can be readily adapted to function as a large-scale in vivo cloning procedure, in which the region excised from the genome is captured as a replicative plasmid. We next provide a procedure for the metabolic analysis of bacterial large-scale genome deletion mutants using the Biolog Phenotype MicroArray™ system. Finally, a pipeline is described, and a sample Matlab script is provided, for the integration of the obtained data with a draft metabolic reconstruction for the refinement of the reactions and gene-protein-reaction relationships in a metabolic reconstruction.

Yeast MRX deletions have short chronological life span and more triacylglycerols.

PubMed

Kanagavijayan, Dhanabalan; Rajasekharan, Ram; Srinivasan, Malathi

2016-02-01

Saccharomyces cerevisiae is an excellent model organism for lipid research. Here, we have used yeast haploid RAdiation Damage (RAD) deletion strains to study life span and lipid storage patterns. RAD genes are mainly involved in DNA repair mechanism and hence, their deletions have resulted in shorter life span. Viable RAD mutants were screened for non-polar lipid content, and some of the mutants showed significantly high amounts of triacylglycerol (TAG) and steryl ester, besides short chronological life span. Among these, RAD50, MRE11 and XRS2 form a complex, MRX that is involved in homologous recombination that showed an increase in the amount of TAG. Microarray data of single MRX deletions revealed that besides DNA damage signature genes, lipid metabolism genes are also differentially expressed. Lipid biosynthetic genes (LPP1, SLC1) were upregulated and lipid hydrolytic gene (TGL3) was downregulated. We observed that rad50Δ, mre11Δ, xrs2Δ and mrxΔ strains have high number of lipid droplets (LDs) with fragmented mitochondria. These mutants have a short chronological life span compared to wild type. Aged wild-type cells also accumulated TAG with LDs of ∼2.0 μm in diameter. These results suggest that TAG accumulation and big size LDs could be possible markers for premature or normal aging. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

rhEGF-containing thermosensitive and mucoadhesive polymeric sol-gel for endoscopic treatment of gastric ulcer and bleeding.

PubMed

Maeng, Jin Hee; So, Jung Won; Kim, Jungju; Kim, In Ae; Jung, Ji Hoon; Min, Kyunghyun; Lee, Don Haeng; Yang, Su-Geun

2014-03-01

Gastrointestinal endoscopy is a standard diagnostic tool for gastrointestinal ulcers and cancer. In this study, we have developed recombinant human epidermal growth factor-containing ulcer-coating polymeric sol-gel for endoscopic application. Chitosan and pluronic F127 were employed for their thermoresponsive and bioadhesive properties. At temperatures below 21, polymeric sol-gel remains liquid during endoscopic application and transforms to gel at body temperature after application on ulcers. In an in vitro cellular wounding assay, recombinant human epidermal growth factor sol-gel significantly enhanced the cell migration and decreased the wounding area (68%) compared to nontreated, recombinant human epidermal growth factor solution, and sol-gel without recombinant human epidermal growth factor (42, 49, and 32 % decreased at day 1). The in vivo ulcer-healing study was performed in an acetic acid-induced gastric ulcer rat model and proved that our recombinant human epidermal growth factor endoscopic sol-gel facilitated the ulcer-healing process more efficiently than the other treatments. Ulcer sizes in the recombinant human epidermal growth factor sol-gel group were decreased 2.9- and 2.1-fold compared with those in the nontreated group on days 1 and 3 after ulceration, respectively. The mucosal thickness in the recombinant human epidermal growth factor sol-gel group was significantly increased compared to that in the nontreated group (3.2- and 6.9-fold on days 1 and 3 after ulceration, respectively). In a gastric retention study, recombinant human epidermal growth factor sol-gel stayed on the gastric mucosa more than 2 h after application. The present study suggests that recombinant human epidermal growth factor sol-gel is a prospective candidate for treating gastric ulcers via endoscopic application.

Evolution and Emergence of Enteroviruses through Intra- and Inter-species Recombination: Plasticity and Phenotypic Impact of Modular Genetic Exchanges in the 5’ Untranslated Region

PubMed Central

Muslin, Claire; Joffret, Marie-Line; Pelletier, Isabelle; Blondel, Bruno; Delpeyroux, Francis

2015-01-01

Genetic recombination shapes the diversity of RNA viruses, including enteroviruses (EVs), which frequently have mosaic genomes. Pathogenic circulating vaccine-derived poliovirus (cVDPV) genomes consist of mutated vaccine poliovirus (PV) sequences encoding capsid proteins, and sequences encoding nonstructural proteins derived from other species’ C EVs, including certain coxsackieviruses A (CV-A) in particular. Many cVDPV genomes also have an exogenous 5’ untranslated region (5’ UTR). This region is involved in virulence and includes the cloverleaf (CL) and the internal ribosomal entry site, which play major roles in replication and the initiation of translation, respectively. We investigated the plasticity of the PV genome in terms of recombination in the 5’ UTR, by developing an experimental model involving the rescue of a bipartite PV/CV-A cVDPV genome rendered defective by mutations in the CL, following the co-transfection of cells with 5’ UTR RNAs from each of the four human EV species (EV-A to -D). The defective cVDPV was rescued by recombination with 5’ UTR sequences from the four EV species. Homologous and nonhomologous recombinants with large deletions or insertions in three hotspots were isolated, revealing a striking plasticity of the 5’ UTR. By contrast to the recombination of the cVDPV with the 5’ UTR of group II (EV-A and -B), which can decrease viral replication and virulence, recombination with the 5’ UTRs of group I (EV-C and -D) appeared to be evolutionarily neutral or associated with a gain in fitness. This study illustrates how the genomes of positive-strand RNA viruses can evolve into mosaic recombinant genomes through intra- or inter-species modular genetic exchanges, favoring the emergence of new recombinant lineages. PMID:26562151

Human common acute lymphoblastic leukemia-derived cell lines are competent to recombine their T-cell receptor delta/alpha regions along a hierarchically ordered pathway.

PubMed

Hansen-Hagge, T E; Yokota, S; Reuter, H J; Schwarz, K; Bartram, C R

1992-11-01

Rearrangements of the T-cell receptor (TCR) delta locus are observed in the majority of human B-cell precursor acute lymphoblastic leukemias (ALL) with a striking predominance of V delta 2(D)D delta 3 recombinations in common ALL (cALL) patients. Recently, we and others showed that almost 20% of cALL cases are characterized by further recombination of V delta 2(D)D delta 3 segments to J alpha elements, thereby deleting the TCR delta locus in analogy to the delta Rec/psi J alpha pathway in differentiating alpha/beta-positive T cells. We report here that two human cALL-derived cell lines, REH and Nalm-6, are competent to recombine the TCR delta/alpha locus under standard tissue culture conditions. Analysis of different REH subclones obtained by limiting dilution of the initial culture showed a biased recombination of V delta 2D delta 3 to distinct J alpha elements. During prolonged tissue culture, a subclone acquired growth advantage and displaced parental cells as well as other subclones. Frequently, the DJ junctions of REH subclones contained extended stretches of palindromic sequences derived from modified D delta 3 coding elements. The other cell line, Nalm-6, started the TCR delta/alpha recombination with an unusual signal joint of a cryptic recombinase signal sequence (RSS) upstream of D delta 3 to the 3' RSS of D delta 3. The RSS dimer was subsequently rearranged in all investigated subclones to an identical J alpha element. Both cell lines might become valuable tools to unravel the complex regulation of TCR delta/alpha recombination pathways in malignant and normal lymphopoiesis.

Genetic modifiers of Velo- cardio- facial syndrome/DiGeorge syndrome

PubMed Central

Aggarwal, Vimla S.; Morrow, Bernice E.

2009-01-01

Velo-cardio-facial syndrome/DiGeorge syndrome (VCFS/DGS), the most common micro-deletion disorder in humans, is characterized by craniofacial, parathyroid and thymic defects as well as cardiac outflow tract malformations. Most patients have a similar hemizygous 3 million base pair deletion on 22q11.2. Studies in mouse have shown that Tbx1, a T- box containing transcription factor present on the deleted region, is likely responsible for the etiology of the syndrome. Furthermore, mutations in TBX1 have been found in rare non-deleted patients. Despite having the same sized deletion, most VCFS/DGS patients exhibit significant clinical variability. Stochastic, environmental and genetic factors likely modify the phenotype of patients with the disorder. Here, we review mouse genetics studies which may help identify genetic modifiers for VCFS/DGS. PMID:18636633

Vowel Deletion in Latvian.

ERIC Educational Resources Information Center

Karins, A. Krisjanis

1995-01-01

Investigates variable deletion of short vowels in word-final unstressed syllables in Latvian spoken in Riga. Affected vowels were almost always inflectional endings and results indicated that internal phonological and prosodic factors (especially distance from main word stress) were the strongest constraints on vowel deletion, along with the…

Off-Target V(D)J Recombination Drives Lymphomagenesis and Is Escalated by Loss of the Rag2 C Terminus.

PubMed

Mijušković, Martina; Chou, Yi-Fan; Gigi, Vered; Lindsay, Cory R; Shestova, Olga; Lewis, Susanna M; Roth, David B

2015-09-22

Genome-wide analysis of thymic lymphomas from Tp53(-/-) mice with wild-type or C-terminally truncated Rag2 revealed numerous off-target, RAG-mediated DNA rearrangements. A significantly higher fraction of these errors mutated known and suspected oncogenes/tumor suppressor genes than did sporadic rearrangements (p < 0.0001). This tractable mouse model recapitulates recent findings in human pre-B ALL and allows comparison of wild-type and mutant RAG2. Recurrent, RAG-mediated deletions affected Notch1, Pten, Ikzf1, Jak1, Phlda1, Trat1, and Agpat9. Rag2 truncation substantially increased the frequency of off-target V(D)J recombination. The data suggest that interactions between Rag2 and a specific chromatin modification, H3K4me3, support V(D)J recombination fidelity. Oncogenic effects of off-target rearrangements created by this highly regulated recombinase may need to be considered in design of site-specific nucleases engineered for genome modification. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Combined deficiency of MSH2 and Sμ region abolishes class switch recombination.

PubMed

Leduc, Claire; Haddad, Dania; Laviolette-Malirat, Nathalie; Nguyen Huu, Ngoc-Sa; Khamlichi, Ahmed Amine

2010-10-01

Class switch recombination (CSR) is mediated by G-rich tandem repeated sequences termed switch regions. Transcription of switch regions generates single-stranded R loops that provide substrates for activation-induced cytidine deaminase. Mice deficient in MSH2 have a mild defect in CSR and analysis of their switch junctions has led to a model in which MSH2 is more critical for switch recombination events outside than within the tandem repeats. It is also known that deletion of the whole Sμ region severely impairs but does not abrogate CSR despite the lack of detectable R loops. Here, we demonstrate that deficiency of both MSH2 and the Sμ region completely abolishes CSR and that the abrogation occurs at the genomic level. This finding further supports the crucial role of MSH2 outside the tandem repeats. It also indicates that during CSR, MSH2 has access to activation-induced cytidine deaminase targets in R-loop-deficient Iμ-Cμ sequences rarely used in CSR, suggesting an MSH2-dependent DNA processing activity at the Iμ exon that may decrease with transcription elongation across the Sμ region.

Camphor Plasmid-Mediated Chromosomal Transfer in Pseudomonas putida

PubMed Central

Shaham, M.; Chakrabarty, A. M.; Gunsalus, I. C.

1973-01-01

Camphor-utilizing strains of Pseudomonas putida have been shown to carry the genetic information required for camphor degradation on a plasmid. The plasmid-carrying strains can serve as donors of both plasmid-borne and chromosomal genes. As recipients, plasmid-deleted strains are much superior to those carrying the camphor pathway genes. The transfer frequency of chromosomal, but not plasmid-borne, genes is markedly enhanced if the donor cells are irradiated with ultraviolet light followed by 3-h of growth on a rich medium in the dark. Recombinants selected for prototrophy are stable and most acquire the camphor (CAM) plasmid concomitantly; only a few of the Cam+ recombinants inherit the donor's ability to transfer chromosomal genes at a high frequency. Transfer-defective mutations occur on the CAM plasmid, affecting both CAM and chromosomal gene transfer. PMID:4745436

Four rice seed cDNA clones belonging to the alpha-amylase/trypsin inhibitor gene family encode potential rice allergens.

PubMed

Alvarez, A M; Fukuhara, E; Nakase, M; Adachi, T; Aoki, N; Nakamura, R; Matsuda, T

1995-07-01

Four rice seed proteins encoded by cDNAs belonging to the alpha-amylase/trypsin inhibitor gene family were overexpressed as TrpE-fusion proteins in E. coli. The expressed rice proteins were detected by SDS-PAGE as major proteins in bacterial cell lysates. Western blot analyses showed that all the recombinant proteins were immunologically reactive to rabbit polyclonal antibodies and to a mouse monoclonal antibody (25B9) specific for a previously isolated rice allergen of 16 kDa. Some truncated proteins from deletion mutants of the cDNAs retained their reactivity to the specific antibodies. These results suggest that the cDNAs encode potential rice allergens and that some epitopes of the recombinant proteins are still immunoreactive when they are expressed as their fragments.

Identification of a new hepatitis B virus recombinant D2/D3 in the city of São Paulo, Brazil.

PubMed

Santana, Luiz Claudio; Mantovani, Nathalia Pena; Ferreira, Maira Cicero; Arnold, Rafael; Duro, Rodrigo Lopes Sanz; Ferreira, Paulo Roberto Abrão; Hunter, James Richard; Leal, Élcio; Diaz, Ricardo Sobhie; Komninakis, Shirley Vasconcelos

2017-02-01

Two hundred forty million people are chronically infected with hepatitis B virus (HBV) worldwide. The rise of globalization has facilitated the emergence of novel HBV recombinants and genotypes. We evaluated HBV genotypes and recombinants, mutations associated with resistance to antivirals (AVs), progression of hepatic illness, and inefficient hepatitis B vaccination responses in chronically infected individuals in the city of São Paulo, Brazil. Forty-five full-length and 24 partial-length sequences were obtained. The genotype distribution was as follows: A (66.7%), D (15.9%), F (11.6%) and C (4.3%). We describe a new recombinant (D2/D3), confirmed through next-generation sequencing (NGS) and reconstruction of the quasispecies sequences in silico. Primary resistance and major vaccine escape mutations were not found. We did, however, find mutations in the S region that might may be related to HBV antigenicity changes, as well as Pre-S deletions. The precore/core mutations A1762T + G1764A (40.9%) were found mostly in genotypes A and D, and G1896A (29.55%) was more frequent in genotype D than in genotype A. The genotypic distribution reflects the history of Brazilian immigration. This is the first description of recombination between genotypes D2 and D3 in Brazil. It is also the first confirmation through NGS and reconstruction of the quasispecies in silico. However, little is known about the response to treatment of recombinants. This demonstrates the need for molecular epidemiology studies involving the analysis of full-length HBV sequences.

Interallelic class switch recombination contributes significantly to class switching in mouse B cells.

PubMed

Reynaud, Stéphane; Delpy, Laurent; Fleury, Laurence; Dougier, Hei-Lanne; Sirac, Christophe; Cogné, Michel

2005-05-15

Except for the expression of IgM and IgD, DNA recombination is constantly needed for the expression of other Ig classes and subclasses. The predominant path of class switch recombination (CSR) is intrachromosomal, and the looping-out and deletion model has been abundantly documented. However, switch regions also occasionally constitute convenient substrates for interchromosomal recombination, since it is noticeably the case in a number of chromosomal translocations causing oncogene deregulation in the course of lymphoma and myeloma. Although asymmetric accessibility of Ig alleles should theoretically limit its occurrence, interallelic CSR was shown to occur at low levels during IgA switching in rabbit, where the definition of allotypes within both V and C regions helped identify interchromosomally derived Ig. Thus, we wished to evaluate precisely interallelic CSR frequency in mouse B cells, by using a system in which only one allele (of b allotype) could express a functional VDJ region, whereas only interallelic CSR could restore expression of an excluded (a allotype) allele. In our study, we show that interchromosomal recombination of V(H) and Cgamma or Calpha occurs in vivo in B cells at a frequency that makes a significant contribution to physiological class switching: trans-association of V(H) and C(H) genes accounted for 7% of all alpha mRNA, and this frequency was about twice higher for the gamma3 transcripts, despite the much shorter distance between the J(H) region and the Cgamma3 gene, thus confirming that this phenomenon corresponded to site-specific switching and not to random recombination between long homologous loci.

Immune responses in pigs induced by recombinant canine adenovirus 2 expressing the glycoprotein 5 of porcine reproductive and respiratory syndrome virus.

PubMed

Zhou, J-X; Xue, J-D; Yu, T; Zhang, J-B; Liu, Y; Jiang, N; Li, Y-L; Hu, R-L

2010-04-01

To develop a new type vaccine for porcine reproductive and respiratory syndrome (PRRS) prevention by using canine adenovirus 2(CAV-2) as vector, the Glycoprotein 5(GP5) gene from PRRSV strain JL was amplified by RT-PCR, and the expression cassette of GP5 was constructed using the human cytomegalovirus (HCMV) promoter and the simian virus 40 (SV40) early mRNA polyadenylation signal. The expression cassette of Glycoprotein 5 was cloned into the CAV-2 genome in which E3 region had been partly deleted, and the recombinant virus (CAV-2-GP5) was obtained by transfecting the recombinant CAV-2-GP5 genome into MDCK cells together with Lipofectamine 2000. Immunization trial in pigs with the recombinant virus CAV-2-GP5 showed that CAV-2-GP5 could stimulate a specific immune response to PRRSV. Immune response to the GP5 and PRRSV was confirmed by ELISA, neutralization test and lymphocyte proliferative responses, and western blotting confirmed expression of GP5 by the vector in cells. These results indicated that CAV-2 may serve as a vector for development of PRRSV vaccine in pigs, and the CAV-2-GP5 might be a candidate vaccine to be tested for preventing PRRSV infection.

Identification of a Novel Virulence Determinant with Serum Opacification Activity in Streptococcus suis

PubMed Central

Baums, Christoph G.; Kaim, Ute; Fulde, Marcus; Ramachandran, Girish; Goethe, Ralph; Valentin-Weigand, Peter

2006-01-01

Streptococcus suis serotype 2 is a porcine and human pathogen with adhesive and invasive properties. In other streptococci, large surface-associated proteins (>100 kDa) of the MSCRAMM family (microbial surface components recognizing adhesive matrix molecules) are key players in interactions with host tissue. In this study, we identified a novel opacity factor of S. suis (OFS) with structural homology to members of the MSCRAMM family. The N-terminal region of OFS is homologous to the respective regions of fibronectin-binding protein A (FnBA) of Streptococcus dysgalactiae and the serum opacity factor (SOF) of Streptococcus pyogenes. Similar to these two proteins, the N-terminal domain of OFS opacified horse serum. Serum opacification activity was detectable in sodium dodecyl sulfate extracts of wild-type S. suis but not in extracts of isogenic ofs knockout mutants. Heterologous expression of OFS in Lactococcus lactis demonstrated that a high level of expression of OFS is sufficient to provide surface-associated serum opacification activity. Furthermore, serum opacification could be inhibited by an antiserum against recombinant OFS. The C-terminal repetitive sequence elements of OFS differed significantly from the respective repeat regions of FnBA and SOF as well as from the consensus sequence of the fibronectin-binding repeats of MSCRAMMs. Accordingly, fibronectin binding was not detectable in recombinant OFS. To investigate the putative function of OFS in the pathogenesis of invasive S. suis diseases, piglets were experimentally infected with an isogenic mutant strain in which the ofs gene had been knocked out by an in-frame deletion. The mutant was severely attenuated in virulence but not in colonization, demonstrating that OFS represents a novel virulence determinant of S. suis. PMID:17057090

Polygalacturonase gene pgxB in Aspergillus niger is a virulence factor in apple fruit

PubMed Central

Yang, Ying; Yang, Feng; Li, Yan-Hong; Liu, He-Ping; Chen, Xiao-Yan

2017-01-01

Aspergillus niger, a saprophytic fungus, is widely distributed in soil, air and cereals, and can cause postharvest diseases in fruit. Polygalacturonase (PG) is one of the main enzymes in fungal pathogens to degrade plant cell wall. To evaluate whether the deletion of an exo-polygalacturonase gene pgxB would influence fungal pathogenicity to fruit, pgxB gene was deleted in Aspergillus niger MA 70.15 (wild type) via homologous recombination. The ΔpgxB mutant showed similar growth behavior compared with the wild type. Pectin medium induced significant higher expression of all pectinase genes in both wild type and ΔpgxB in comparison to potato dextrose agar medium. However, the ΔpgxB mutant was less virulent on apple fruits as the necrosis diameter caused by ΔpgxB mutant was significantly smaller than that of wild type. Results of quantitive-PCR showed that, in the process of infection in apple fruit, gene expressions of polygalacturonase genes pgaI, pgaII, pgaA, pgaC, pgaD and pgaE were enhanced in ΔpgxB mutant in comparison to wild type. These results prove that, despite the increased gene expression of other polygalacturonase genes in ΔpgxB mutant, the lack of pgxB gene significantly reduced the virulence of A. niger on apple fruit, suggesting that pgxB plays an important role in the infection process on the apple fruit. PMID:28257463

The SaeRS Two-Component System Controls Survival of Staphylococcus aureus in Human Blood through Regulation of Coagulase

PubMed Central

Guo, Haiyong; Hall, Jeffrey W.; Yang, Junshu; Ji, Yinduo

2017-01-01

The SaeRS two-component system plays important roles in regulation of key virulence factors and pathogenicity. In this study, however, we found that the deletion mutation of saeRS enhanced bacterial survival in human blood, whereas complementation of the mutant with SaeRS returned survival to wild-type levels. Moreover, these phenomena were observed in different MRSA genetic background isolates, including HA-MRSA WCUH29, CA-MRSA 923, and MW2. To elucidate which gene(s) regulated by SaeRS contribute to the effect, we conducted a series of complementation studies with selected known SaeRS target genes in trans. We found coagulase complementation abolished the enhanced survival of the SaeRS mutant in human blood. The coa and saeRS deletion mutants exhibited a similar survival phenotype in blood. Intriguingly, heterologous expression of coagulase decreased survival of S. epidermidis in human blood. Further, the addition of recombinant coagulase to blood significantly decreased the survival of S. aureus. Further, analysis revealed staphylococcal resistance to killing by hydrogen peroxide was partially dependent on the presence or absence of coagulase. Furthermore, complementation with coagulase, but not SaeRS, returned saeRS/coa double mutant survival in blood to wild-type levels. These data indicate SaeRS modulates bacterial survival in blood in coagulase-dependent manner. Our results provide new insights into the role of staphylococcal SaeRS and coagulase on bacterial survival in human blood. PMID:28611950

Screening and identification of genetic loci involved in producing more/denser inclusion bodies in Escherichia coli

PubMed Central

2013-01-01

Background Many proteins and peptides have been used in therapeutic or industrial applications. They are often produced in microbial production hosts by fermentation. Robust protein production in the hosts and efficient downstream purification are two critical factors that could significantly reduce cost for microbial protein production by fermentation. Producing proteins/peptides as inclusion bodies in the hosts has the potential to achieve both high titers in fermentation and cost-effective downstream purification. Manipulation of the host cells such as overexpression/deletion of certain genes could lead to producing more and/or denser inclusion bodies. However, there are limited screening methods to help to identify beneficial genetic changes rendering more protein production and/or denser inclusion bodies. Results We report development and optimization of a simple density gradient method that can be used for distinguishing and sorting E. coli cells with different buoyant densities. We demonstrate utilization of the method to screen genetic libraries to identify a) expression of glyQS loci on plasmid that increased expression of a peptide of interest as well as the buoyant density of inclusion body producing E. coli cells; and b) deletion of a host gltA gene that increased the buoyant density of the inclusion body produced in the E. coli cells. Conclusion A novel density gradient sorting method was developed to screen genetic libraries. Beneficial host genetic changes could be exploited to improve recombinant protein expression as well as downstream protein purification. PMID:23638724

Screening and identification of genetic loci involved in producing more/denser inclusion bodies in Escherichia coli.

PubMed

Pandey, Neeraj; Sachan, Annapurna; Chen, Qi; Ruebling-Jass, Kristin; Bhalla, Ritu; Panguluri, Kiran Kumar; Rouviere, Pierre E; Cheng, Qiong

2013-05-02

Many proteins and peptides have been used in therapeutic or industrial applications. They are often produced in microbial production hosts by fermentation. Robust protein production in the hosts and efficient downstream purification are two critical factors that could significantly reduce cost for microbial protein production by fermentation. Producing proteins/peptides as inclusion bodies in the hosts has the potential to achieve both high titers in fermentation and cost-effective downstream purification. Manipulation of the host cells such as overexpression/deletion of certain genes could lead to producing more and/or denser inclusion bodies. However, there are limited screening methods to help to identify beneficial genetic changes rendering more protein production and/or denser inclusion bodies. We report development and optimization of a simple density gradient method that can be used for distinguishing and sorting E. coli cells with different buoyant densities. We demonstrate utilization of the method to screen genetic libraries to identify a) expression of glyQS loci on plasmid that increased expression of a peptide of interest as well as the buoyant density of inclusion body producing E. coli cells; and b) deletion of a host gltA gene that increased the buoyant density of the inclusion body produced in the E. coli cells. A novel density gradient sorting method was developed to screen genetic libraries. Beneficial host genetic changes could be exploited to improve recombinant protein expression as well as downstream protein purification.

Genetics of Bone Mineralization and Morphology in Inbred Mice: Analysis of the HcB/Dem Recombinant Congenic Strains

DTIC Science & Technology

2005-04-01

manuscript. RDB also thanks Dr. Barry that Kastl and colleagues (10) observed performance differences Rickman, Jon Deegan , Don Settergren, and Greg Bange for...11(5):547-53. Meitinger T. Pex gene deletions in Gy and Hyp mice provide mouse 18. Berndt T, Craig TA, Bowe AE, models for X-linked Vassiliadis J...manuscript, and Dr. S. Barry Rickman, between anatomic sites than ipsilateral data and that the Jon Deegan , and Greg Bange for helpful discussions. Dr

Expression and Purification of Soluble STAT5b/STAT3 Proteins for SH2 Domain Binding Assay.

PubMed

Asai, Akira; Takakuma, Kazuyuki

2017-01-01

When a large hydrophobic full-length protein is expressed in bacteria, it is often challenging to obtain recombinant proteins in the soluble fraction. One way to overcome this challenge is expression of deletion mutants that have improved solubility while maintaining biological activity. In this chapter, we describe a protocol for expression of truncated forms of STAT5b and STAT3 proteins that are soluble and retain SH2-mediated activity for phospho-Tyr peptide recognition.

Antigen expression level threshold tunes the fate of CD8 T cells during primary hepatic immune responses.

PubMed

Tay, Szun Szun; Wong, Yik Chun; McDonald, David M; Wood, Nicole A W; Roediger, Ben; Sierro, Frederic; Mcguffog, Claire; Alexander, Ian E; Bishop, G Alex; Gamble, Jennifer R; Weninger, Wolfgang; McCaughan, Geoffrey W; Bertolino, Patrick; Bowen, David G

2014-06-24

CD8 T-cell responses to liver-expressed antigens range from deletional tolerance to full effector differentiation resulting in overt hepatotoxicity. The reasons for these heterogeneous outcomes are not well understood. To identify factors that govern the fate of CD8 T cells activated by hepatocyte-expressed antigen, we exploited recombinant adenoassociated viral vectors that enabled us to vary potential parameters determining these outcomes in vivo. Our findings reveal a threshold of antigen expression within the liver as the dominant factor determining T-cell fate, irrespective of T-cell receptor affinity or antigen cross-presentation. Thus, when a low percentage of hepatocytes expressed cognate antigen, high-affinity T cells developed and maintained effector function, whereas, at a high percentage, they became functionally exhausted and silenced. Exhaustion was not irreversibly determined by initial activation, but was maintained by high intrahepatic antigen load during the early phase of the response; cytolytic function was restored when T cells primed under high antigen load conditions were transferred into an environment of low-level antigen expression. Our study reveals a hierarchy of factors dictating the fate of CD8 T cells during hepatic immune responses, and provides an explanation for the different immune outcomes observed in a variety of immune-mediated liver pathologic conditions.

Suppressed prostate epithelial development with impaired branching morphogenesis in mice lacking stromal fibromuscular androgen receptor.

PubMed

Lai, Kuo-Pao; Yamashita, Shinichi; Vitkus, Spencer; Shyr, Chih-Rong; Yeh, Shuyuan; Chang, Chawnshang

2012-01-01

Using the cre-loxP system, we generated a new mouse model [double stromal androgen receptor knockout (dARKO)] with selectively deleted androgen receptor (AR) in both stromal fibroblasts and smooth muscle cells, and found the size of the anterior prostate (AP) lobes was significantly reduced as compared with those from wild-type littermate controls. The reduction in prostate size of the dARKO mouse was accompanied by impaired branching morphogenesis and partial loss of the infolding glandular structure. Further dissection found decreased proliferation and increased apoptosis of the prostate epithelium in the dARKO mouse AP. These phenotype changes were further confirmed with newly established immortalized prostate stromal cells (PrSC) from wild-type and dARKO mice. Mechanistically, IGF-1, placental growth factor, and secreted phosphoprotein-1 controlled by stromal AR were differentially expressed in PrSC-wt and PrSC-ARKO. Moreover, the conditioned media (CM) from PrSC-wt promoted prostate epithelium growth significantly as compared with CM from PrSC-dARKO. Finally, adding IGF-1/placental growth factor recombinant proteins into PrSC-dARKO CM was able to partially rescue epithelium growth. Together, our data concluded that stromal fibromuscular AR could modulate epithelium growth and maintain cellular homeostasis through identified growth factors.

Genomic deletion of a long-range bone enhancer misregulatessclerostin in Van Buchem disease

DOE Office of Scientific and Technical Information (OSTI.GOV)

Loots, Gabriela G.; Kneissel, Michaela; Keller, Hansjoerg

2005-04-15

Mutations in distant regulatory elements can negatively impact human development and health, yet due to the difficulty of detecting these critical sequences we predominantly focus on coding sequences for diagnostic purposes. We have undertaken a comparative sequence-based approach to characterize a large noncoding region deleted in patients affected by Van Buchem disease (VB), a severe sclerosing bone dysplasia. Using BAC recombination and transgenesis we characterized the expression of human sclerostin (sost) from normal (hSOSTwt) or Van Buchem(hSOSTvb D) alleles. Only the hSOSTwt allele faithfully expressed high levels of human sost in the adult bone and impacted bone metabolism, consistent withmore » the model that the VB noncoding deletion removes a sost specific regulatory element. By exploiting cross-species sequence comparisons with in vitro and in vivo enhancer assays we were able to identify a candidate enhancer element that drives human sost expression in osteoblast-like cell lines in vitro and in the skeletal anlage of the E14.5 mouse embryo, and discovered a novel function for sclerostin during limb development. Our approach represents a framework for characterizing distant regulatory elements associated with abnormal human phenotypes.« less

Deletion of lactate dehydrogenase in Enterobacter aerogenes to enhance 2,3-butanediol production.

PubMed

Jung, Moo-Young; Ng, Chiam Yu; Song, Hyohak; Lee, Jinwon; Oh, Min-Kyu

2012-07-01

2,3-Butanediol is an important bio-based chemical product, because it can be converted into several C4 industrial chemicals. In this study, a lactate dehydrogenase-deleted mutant was constructed to improve 2,3-butanediol productivity in Enterobacter aerogenes. To delete the gene encoding lactate dehydrogenase, λ Red recombination method was successfully adapted for E. aerogenes. The resulting strain produced a very small amount of lactate and 16.7% more 2,3-butanediol than that of the wild-type strain in batch fermentation. The mutant and its parental strain were then cultured with six different carbon sources, and the mutant showed higher carbon source consumption and microbial growth rates in all media. The 2,3-butanediol titer reached 69.5 g/l in 54 h during fed-batch fermentation with the mutant,which was 27.4% higher than that with the parental strain.With further optimization of the medium and aeration conditions,118.05 g/l 2,3-butanediol was produced in 54 h during fed-batch fermentation with the mutant. This is by far the highest titer of 2,3-butanediol with E. aerogenes achieved by metabolic pathway engineering.

A mitochondrial locus is necessary for the synthesis of mitochondrial tRNA in the yeast Saccharomyces cerevisiae.

PubMed Central

Martin, N C; Underbrink-Lyon, K

1981-01-01

We have used a cloned yeast mitochondrial tRNAUCNSer gene as a probe to detect RNA species that are transcripts from this gene in wild-type Saccharomyces cerevisiae and in petite deletion mutants. In RNA from wild-type cells, the tRNA is the most prominent transcript of the gene. In RNA from deletion mutants that retain this gene but have lost other regions of mtDNA, high molecular weight transcripts containing the tRNAUCNSer sequences accumulate but tRNAUCNSer is not made. tRNAUCNSer synthesis can be restored in these mutants when they are mated to other deletion mutants that retain a different portion of the mitochondrial genome. Protein synthesis is not necessary for the restoration, and the restoration is not due to a nuclear effect or to an effect of mating alone, because strains without mtDNA are not able to restore tRNA synthesis. These results definitively demonstrate the existence of a yeast mitochondrial locus that is necessary for tRNA synthesis and, because the restoration of tRNAUCNSer synthesis appears to result from intergenic complementation, not recombination, indicate that this locus acts in trans. Images PMID:6795621

Ablation of TrkB expression in RGS9-2 cells leads to hyperphagic obesity★

PubMed Central

Liao, Guey-Ying; Li, Yuqing; Xu, Baoji

2013-01-01

Brain-derived neurotrophic factor (BDNF) and its cognate receptor, TrkB (tropomyosin receptor kinase B), are widely expressed in the brain where they regulate a wide variety of biological processes, including energy homeostasis. However, the specific population(s) of TrkB-expressing neurons through which BDNF governs energy homeostasis remain(s) to be determined. Using the Cre-loxP recombination system, we deleted the mouse TrkB gene in RGS9-2-expressing cells. In this mouse mutant, TrkB expression was abolished in several hypothalamic nuclei, including arcuate nucleus, dorsomedial hypothalamus, and lateral hypothalamus. TrkB expression was also abolished in a small number of cells in other brain regions, including the cerebral cortex and striatum. The mutant animals developed hyperphagic obesity with normal energy expenditure. Despite hyperglycemia under fed conditions, these animals exhibited normal fasting blood glucose levels and normal glucose tolerance. These results suggest that BDNF regulates energy homeostasis in part through TrkB-expressing neurons in the hypothalamus. PMID:24327964

Evolution of sfbI Encoding Streptococcal Fibronectin-Binding Protein I: Horizontal Genetic Transfer and Gene Mosaic Structure

PubMed Central

Towers, Rebecca J.; Fagan, Peter K.; Talay, Susanne R.; Currie, Bart J.; Sriprakash, Kadaba S.; Walker, Mark J.; Chhatwal, Gursharan S.

2003-01-01

Streptococcal fibronectin-binding protein is an important virulence factor involved in colonization and invasion of epithelial cells and tissues by Streptococcus pyogenes. In order to investigate the mechanisms involved in the evolution of sfbI, the sfbI genes from 54 strains were sequenced. Thirty-four distinct alleles were identified. Three principal mechanisms appear to have been involved in the evolution of sfbI. The amino-terminal aromatic amino acid-rich domain is the most variable region and is apparently generated by intergenic recombination of horizontally acquired DNA cassettes, resulting in a genetic mosaic in this region. Two distinct and divergent sequence types that shared only 61 to 70% identity were identified in the central proline-rich region, while variation at the 3′ end of the gene is due to deletion or duplication of defined repeat units. Potential antigenic and functional variabilities in SfbI imply significant selective pressure in vivo with direct implications for the microbial pathogenesis of S. pyogenes. PMID:14662917

New technology and resources for cryptococcal research

PubMed Central

Zhang, Nannan; Park, Yoon-Dong; Williamson, Peter R.

2014-01-01

Rapid advances in molecular biology and genome sequencing have enabled the generation of new technology and resources for cryptococcal research. RNAi-mediated specific gene knock down has become routine and more efficient by utilizing modified shRNA plasmids and convergent promoter RNAi constructs. This system was recently applied in a high-throughput screen to identify genes involved in host-pathogen interactions. Gene deletion efficiencies have also been improved by increasing rates of homologous recombination through a number of approaches, including a combination of double-joint PCR with split-marker transformation, the use of dominant selectable markers and the introduction of Cre-Loxp systems into Cryptococcus. Moreover, visualization of cryptococcal proteins has become more facile using fusions with codon-optimized fluorescent tags, such as green or red fluorescent proteins or, mCherry. Using recent genome-wide analytical tools, new transcriptional factors and regulatory proteins have been identified in novel virulence-related signaling pathways by employing microarray analysis, RNA-sequencing and proteomic analysis. PMID:25460849

Sequence family variant loss from the AZFc interval of the human Y chromosome, but not gene copy loss, is strongly associated with male infertility

PubMed Central

Machev, N; Saut, N; Longepied, G; Terriou, P; Navarro, A; Levy, N; Guichaoua, M; Metzler-Guillemai..., C; Collignon, P; Frances, A; Belougne, J; Clemente, E; Chiaroni, J; Chevillard, C; Durand, C; Ducourneau, A; Pech, N; McElreavey, K; Mattei, M; Mitchell, M

2004-01-01

Background: Complete deletion of the complete AZFc interval of the Y chromosome is the most common known genetic cause of human male infertility. Two partial AZFc deletions (gr/gr and b1/b3) that remove some copies of all AZFc genes have recently been identified in infertile and fertile populations, and an association study indicates that the resulting gene dose reduction represents a risk factor for spermatogenic failure. Methods: To determine the incidence of various partial AZFc deletions and their effect on fertility, we combined quantitative and qualitative analyses of the AZFc interval at the DAZ and CDY1 loci in 300 infertile men and 399 control men. Results: We detected 34 partial AZFc deletions (32 gr/gr deletions), arising from at least 19 independent deletion events, and found gr/gr deletion in 6% of infertile and 3.5% of control men (p>0.05). Our data provide evidence for two large AZFc inversion polymorphisms, and for relative hot and cold spots of unequal crossing over within the blocks of homology that mediate gr/gr deletion. Using SFVs (sequence family variants), we discriminate DAZ1/2, DAZ3/4, CDY1a (proximal), and CDY1b (distal) and define four types of DAZ-CDY1 gr/gr deletion. Conclusions: The only deletion type to show an association with infertility was DAZ3/4-CDY1a (p = 0.042), suggesting that most gr/gr deletions are neutral variants. We see a stronger association, however, between loss of the CDY1a SFV and infertility (p = 0.002). Thus, loss of this SFV through deletion or gene conversion could be a major risk factor for male infertility. PMID:15520406

A RecET-assisted CRISPR-Cas9 genome editing in Corynebacterium glutamicum.

PubMed

Wang, Bo; Hu, Qitiao; Zhang, Yu; Shi, Ruilin; Chai, Xin; Liu, Zhe; Shang, Xiuling; Zhang, Yun; Wen, Tingyi

2018-04-23

Extensive modification of genome is an efficient manner to regulate the metabolic network for producing target metabolites or non-native products using Corynebacterium glutamicum as a cell factory. Genome editing approaches by means of homologous recombination and counter-selection markers are laborious and time consuming due to multiple round manipulations and low editing efficiencies. The current two-plasmid-based CRISPR-Cas9 editing methods generate false positives due to the potential instability of Cas9 on the plasmid, and require a high transformation efficiency for co-occurrence of two plasmids transformation. Here, we developed a RecET-assisted CRISPR-Cas9 genome editing method using a chromosome-borne Cas9-RecET and a single plasmid harboring sgRNA and repair templates. The inducible expression of chromosomal RecET promoted the frequencies of homologous recombination, and increased the efficiency for gene deletion. Due to the high transformation efficiency of a single plasmid, this method enabled 10- and 20-kb region deletion, 2.5-, 5.7- and 7.5-kb expression cassette insertion and precise site-specific mutation, suggesting a versatility of this method. Deletion of argR and farR regulators as well as site-directed mutation of argB and pgi genes generated the mutant capable of accumulating L-arginine, indicating the stability of chromosome-borne Cas9 for iterative genome editing. Using this method, the model-predicted target genes were modified to redirect metabolic flux towards 1,2-propanediol biosynthetic pathway. The final engineered strain produced 6.75 ± 0.46 g/L of 1,2-propanediol that is the highest titer reported in C. glutamicum. Furthermore, this method is available for Corynebacterium pekinense 1.563, suggesting its universal applicability in other Corynebacterium species. The RecET-assisted CRISPR-Cas9 genome editing method will facilitate engineering of metabolic networks for the synthesis of interested bio-based products from renewable biomass using Corynebacterium species as cell factories.

The mitochondrial SIR2 related protein 2 (SIR2RP2) impacts Leishmania donovani growth and infectivity

PubMed Central

Mittal, Nimisha; Muthuswami, Rohini

2017-01-01

Background Leishmania donovani, a protozoan parasite is the major causative agent of visceral leishmaniasis. Increased toxicity and resistance to the existing repertoire of drugs has been reported. Hence, an urgent need exists for identifying newer drugs and drug targets. Previous reports have shown sirtuins (Silent Information Regulator) from kinetoplastids as promising drug targets. Leishmania species code for three SIR2 (Silent Information Regulator) related proteins. Here, we for the first time report the functional characterization of SIR2 related protein 2 (SIR2RP2) of L. donovani. Methodology Recombinant L. donovani SIR2RP2 was expressed in E. coli and purified. The enzymatic functions of SIR2RP2 were determined. The subcellular localization of LdSIR2RP2 was done by constructing C-terminal GFP-tagged full-length LdSIR2RP2. Deletion mutants of LdSIR2RP2 were generated in Leishmania by double targeted gene replacement methodology. These null mutants were tested for their proliferation, virulence, cell cycle defects, mitochondrial functioning and sensitivity to known SIR2 inhibitors. Conclusion Our data suggests that LdSIR2RP2 possesses NAD+-dependent ADP-ribosyltransferase activity. However, NAD+-dependent deacetylase and desuccinylase activities were not detected. The protein localises to the mitochondrion of the promastigotes. Gene deletion studies showed that ΔLdSIR2RP2 null mutants had restrictive growth phenotype associated with accumulation of cells in the G2/M phase and compromised mitochondrial functioning. The null mutants had attenuated infectivity. Deletion of LdSIR2RP2 resulted in increased sensitivity of the parasites to the known SIR2 inhibitors. The sirtuin inhibitors inhibited the ADP-ribosyltransferase activity of recombinant LdSIR2RP2. In conclusion, sirtuins could be used as potential new drug targets for visceral leishmaniasis. PMID:28493888

Preventive effects of the deleted form of hepatocyte growth factor against various liver injuries.

PubMed

Masunaga, H; Fujise, N; Shiota, A; Ogawa, H; Sato, Y; Imai, E; Yasuda, H; Higashio, K

1998-01-26

The effects of a naturally occurring deleted form of hepatocyte growth factor (HGF) on hepatic disorder were studied in various models of hepatic failure. The pretreatment of rats and mice with the deleted form of HGF prevented the liver injuries and coagulopathy induced by endotoxin, dimethylnitrosamine and acetaminophen and reduced the mortality due to hepatic dysfunction induced by these hepatotoxins. The concurrent administration of the deleted form of HGF also prevented the liver injury and hepatic fibrosis in mice treated with alpha-naphthylisothiocyanate and in rats treated with dimethylnitrosamine. Moreover, the deleted form of HGF normalized the results of the bromosulphalein-clearance test and ameliorated jaundice in rats with periportal cholangiolitic hepatopathy induced by alpha-naphthylisothiocyanate. The deleted form of HGF also reversed the coagulopathy in rats with hepatic disorder induced by dimethylnitrosamine or by 70% resection of cirrhotic liver (induced by carbon tetrachloride). In Long Evans cinnamon rats receiving vehicle, 20 out of 21 animals died within 4 days after the onset of jaundice. After infusion of the deleted form of HGF for 4 days, 7 out of 20 Long-Evans cinnamon rats survived. These results indicate that the deleted form of HGF could have therapeutic potency in patients with severe hepatic failure.

Characterization of a Diffusible Signaling Factor from Xylella fastidiosa

PubMed Central

Beaulieu, Ellen D.; Ionescu, Michael; Chatterjee, Subhadeep; Yokota, Kenji; Trauner, Dirk; Lindow, Steven

2013-01-01

ABSTRACT Cell-cell signaling in Xylella fastidiosa has been implicated in the coordination of traits enabling colonization in plant hosts as well as insect vectors. This cell density-dependent signaling has been attributed to a diffusible signaling factor (DSF) produced by the DSF synthase RpfF. DSF produced by related bacterial species are unsaturated fatty acids, but that of X. fastidiosa was thought to be different from those of other taxa. We describe here the isolation and characterization of an X. fastidiosa DSF (XfDSF) as 2(Z)-tetradecenoic acid. This compound was isolated both from recombinant Erwinia herbicola expressing X. fastidiosa rpfF and from an X. fastidiosa rpfC deletion mutant that overproduces DSF. Since an rpfF mutant is impaired in biofilm formation and underexpresses the hemagglutinin-like protein-encoding genes hxfA and hxfB, we demonstrate that these traits can be restored by ca. 0.5 µM XfDSF but not by myristic acid, the fully saturated tetradecenoic acid. A phoA-based X. fastidiosa biosensor that assesses DSF-dependent expression of hxfA or hxfB revealed a high level of molecular specificity of DSF signaling. PMID:23300249

Bombyx mori nucleopolyhedrovirus ORF79 is a per os infectivity factor associated with the PIF complex.

PubMed

Dong, Zhan-Qi; Zhang, Jun; Chen, Xue-Mei; He, Qian; Cao, Ming-Ya; Wang, La; Li, Hai-Qing; Xiao, Wen-Fu; Pan, Cai-Xia; Lu, Cheng; Pan, Min-Hui

2014-05-12

Bombyx mori nucleopolyhedrovirus (BmNPV) ORF79 (Bm79) encodes an occlusion-derived virus (ODV)-specific envelope protein, which is a homologue of the per os infectivity factor 4 (PIF4) of Autographa californica multiple nucleopolyhedrovirus (AcMNPV). To investigate the role of ORF79 in the BmNPV life cycle, a Bm79 knockout virus (vBm(Bm79KO)) was constructed through homologous recombination in Escherichia coli. Viral DNA replication, budded virus (BV) production and polyhedra formation were unaffected by the absence of BM79. However, results of the larval bioassay demonstrated that the Bm79 deletion resulted in a complete loss of per os infection. Immunofluorescence analysis showed that BM79 localized at the innernuclear membrane of infected cells through its N-terminal sorting motif (SM). Further bimolecular fluorescence protein complementation and co-immunoprecipitation assays demonstrated the interaction of BM79 with PIF1, PIF2, PIF3 and ODV-E66. Thus, BM79 plays an important role in per os infection and is associated with the viral PIF complex of BmNPV. Copyright © 2014 Elsevier B.V. All rights reserved.

PecS regulates the urate-responsive expression of type 1 fimbriae in Klebsiella pneumoniae CG43.

PubMed

Wang, Zhe-Chong; Liu, Chia-Jui; Huang, Ying-Jung; Wang, Yu-Seng; Peng, Hwei-Ling

2015-12-01

In the Klebsiella pneumoniae CG43 genome, the divergently transcribed genes coding for PecS, the MarR-type transcription factor, and PecM, the drug metabolite transporter, are located between the type 1 and type 3 fimbrial gene clusters. The intergenic sequence pecO between pecS and pecM contains three putative PecS binding sites and a CpxR box. Electrophoretic mobility shift assay revealed that the recombinant PecS and CpxR could specifically bind to the pecO sequence, and the specific interaction of PecS and pecO could be attenuated by urate. The expression of pecS and pecM was negatively regulated by CpxAR and PecS, and was inducible by exogenous urate in the absence of cpxAR. Compared with CG43S3ΔcpxAR, the derived mutants CG43S3ΔcpxARΔpecS and CG43S3ΔcpxARΔpecSΔpecM exerted similar levels of sensitivity to H2O2 or paraquat, but higher levels of mannose-sensitive yeast agglutination activity and FimA production. The promoter activity and transcript levels of fimA in CG43S3ΔcpxAR were also increased by deleting pecS. However, no binding activity between PecS and the fimA promoter could be observed. Nevertheless, PecS deletion could reduce the expression of the global regulator HNS and release the negative effect of HNS on FimA expression. In CG43S3ΔcpxAR, the expression of FimA as well as PecS was inducible by urate, whilst urate-induced FimA expression was inhibited by the deletion of pecS. Taken together, we propose that K. pneumoniae PecS indirectly and negatively regulates the expression of type 1 fimbriae, and the regulation is urate-inducible in the absence of CpxAR.

EPHA4 haploinsufficiency is responsible for the short stature of a patient with 2q35-q36.2 deletion and Waardenburg syndrome.

PubMed

Li, Chuan; Chen, Rongyu; Fan, Xin; Luo, Jingsi; Qian, Jiale; Wang, Jin; Xie, Bobo; Shen, Yiping; Chen, Shaoke

2015-04-11

Waardenburg syndrome type I (WS1), an auditory-pigmentary genetic disorder, is caused by heterozygous loss-of-function mutations in PAX3. Abnormal physical signs such as dystopia canthorum, patchy hypopigmentation and sensorineural hearing loss are common, but short stature is not associated with WS1. We reported a 4-year and 6 month-old boy with a rare combination of WS1 and severe short stature (83.5 cm (-5.8SD)). His facial features include dystopia canthorum, mild synophrys, slightly up-slanted palpebral fissure, posteriorly rotated ears, alae nasi hypoplasia and micrognathia. No heterochromia was noticed. He had a normal intelligence quotient and hearing. Insulin-like growth factor-1 (IGF-1) was 52.7 ng/ml, lower than the normal range (55 ~ 452 ng/ml) and the peak growth hormone level was 7.57 ng/ml at 90 minutes after taking moderate levodopa and pyridostigmine bromide. The patient exhibited a good response to human growth hormone (rhGH) replacement therapy, showing a 9.2 cm/year growth rate and an improvement of 1 standard deviation (SD) of height after one year treatment. CMA test of patient's DNA revealed a 4.46 Mb de novo deletion at 2q35-q36.2 (hg19; chr2:221,234,146-225,697,363). PAX3 haploinsufficiency is known to cause Waardenburg syndrome. Examining overlapping deletions in patients led to the conclusion that EPHA4 is a novel short stature gene. The finding is supported by the splotch-retarded and epha4 knockout mouse models which both showed growth retardation. We believe this rare condition is caused by the haploinsufficiency of both PAX3 and EPH4 genes. We further reported a growth response to recombinant human growth hormone treatment in this patient.

Further delineation of nonhomologous-based recombination and evidence for subtelomeric segmental duplications in 1p36 rearrangements.

PubMed

D'Angelo, Carla S; Gajecka, Marzena; Kim, Chong A; Gentles, Andrew J; Glotzbach, Caron D; Shaffer, Lisa G; Koiffmann, Célia P

2009-06-01

The mechanisms involved in the formation of subtelomeric rearrangements are now beginning to be elucidated. Breakpoint sequencing analysis of 1p36 rearrangements has made important contributions to this line of inquiry. Despite the unique architecture of segmental duplications inherent to human subtelomeres, no common mechanism has been identified thus far and different nonexclusive recombination-repair mechanisms seem to predominate. In order to gain further insights into the mechanisms of chromosome breakage, repair, and stabilization mediating subtelomeric rearrangements in humans, we investigated the constitutional rearrangements of 1p36. Cloning of the breakpoint junctions in a complex rearrangement and three non-reciprocal translocations revealed similarities at the junctions, such as microhomology of up to three nucleotides, along with no significant sequence identity in close proximity to the breakpoint regions. All the breakpoints appeared to be unique and their occurrence was limited to non-repetitive, unique DNA sequences. Several recombination- or cleavage-associated motifs that may promote non-homologous recombination were observed in close proximity to the junctions. We conclude that NHEJ is likely the mechanism of DNA repair that generates these rearrangements. Additionally, two apparently pure terminal deletions were also investigated, and the refinement of the breakpoint regions identified two distinct genomic intervals ~25-kb apart, each containing a series of 1p36 specific segmental duplications with 90-98% identity. Segmental duplications can serve as substrates for ectopic homologous recombination or stimulate genomic rearrangements.

Induction of homologous recombination following in utero exposure to DNA-damaging agents.

PubMed

Karia, Bijal; Martinez, Jo Ann; Bishop, Alexander J R

2013-11-01

Much of our understanding of homologous recombination, as well as the development of the working models for these processes, has been derived from extensive work in model organisms, such as yeast and fruit flies, and mammalian systems by studying the repair of induced double strand breaks or repair following exposure to genotoxic agents in vitro. We therefore set out to expand this in vitro work to ask whether DNA-damaging agents with varying modes of action could induce somatic change in an in vivo mouse model of homologous recombination. We exposed pregnant dams to DNA-damaging agents, conferring a variety of lesions at a specific time in embryo development. To monitor homologous recombination frequency, we used the well-established retinal pigment epithelium pink-eyed unstable assay. Homologous recombination resulting in the deletion of a duplicated 70 kb fragment in the coding region of the Oca2 gene renders this gene functional and can be visualized as a pigmented eyespot in the retinal pigment epithelium. We observed an increased frequency of pigmented eyespots in resultant litters following exposure to cisplatin, methyl methanesulfonate, ethyl methanesulfonate, 3-aminobenzamide, bleomycin, and etoposide with a contrasting decrease in the frequency of detectable reversion events following camptothecin and hydroxyurea exposure. The somatic genomic rearrangements that result from such a wide variety of differently acting damaging agents implies long-term potential effects from even short-term in utero exposures. Copyright © 2013 Elsevier B.V. All rights reserved.

The use of recombinant factor VIIa in a patient with Noonan syndrome and life-threatening bleeding.

PubMed

Tofil, Nancy M; Winkler, Margaret K; Watts, Raymond G; Noonan, Jacqueline

2005-05-01

To present a case report of a patient with Noonan syndrome who developed life-threatening gastrointestinal bleeding shortly after cardiac surgery that was successfully treated with recombinant factor VIIa. Case report. Pediatric intensive care unit of a children's hospital. Ten-month-old with Noonan syndrome and massive gastrointestinal bleeding resulting in severe hypovolemic shock. Recombinant factor VIIa was used in this patient's severe bleeding associated with Noonan syndrome after no other supportive measures were successful. Recombinant Factor VIIa significantly decreased the patient's bleeding and allowed his hypovolemic shock to improve. Ultimately, the patient made a complete recovery. Noonan syndrome has a constellation of both cardiac and noncardiac malformations including an increased risk of bleeding, and recombinant factor VIIa is an important agent in the treatment of significant bleeding.

Transcriptional requirements of the distal heavy-strand promoter of mtDNA

PubMed Central

Zollo, Ornella; Tiranti, Valeria; Sondheimer, Neal

2012-01-01

The heavy strand of mtDNA contains two promoters with nonoverlapping functions. The role of the minor heavy-strand promoter (HSP2) is controversial, because the promoter has been difficult to activate in an in vitro system. We have isolated HSP2 by excluding its interaction with the more powerful HSP1 promoter, and we find that it is transcribed efficiently by recombinant mtRNA polymerase and mitochondrial transcription factor B2. The mitochondrial transcription factor A is not required for initiation, but it has the ability to alternatively activate and repress the HSP2 transcriptional unit depending on the ratio between mitochondrial transcription factor A and other transcription factors. The positioning of transcriptional initiation agrees with our current understanding of HSP2 activity in vivo. Serial deletion of HSP2 shows that only proximal sequences are required. Several mutations, including the disruption of a polycytosine track upstream of the HSP2 initiation site, influence transcriptional activity. Transcription from HSP2 is also observed when HeLa cell mitochondrial extract is used as the source of mitochondrial polymerase, and this transcription is maintained when HSP2 is provided in proper spacing and context to the HSP1 promoter. Studies of the linked heavy-strand promoters show that they are differentially regulated by ATP dosage. We conclude that HSP2 is transcribed and has features that allow it to regulate mitochondrial mRNA synthesis. PMID:22454497

Genetic engineering approach to develop next-generation reagents for endotoxin quantification.

PubMed

Mizumura, Hikaru; Ogura, Norihiko; Aketagawa, Jun; Aizawa, Maki; Kobayashi, Yuki; Kawabata, Shun-Ichiro; Oda, Toshio

2017-02-01

The bacterial endotoxin test, which uses amebocyte lysate reagents of horseshoe crab origin, is a sensitive, reproducible and simple assay to measure endotoxin concentration. To develop sustainable raw materials for lysate reagents that do not require horseshoe crabs, three recombinant protease zymogens (factor C, derived from mammalian cells; factor B; and the proclotting enzyme derived from insect cells) were prepared using a genetic engineering technique. Recombinant cascade reagents (RCRs) were then prepared to reconstruct the reaction cascade in the amebocyte lysate reagent. The protease activity of the RCR containing recombinant factor C was much greater than that of recombinant factor C alone, indicating the efficiency of signal amplification in the cascade. Compared with the RCR containing the insect cell-derived factor C, those containing mammalian cell-derived factor C, which features different glycosylation patterns, were less susceptible to interference by the injectable drug components. The standard curve of the RCR containing mammalian cell-derived recombinant factor C had a steeper slope than the curves for those containing natural lysate reagents, suggesting a greater sensitivity to endotoxin. The present study supports the future production of recombinant reagents that do not require the use of natural resources.

Genetic engineering approach to develop next-generation reagents for endotoxin quantification

PubMed Central

Ogura, Norihiko; Aketagawa, Jun; Aizawa, Maki; Kobayashi, Yuki; Kawabata, Shun-ichiro; Oda, Toshio

2016-01-01

The bacterial endotoxin test, which uses amebocyte lysate reagents of horseshoe crab origin, is a sensitive, reproducible and simple assay to measure endotoxin concentration. To develop sustainable raw materials for lysate reagents that do not require horseshoe crabs, three recombinant protease zymogens (factor C, derived from mammalian cells; factor B; and the proclotting enzyme derived from insect cells) were prepared using a genetic engineering technique. Recombinant cascade reagents (RCRs) were then prepared to reconstruct the reaction cascade in the amebocyte lysate reagent. The protease activity of the RCR containing recombinant factor C was much greater than that of recombinant factor C alone, indicating the efficiency of signal amplification in the cascade. Compared with the RCR containing the insect cell-derived factor C, those containing mammalian cell-derived factor C, which features different glycosylation patterns, were less susceptible to interference by the injectable drug components. The standard curve of the RCR containing mammalian cell-derived recombinant factor C had a steeper slope than the curves for those containing natural lysate reagents, suggesting a greater sensitivity to endotoxin. The present study supports the future production of recombinant reagents that do not require the use of natural resources. PMID:27913792

Noncore RAG1 regions promote Vβ rearrangements and αβ T cell development by overcoming inherent inefficiency of Vβ recombination signal sequences.

PubMed

Horowitz, Julie E; Bassing, Craig H

2014-02-15

The RAG proteins are comprised of core endonuclease domains and noncore regions that modulate endonuclease activity. Mutation or deletion of noncore RAG regions in humans causes immunodeficiency and altered TCR repertoire, and mice expressing core but not full-length Rag1 (Rag1(C/C)) or Rag2 (Rag2(C/C)) exhibit lymphopenia, reflecting impaired V(D)J recombination and lymphocyte development. Rag1(C/C) mice display reduced D-to-J and V-to-DJ rearrangements of TCRβ and IgH loci, whereas Rag2(C/C) mice show decreased V-to-DJ rearrangements and altered Vβ/VH repertoire. Because Vβs/VHs only recombine to DJ complexes, the Rag1(C/C) phenotype could reflect roles for noncore RAG1 regions in promoting recombination during only the D-to-J step or during both steps. In this study, we demonstrate that a preassembled TCRβ gene, but not a preassembled DβJβ complex or the prosurvival BCL2 protein, completely rescues αβ T cell development in Rag1(C/C) mice. We find that Rag1(C/C) mice exhibit altered Vβ utilization in Vβ-to-DJβ rearrangements, increased usage of 3'Jα gene segments in Vα-to-Jα rearrangements, and abnormal changes in Vβ repertoire during αβ TCR selection. Inefficient Vβ/VH recombination signal sequences (RSSs) have been hypothesized to cause impaired V-to-DJ recombination on the background of a defective recombinase as in core-Rag mice. We show that replacement of the Vβ14 RSS with a more efficient RSS increases Vβ14 recombination and rescues αβ T cell development in Rag1(C/C) mice. Our data indicate that noncore RAG1 regions establish a diverse TCR repertoire by overcoming Vβ RSS inefficiency to promote Vβ recombination and αβ T cell development, and by modulating TCRβ and TCRα gene segment utilization.

Molecular characterization of a novel Muscovy duck parvovirus isolate: evidence of recombination between classical MDPV and goose parvovirus strains.

PubMed

Wang, Jianye; Ling, Jueyi; Wang, Zhixian; Huang, Yu; Zhu, Jianzhong; Zhu, Guoqiang

2017-11-09

Muscovy duck parvovirus (MDPV) and Goose parvovirus (GPV) are important etiological agents for Muscovy duck parvoviral disease and Derzsy's disease, respectively; both of which can cause substantial economic losses in waterfowl industry. In contrast to GPV, the complete genomic sequence data of MDPV isolates are still limited and their phylogenetic relationships largely remain unknown. In this study, the entire genome of a pathogenic MDPV strain ZW, which was isolated from a deceased Muscovy duckling in 2006 in China, was cloned, sequenced, and compared with that of other classical MDPV and GPV strains. The genome of strain ZW comprises of 5071 nucleotides; this genome was shorter than that of the pathogenic MDPV strain YY (5075 nt). All the four deleted nucleotides produced in strain ZW are located at the base-pairing positions in the palindromic stem of inverted terminal repeats (ITR) without influencing the formation of a hairpin structure. Recombination analysis revealed that strain ZW originated from genetic recombination between the classical MDPV and GPV strain. The YY strain of MDPV acts as the major parent, whereas the virulent strains YZ99-6 and B and the vaccine strain SYG61v of GPV act as the minor parents in varying degrees. Two recombination sites were detected in strain ZW, with the small recombination site surrounding the P9 promoter, and the large recombination site situated in the middle of the VP3 gene. The SYG61V strain is a vaccine strain used for preventing goose parvoviral disease. This strain was found to be solely involved in the recombination event detected in the P9 promoter region. Phylogenetic analyses between strain ZW and other classical strains of MDPV and GPV were performed. The results supported the in silico recombination analysis conclusion. MDPV Strain ZW is a novel recombinant parvovirus, and the bulk of its genome originates from the classical MDPV strain. Two virulent strains and a vaccine strain of GPV were involved in the recombination process in varying degrees.

Trap-assisted and Langevin-type recombination in organic light-emitting diodes

NASA Astrophysics Data System (ADS)

Wetzelaer, G. A. H.; Kuik, M.; Nicolai, H. T.; Blom, P. W. M.

2011-04-01

Trapping of charges is known to play an important role in the charge transport of organic semiconductors, but the role of traps in the recombination process has not been addressed. Here we show that the ideality factor of the current of organic light-emitting diodes (OLEDs) in the diffusion-dominated regime has a temperature-independent value of 2, which reveals that nonradiative trap-assisted recombination dominates the current. In contrast, the ideality factor of the light output approaches unity, demonstrating that luminance is governed by recombination of the bimolecular Langevin type. This apparent contradiction can be resolved by measuring the current and luminance ideality factor for a white-emitting polymer, where both free and trapped charge carriers recombine radiatively. With increasing bias voltage, Langevin recombination becomes dominant over trap-assisted recombination due to its stronger dependence on carrier density, leading to an enhancement in OLED efficiency.

Response to Growth Hormone Treatment in a Patient with Insulin-Like Growth Factor 1 Receptor Deletion

PubMed Central

Mahmoud, Ranim; Naidu, Ajanta; Risheg, Hiba; Kimonis, Virginia

2017-01-01

We report a six-year-old boy who presented with short stature, microcephaly, dysmorphic features, and developmental delay and who was identified with a terminal deletion of 15q26.2q26.3 containing the insulin-like growth factor receptor (IGF1R) gene in addition to a terminal duplication of the 4q35.1q35.2 region. We compare our case with other reports of deletions and mutations affecting the IGF1R gene associated with pre-and postnatal growth restriction. We report the dramatic response to growth hormone therapy in this patient which highlights the importance of identifying patients with IGF1R deletion and treating them early. PMID:28720553

Response to Growth Hormone Treatment in a Patient with Insulin-Like Growth Factor 1 Receptor Deletion.

PubMed

Mahmoud, Ranim; Naidu, Ajanta; Risheg, Hiba; Kimonis, Virginia

2017-12-15

We report a six-year-old boy who presented with short stature, microcephaly, dysmorphic features, and developmental delay and who was identified with a terminal deletion of 15q26.2q26.3 containing the insulin-like growth factor receptor (IGF1R) gene in addition to a terminal duplication of the 4q35.1q35.2 region. We compare our case with other reports of deletions and mutations affecting the IGF1R gene associated with pre-and postnatal growth restriction. We report the dramatic response to growth hormone therapy in this patient which highlights the importance of identifying patients with IGF1R deletion and treating them early.

Analysis of chromosome 22 deletions in neurofibromatosis type 2-related tumors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wolff, R.K.; Frazer, K.A.; Jackler, R.K.

The neurofibromatosis type 2 (NF2) gene has been hypothesized to be a recessive tumor suppressor, with mutations at the same locus on chromosome 22 that lead to NF2 also leading to sporadic tumors of the types seen in NF2. Flanking markers for this gene have previously been defined as D22S1 centromeric and D22S28 telomeric. Identification of subregions of this interval that are consistently rearranged in the NF2-related tumors would aid in better defining the disease locus. To this end, the authors have compared tumor and constitutional DNAs, isolated from 39 unrelated patients with sporadic and NF2-associated acoustic neuromas, meningiomas, schwannomas,more » and ependymomas, at eight polymorphic loci on chromosome 22. Two of the tumors studied revealed loss-of-heterozygosity patterns, which is consistent with the presence of chromosome 22 terminal deletions. By using additional polymorphic markers, the terminal deletion breakpoint found in one of the tumors, an acoustic neuroma from an NF2 patient, was mapped within the previously defined NF2 region. The breakpoint occurred between the haplotyped markers D22S41/D22S46 and D22S56. This finding redefines the proximal flanking marker and localizes the NF2 gene between markers D22S41/D22S46 and D22S28. In addition, the authors identified a sporadic acoustic neuroma that reveals a loss-of-heterozygosity pattern consistent with mitotic recombination or deletion and reduplication, which are mechanisms not previously seen in studies of these tumors. This finding, while inconsistent with models of tumorigenesis that invoke single deletions and their gene-dosage effects, lends further support to the recessive tumor-suppressor model. 33 refs., 2 figs., 1 tab.« less

Boosting Anaplerotic Reactions by Pyruvate Kinase Gene Deletion and Phosphoenolpyruvate Carboxylase Desensitization for Glutamic Acid and Lysine Production in Corynebacterium glutamicum.

PubMed

Yokota, Atsushi; Sawada, Kazunori; Wada, Masaru

In the 1980s, Shiio and coworkers demonstrated using random mutagenesis that the following three phenotypes were effective for boosting lysine production by Corynebacterium glutamicum: (1) low-activity-level citrate synthase (CS L ), (2) phosphoenolpyruvate carboxylase (PEPC) resistant to feedback inhibition by aspartic acid (PEPC R ), and (3) pyruvate kinase (PYK) deficiency. Here, we reevaluated these phenotypes and their interrelationship in lysine production using recombinant DNA techniques.The pyk deletion and PEPC R (D299N in ppc) independently showed marginal effects on lysine production, but both phenotypes synergistically increased lysine yield, demonstrating the importance of PEPC as an anaplerotic enzyme in lysine production. Similar effects were also found for glutamic acid production. CS L (S252C in gltA) further increased lysine yield. Thus, using molecular techniques, the combination of these three phenotypes was reconfirmed to be effective for lysine production. However, a simple CS L mutant showed instabilities in growth and lysine yield.Surprisingly, the pyk deletion was found to increase biomass production in wild-type C. glutamicum ATCC13032 under biotin-sufficient conditions. The mutant showed a 37% increase in growth (based on OD 660 ) compared with the ATCC13032 strain in a complex medium containing 100 g/L glucose. Metabolome analysis revealed the intracellular accumulation of excess precursor metabolites. Thus, their conversion into biomass was considered to relieve the metabolic distortion in the pyk-deleted mutant. Detailed physiological studies of various pyk-deleted mutants also suggested that malate:quinone oxidoreductase (MQO) is important to control both the intracellular oxaloacetic acid (OAA) level and respiration rate. These findings may facilitate the rational use of C. glutamicum in fermentation industries.

Construction of brewing-wine Aspergillus oryzae pyrG- mutant by pyrG gene deletion and its application in homology transformation.

PubMed

Du, Yu; Xie, Guizhen; Yang, Chunfa; Fang, Baishan; Chen, Hongwen

2014-06-01

pyrG(-) host cells are indispensable for pyrG(-) based transformation system. Isolations of pyrG(-) host cells by random mutations are limited by time-consuming, unclear genetic background and potential interferences of homogenous recombination. The purpose of this study was to construct brewing-wine Aspergillus oryzae pyrG(-) mutant by site-directed mutation of pyrG gene deletion which would be used as a host for further transformation. pMD-pyrGAB, a vector carrying pyrG deletion cassette, was used to construct pyrG(-) mutant of A. oryzae. Three stable pyrG deletion mutants of A. oryzae were isolated by resistant to 5-fluoroorotic acid and confirmed by polymerase chain reaction analysis, indicating that pyrG was completely excised. The ΔpyrG mutants were applied as pyrG(-) host cells to disrupt xdh gene encoding xylitol dehydrogenase, which involves in xylitol production of A. oryzae. The xdh disruption mutants were efficiently constructed by transforming a pMD-pyrG-xdh disruption plasmid carrying pyrG, and the produced xylitol concentration of the Δxdh mutant was three times as much as that of the ΔpyrG recipient. Site-directed pyrG gene deletion is thus an effective way for the isolation of pyrG(-) host cells, and the established host-vector system could be applied in further functional genomics analysis and molecular breeding of A. oryzae. © The Author 2014. Published by ABBS Editorial Office in association with Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.

Increased metabolite production by deletion of an HDA1-type histone deacetylase in the phytopathogenic fungi, Magnaporthe oryzae (Pyricularia oryzae) and Fusarium asiaticum.

PubMed

Maeda, K; Izawa, M; Nakajima, Y; Jin, Q; Hirose, T; Nakamura, T; Koshino, H; Kanamaru, K; Ohsato, S; Kamakura, T; Kobayashi, T; Yoshida, M; Kimura, M

2017-11-01

Histone deacetylases (HDACs) play an important role in the regulation of chromatin structure and gene expression. We found that dark pigmentation of Magnaporthe oryzae (anamorph Pyricularia oryzae) ΔMohda1, a mutant strain in which an orthologue of the yeast HDA1 was disrupted by double cross-over homologous recombination, was significantly stimulated in liquid culture. Analysis of metabolites in a ΔMohda1 mutant culture revealed that the accumulation of shunt products of the 1,8-dihydroxynaphthalene melanin and ergosterol pathways were significantly enhanced compared to the wild-type strain. Northern blot analysis of the ΔMohda1 mutant revealed transcriptional activation of three melanin genes that are dispersed throughout the genome of M. oryzae. The effect of deletion of the yeast HDA1 orthologue was also observed in Fusarium asiaticum from the Fusarium graminearum species complex; the HDF2 deletion mutant produced increased levels of nivalenol-type trichothecenes. These results suggest that histone modification via HDA1-type HDAC regulates the production of natural products in filamentous fungi. Natural products of fungi have significant impacts on human welfare, in both detrimental and beneficial ways. Although HDA1-type histone deacetylase is not essential for vegetative growth, deletion of the gene affects the expression of clustered secondary metabolite genes in some fungi. Here, we report that such phenomena are also observed in physically unlinked genes required for melanin biosynthesis in the rice blast fungus. In addition, production of Fusarium trichothecenes, previously reported to be unaffected by HDA1 deletion, was significantly upregulated in another Fusarium species. Thus, the HDA1-inactivation strategy may be regarded as a general approach for overproduction and/or discovery of fungal metabolites. © 2017 The Society for Applied Microbiology.

Analysis of chromosome 22 deletions in neurofibromatosis type 2-related tumors.

PubMed

Wolff, R K; Frazer, K A; Jackler, R K; Lanser, M J; Pitts, L H; Cox, D R

1992-09-01

The neurofibromatosis type 2 (NF2) gene has been hypothesized to be a recessive tumor suppressor, with mutations at the same locus on chromosome 22 that lead to NF2 also leading to sporadic tumors of the types seen in NF2. Flanking markers for this gene have previously been defined as D22S1 centromeric and D22S28 telomeric. Identification of subregions of this interval that are consistently rearranged in the NF2-related tumors would aid in better defining the disease locus. To this end, we have compared tumor and constitutional DNAs, isolated from 39 unrelated patients with sporadic and NF2-associated acoustic neuromas, meningiomas, schwannomas, and ependymomas, at eight polymorphic loci on chromosome 22. Two of the tumors studied revealed loss-of-heterozygosity patterns, which is consistent with the presence of chromosome 22 terminal deletions. By using additional polymorphic markers, the terminal deletion breakpoint found in one of the tumors, an acoustic neuroma from an NF2 patient, was mapped within the previously defined NF2 region. The breakpoint occurred between the haplotyped markers D22S41/D22S46 and D22S56. This finding redefines the proximal flanking marker and localizes the NF2 gene between markers D22S41/D22S46 and D22S28. In addition, we identified a sporadic acoustic neuroma that reveals a loss-of-heterozygosity pattern consistent with mitotic recombination or deletion and reduplication, which are mechanisms not previously seen in studies of these tumors. This finding, while inconsistent with models of tumorigenesis that invoke single deletions and their gene-dosage effects, lends further support to the recessive tumor-suppressor model.

Analysis of chromosome 22 deletions in neurofibromatosis type 2-related tumors.

PubMed Central

Wolff, R K; Frazer, K A; Jackler, R K; Lanser, M J; Pitts, L H; Cox, D R

1992-01-01

The neurofibromatosis type 2 (NF2) gene has been hypothesized to be a recessive tumor suppressor, with mutations at the same locus on chromosome 22 that lead to NF2 also leading to sporadic tumors of the types seen in NF2. Flanking markers for this gene have previously been defined as D22S1 centromeric and D22S28 telomeric. Identification of subregions of this interval that are consistently rearranged in the NF2-related tumors would aid in better defining the disease locus. To this end, we have compared tumor and constitutional DNAs, isolated from 39 unrelated patients with sporadic and NF2-associated acoustic neuromas, meningiomas, schwannomas, and ependymomas, at eight polymorphic loci on chromosome 22. Two of the tumors studied revealed loss-of-heterozygosity patterns, which is consistent with the presence of chromosome 22 terminal deletions. By using additional polymorphic markers, the terminal deletion breakpoint found in one of the tumors, an acoustic neuroma from an NF2 patient, was mapped within the previously defined NF2 region. The breakpoint occurred between the haplotyped markers D22S41/D22S46 and D22S56. This finding redefines the proximal flanking marker and localizes the NF2 gene between markers D22S41/D22S46 and D22S28. In addition, we identified a sporadic acoustic neuroma that reveals a loss-of-heterozygosity pattern consistent with mitotic recombination or deletion and reduplication, which are mechanisms not previously seen in studies of these tumors. This finding, while inconsistent with models of tumorigenesis that invoke single deletions and their gene-dosage effects, lends further support to the recessive tumor-suppressor model. Images Figure 1 Figure 2 PMID:1496981

Molecular characterization of a polymorphic 3-Mb deletion at chromosome Yp11.2 containing the AMELY locus in Singapore and Malaysia populations.

PubMed

Yong, Rita Y Y; Gan, Linda S H; Chang, Yuet Meng; Yap, Eric P H

2007-11-01

Amelogenin paralogs on Chromosome X (AMELX) and Y (AMELY) are commonly used sexing markers. Interstitial deletion of Yp involving the AMELY locus has previously been reported. The combined frequency of the AMELY null allele in Singapore and Malaysia populations is 2.7%, 0.6% in Indian and Malay ethnic groups respectively. It is absent among 541 Chinese screened. The null allele in this study belongs to 3 Y haplogroups; J2e1 (85.7%), F* (9.5%) and D* (4.8%). Low and high-resolution STS mapping, followed by sequence analysis of breakpoint junction confirmed a large deletion of 3 to 3.7-Mb located at the Yp11.2 region. Both breakpoints were located in TSPY repeat arrays, suggesting a non-allelic homologous recombination (NAHR) mechanism of deletion. All regional null samples shared identical breakpoint sequences according to their haplogroup affiliation, providing molecular evidence of a common ancestry origin for each haplogroup, and at least 3 independent deletion events recurred in history. The estimated ages based on Y-SNP and STR analysis were approximately 13.5 +/- 3.1 kyears and approximately 0.9 +/- 0.9 kyears for the J2e1 and F* mutations, respectively. A novel polymorphism G > A at Y-GATA-H4 locus in complete linkage disequilibrium with J2e1 null mutations is a more recent event. This work re-emphasizes the need to include other sexing markers for gender determination in certain regional populations. The frequency difference among global populations suggests it constitutes another structural variation locus of human chromosome Y. The breakpoint sequences provide further information to a better understanding of the NAHR mechanism and DNA rearrangements due to higher order genomic architecture.

Disparities in visuo-spatial constructive abilities in Williams syndrome patients with typical deletion on chromosome 7q11.23.

PubMed

Muramatsu, Yukako; Tokita, Yoshihito; Mizuno, Seiji; Nakamura, Miho

2017-02-01

Williams syndrome (WS) is known for its uneven cognitive abilities, especially the difficulty in visuo-spatial cognition, though there are some inter-individual phenotypic differences. It has been proposed that the difficulty in visuo-spatial cognition of WS patients can be attributed to a haploinsufficiency of some genes located on the deleted region in 7q11.23, based on an examination of atypical deletions identified in WS patients with atypical cognitive deficits. According to this hypothesis, the inter-individual differences in visuo-spatial cognitive ability arise from variations in deletion. We investigated whether there were inter-individual differences in the visuo-spatial constructive abilities of five unrelated WS patients with the typical deletion on chromosome 7q11.23 that includes the candidate genes contributing visuo-spatial difficulty in WS patients. We used tests with three-dimensional factors such as Benton's three-dimensional block construction test, which are considered to be more sensitive than those with only two-dimensional factors. There were diverse inter-individual differences in the visuo-spatial constructive abilities among the present participants who shared the same typical genomic deletion of WS. One of the participants showed almost equivalent performances to typically developing adults in those tests. In the present study, we found a wide range of cognitive abilities in visuo-spatial construction even among the patients with a common deletion pattern of WS. The findings suggest that attributing differences in the phenotypes entirely to genetic factors such as an atypical deletion may not be always correct. Copyright © 2016 The Japanese Society of Child Neurology. Published by Elsevier B.V. All rights reserved.

Cre/lox-based multiple markerless gene disruption in the genome of the extreme thermophile Thermus thermophilus.

PubMed

Togawa, Yoichiro; Nunoshiba, Tatsuo; Hiratsu, Keiichiro

2018-02-01

Markerless gene-disruption technology is particularly useful for effective genetic analyses of Thermus thermophilus (T. thermophilus), which have a limited number of selectable markers. In an attempt to develop a novel system for the markerless disruption of genes in T. thermophilus, we applied a Cre/lox system to construct a triple gene disruptant. To achieve this, we constructed two genetic tools, a loxP-htk-loxP cassette and cre-expressing plasmid, pSH-Cre, for gene disruption and removal of the selectable marker by Cre-mediated recombination. We found that the Cre/lox system was compatible with the proliferation of the T. thermophilus HB27 strain at the lowest growth temperature (50 °C), and thus succeeded in establishing a triple gene disruptant, the (∆TTC1454::loxP, ∆TTC1535KpnI::loxP, ∆TTC1576::loxP) strain, without leaving behind a selectable marker. During the process of the sequential disruption of multiple genes, we observed the undesired deletion and inversion of the chromosomal region between multiple loxP sites that were induced by Cre-mediated recombination. Therefore, we examined the effects of a lox66-htk-lox71 cassette by exploiting the mutant lox sites, lox66 and lox71, instead of native loxP sites. We successfully constructed a (∆TTC1535::lox72, ∆TTC1537::lox72) double gene disruptant without inducing the undesired deletion of the 0.7-kbp region between the two directly oriented lox72 sites created by the Cre-mediated recombination of the lox66-htk-lox71 cassette. This is the first demonstration of a Cre/lox system being applicable to extreme thermophiles in a genetic manipulation. Our results indicate that this system is a powerful tool for multiple markerless gene disruption in T. thermophilus.

Activation of Dun1 in response to nuclear DNA instability accounts for the increase in mitochondrial point mutations in Rad27/FEN1 deficient S. cerevisiae.

PubMed

Kaniak-Golik, Aneta; Kuberska, Renata; Dzierzbicki, Piotr; Sledziewska-Gojska, Ewa

2017-01-01

Rad27/FEN1 nuclease that plays important roles in the maintenance of DNA stability in the nucleus has recently been shown to reside in mitochondria. Accordingly, it has been established that Rad27 deficiency causes increased mutagenesis, but decreased microsatellite instability and homologous recombination in mitochondria. Our current analysis of mutations leading to erythromycin resistance indicates that only some of them arise in mitochondrial DNA and that the GC→AT transition is a hallmark of the mitochondrial mutagenesis in rad27 null background. We also show that the mitochondrial mutator phenotype resulting from Rad27 deficiency entirely depends on the DNA damage checkpoint kinase Dun1. DUN1 inactivation suppresses the mitochondrial mutator phenotype caused by Rad27 deficiency and this suppression is eliminated at least in part by subsequent deletion of SML1 encoding a repressor of ribonucleotide reductase. We conclude that Rad27 deficiency causes a mitochondrial mutator phenotype via activation of DNA damage checkpoint kinase Dun1 and that a Dun1-mediated increase of dNTP pools contributes to this phenomenon. These results point to the nuclear DNA instability as the source of mitochondrial mutagenesis. Consistently, we show that mitochondrial mutations occurring more frequently in yeast devoid of Rrm3, a DNA helicase involved in rDNA replication, are also dependent on Dun1. In addition, we have established that overproduction of Exo1, which suppresses DNA damage sensitivity and replication stress in nuclei of Rad27 deficient cells, but does not enter mitochondria, suppresses the mitochondrial mutagenesis. Exo1 overproduction restores also a great part of allelic recombination and microsatellite instability in mitochondria of Rad27 deficient cells. In contrast, the overproduction of Exo1 does not influence mitochondrial direct-repeat mediated deletions in rad27 null background, pointing to this homologous recombination pathway as the direct target of Rad27 activity in mitochondria.

Activation of Dun1 in response to nuclear DNA instability accounts for the increase in mitochondrial point mutations in Rad27/FEN1 deficient S. cerevisiae

PubMed Central

Dzierzbicki, Piotr

2017-01-01

Rad27/FEN1 nuclease that plays important roles in the maintenance of DNA stability in the nucleus has recently been shown to reside in mitochondria. Accordingly, it has been established that Rad27 deficiency causes increased mutagenesis, but decreased microsatellite instability and homologous recombination in mitochondria. Our current analysis of mutations leading to erythromycin resistance indicates that only some of them arise in mitochondrial DNA and that the GC→AT transition is a hallmark of the mitochondrial mutagenesis in rad27 null background. We also show that the mitochondrial mutator phenotype resulting from Rad27 deficiency entirely depends on the DNA damage checkpoint kinase Dun1. DUN1 inactivation suppresses the mitochondrial mutator phenotype caused by Rad27 deficiency and this suppression is eliminated at least in part by subsequent deletion of SML1 encoding a repressor of ribonucleotide reductase. We conclude that Rad27 deficiency causes a mitochondrial mutator phenotype via activation of DNA damage checkpoint kinase Dun1 and that a Dun1-mediated increase of dNTP pools contributes to this phenomenon. These results point to the nuclear DNA instability as the source of mitochondrial mutagenesis. Consistently, we show that mitochondrial mutations occurring more frequently in yeast devoid of Rrm3, a DNA helicase involved in rDNA replication, are also dependent on Dun1. In addition, we have established that overproduction of Exo1, which suppresses DNA damage sensitivity and replication stress in nuclei of Rad27 deficient cells, but does not enter mitochondria, suppresses the mitochondrial mutagenesis. Exo1 overproduction restores also a great part of allelic recombination and microsatellite instability in mitochondria of Rad27 deficient cells. In contrast, the overproduction of Exo1 does not influence mitochondrial direct-repeat mediated deletions in rad27 null background, pointing to this homologous recombination pathway as the direct target of Rad27 activity in mitochondria. PMID:28678842

The molecular biology of environmental aromatic hydrocarbons: Progress report for the period September 1, 1986 through July 31, 1987

DOE Office of Scientific and Technical Information (OSTI.GOV)

Weiss, S.B.

Our laboratory has explored the use of short DNA oligomers as targets for activated polycyclic aromatic hydrocarbons, such as benzo(a)pyrene diol epoxide (BPDE), in order to detect alterations in DNA sequence arrangement. In this model system, oligomers alkylated with (+)-BPDE are ligated into M13 viral DNA and used to transfect Escherichia coli. These cells are plated on agar, incubated at 37/sup 0/C, progeny viral clones are selected, amplified, and the viral DNAs isolated are sequenced at the site of oligomer insertion. We have devised a procedure for the preparation of unique duplex DNA oligomers such that the site of oligomermore » alkylation is specific for a single deoxynucleotide species in the two DNA strands. The procedure for oligomer assembly also allows us to vary the position of the alkylated residue in each of the two strands. Using our model system, the results obtained over the past year can be summarized as follows. When nonalkylated oligomer constructs are ligated into M13 viral DNA and used to transfect E. coli, no modifications in DNA sequence arrangement are detected in progeny viral DNAs. On the other hand, with oligomer constructs containing BP-adducts two major types of modifications in DNA sequence arrangement were observed: (1) large deletions, and (2) nonhomologous (illegitimate) recombinants. Both of these DNA modifications result in the complete removal of the oligomer insert. Transfection of E. coli that are recA/sup -/ does not alter these DNA modifications, therefore, it appears that the deletions and recombinants induced by the alkylated inserts are not under control of the RecA gene. As the distance between the alkylated residues in the duplex strands is increased, the number of recombinant events detected is reduced. In addition to the above types of DNA modifications, restoration of the original nucleotide sequence in the alkylated construct was also observed in progeny viral DNAs. 7 refs., 6 figs., 2 tabs.« less

Penicillin production in industrial strain Penicillium chrysogenum P2niaD18 is not dependent on the copy number of biosynthesis genes.

PubMed

Ziemons, Sandra; Koutsantas, Katerina; Becker, Kordula; Dahlmann, Tim; Kück, Ulrich

2017-02-16

Multi-copy gene integration into microbial genomes is a conventional tool for obtaining improved gene expression. For Penicillium chrysogenum, the fungal producer of the beta-lactam antibiotic penicillin, many production strains carry multiple copies of the penicillin biosynthesis gene cluster. This discovery led to the generally accepted view that high penicillin titers are the result of multiple copies of penicillin genes. Here we investigated strain P2niaD18, a production line that carries only two copies of the penicillin gene cluster. We performed pulsed-field gel electrophoresis (PFGE), quantitative qRT-PCR, and penicillin bioassays to investigate production, deletion and overexpression strains generated in the P. chrysogenum P2niaD18 background, in order to determine the copy number of the penicillin biosynthesis gene cluster, and study the expression of one penicillin biosynthesis gene, and the penicillin titer. Analysis of production and recombinant strain showed that the enhanced penicillin titer did not depend on the copy number of the penicillin gene cluster. Our assumption was strengthened by results with a penicillin null strain lacking pcbC encoding isopenicillin N synthase. Reintroduction of one or two copies of the cluster into the pcbC deletion strain restored transcriptional high expression of the pcbC gene, but recombinant strains showed no significantly different penicillin titer compared to parental strains. Here we present a molecular genetic analysis of production and recombinant strains in the P2niaD18 background carrying different copy numbers of the penicillin biosynthesis gene cluster. Our analysis shows that the enhanced penicillin titer does not strictly depend on the copy number of the cluster. Based on these overall findings, we hypothesize that instead, complex regulatory mechanisms are prominently implicated in increased penicillin biosynthesis in production strains.

Analysis of BIM (BCL-2 like 11 gene) deletion polymorphism in Chinese non-small cell lung cancer patients

PubMed Central

Zhong, Jia; Li, Zheng-Xiang; Zhao, Jun; Duan, Jian-Chun; Bai, Hua; An, Tong-Tong; Yang, Xiao-Dan; Wang, Jie

2014-01-01

Background Drug resistance significantly weakens the efficacy of cancer treatment, and the BIM (also known as the BCL2L11 gene) deletion polymorphism has been identified as a potential biomarker for drug resistance. In this retrospective study, we included a total of 290 patients with advanced non-small cell lung cancer (NSCLC) who received treatment with epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) and chemotherapy. Methods The BIM deletion polymorphism of each patient was detected by polymerase chain reaction. EGFR mutations were detected by denaturing high-performance liquid chromatography methods and the amplification refractory mutation system. Results The BIM deletion polymorphism was detected in 45/290 (15.5%) Chinese NSCLC patients. No associations were observed between the BIM deletion and clinic-pathologic characteristics of patients. The BIM deletion polymorphism was predictive of shorter progression-free survival in Chinese patients with EGFR-mutant adenocarcinoma and who were treated with EGFR-TKIs (7.30 vs. 9.53 months, P = 0.034). Additionally, we found that the BIM deletion polymorphism was an effective predictor of short progression-free survival in individuals with EGFR-mutant NSCLC and treated with chemotherapy containing pemetrexed (3.32 vs. 5.30, P = 0.012) or second-/beyond-line chemotherapy containing taxanes (1.53 vs. 2.61 months, P = 0.025). The BIM deletion was not correlated with overall survival. Conclusion The BIM deletion polymorphism occurs in 15.5% of Chinese NSCLC patients, and is a biomarker for resistance to TKIs and chemotherapy. However, BIM deletion was not a decisive factor in overall survival. PMID:26767045

Analysis of BIM (BCL-2 like 11 gene) deletion polymorphism in Chinese non-small cell lung cancer patients.

PubMed

Zhong, Jia; Li, Zheng-Xiang; Zhao, Jun; Duan, Jian-Chun; Bai, Hua; An, Tong-Tong; Yang, Xiao-Dan; Wang, Jie

2014-11-01

Drug resistance significantly weakens the efficacy of cancer treatment, and the BIM (also known as the BCL2L11 gene) deletion polymorphism has been identified as a potential biomarker for drug resistance. In this retrospective study, we included a total of 290 patients with advanced non-small cell lung cancer (NSCLC) who received treatment with epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) and chemotherapy. The BIM deletion polymorphism of each patient was detected by polymerase chain reaction. EGFR mutations were detected by denaturing high-performance liquid chromatography methods and the amplification refractory mutation system. The BIM deletion polymorphism was detected in 45/290 (15.5%) Chinese NSCLC patients. No associations were observed between the BIM deletion and clinic-pathologic characteristics of patients. The BIM deletion polymorphism was predictive of shorter progression-free survival in Chinese patients with EGFR-mutant adenocarcinoma and who were treated with EGFR-TKIs (7.30 vs. 9.53 months, P = 0.034). Additionally, we found that the BIM deletion polymorphism was an effective predictor of short progression-free survival in individuals with EGFR-mutant NSCLC and treated with chemotherapy containing pemetrexed (3.32 vs. 5.30, P = 0.012) or second-/beyond-line chemotherapy containing taxanes (1.53 vs. 2.61 months, P = 0.025). The BIM deletion was not correlated with overall survival. The BIM deletion polymorphism occurs in 15.5% of Chinese NSCLC patients, and is a biomarker for resistance to TKIs and chemotherapy. However, BIM deletion was not a decisive factor in overall survival.

High-Resolution Maps of Mouse Reference Populations

PubMed Central

Simecek, Petr; Forejt, Jiri; Williams, Robert W.; Shiroishi, Toshihiko; Takada, Toyoyuki; Lu, Lu; Johnson, Thomas E.; Bennett, Beth; Deschepper, Christian F.; Scott-Boyer, Marie-Pier; Pardo-Manuel de Villena, Fernando; Churchill, Gary A.

2017-01-01

Genetic reference panels are widely used to map complex, quantitative traits in model organisms. We have generated new high-resolution genetic maps of 259 mouse inbred strains from recombinant inbred strain panels (C57BL/6J × DBA/2J, ILS/IbgTejJ × ISS/IbgTejJ, and C57BL/6J × A/J) and chromosome substitution strain panels (C57BL/6J-Chr#, C57BL/6J-Chr#, and C57BL/6J-Chr#). We genotyped all samples using the Affymetrix Mouse Diversity Array with an average intermarker spacing of 4.3 kb. The new genetic maps provide increased precision in the localization of recombination breakpoints compared to the previous maps. Although the strains were presumed to be fully inbred, we found residual heterozygosity in 40% of individual mice from five of the six panels. We also identified de novo deletions and duplications, in homozygous or heterozygous state, ranging in size from 21 kb to 8.4 Mb. Almost two-thirds (46 out of 76) of these deletions overlap exons of protein coding genes and may have phenotypic consequences. Twenty-nine putative gene conversions were identified in the chromosome substitution strains. We find that gene conversions are more likely to occur in regions where the homologous chromosomes are more similar. The raw genotyping data and genetic maps of these strain panels are available at http://churchill-lab.jax.org/website/MDA. PMID:28839117

High-Resolution Maps of Mouse Reference Populations.

PubMed

Simecek, Petr; Forejt, Jiri; Williams, Robert W; Shiroishi, Toshihiko; Takada, Toyoyuki; Lu, Lu; Johnson, Thomas E; Bennett, Beth; Deschepper, Christian F; Scott-Boyer, Marie-Pier; Pardo-Manuel de Villena, Fernando; Churchill, Gary A

2017-10-05

Genetic reference panels are widely used to map complex, quantitative traits in model organisms. We have generated new high-resolution genetic maps of 259 mouse inbred strains from recombinant inbred strain panels (C57BL/6J × DBA/2J, ILS/IbgTejJ × ISS/IbgTejJ, and C57BL/6J × A/J) and chromosome substitution strain panels (C57BL/6J-Chr#, C57BL/6J-Chr#, and C57BL/6J-Chr#). We genotyped all samples using the Affymetrix Mouse Diversity Array with an average intermarker spacing of 4.3 kb. The new genetic maps provide increased precision in the localization of recombination breakpoints compared to the previous maps. Although the strains were presumed to be fully inbred, we found residual heterozygosity in 40% of individual mice from five of the six panels. We also identified de novo deletions and duplications, in homozygous or heterozygous state, ranging in size from 21 kb to 8.4 Mb. Almost two-thirds (46 out of 76) of these deletions overlap exons of protein coding genes and may have phenotypic consequences. Twenty-nine putative gene conversions were identified in the chromosome substitution strains. We find that gene conversions are more likely to occur in regions where the homologous chromosomes are more similar. The raw genotyping data and genetic maps of these strain panels are available at http://churchill-lab.jax.org/website/MDA. Copyright © 2017 Simecek et al.

Non-classical phase diagram for virus bacterial coevolution mediated by clustered regularly interspaced short palindromic repeats.

PubMed

Han, Pu; Deem, Michael W

2017-02-01

CRISPR is a newly discovered prokaryotic immune system. Bacteria and archaea with this system incorporate genetic material from invading viruses into their genomes, providing protection against future infection by similar viruses. The condition for coexistence of prokaryots and viruses is an interesting problem in evolutionary biology. In this work, we show an intriguing phase diagram of the virus extinction probability, which is more complex than that of the classical predator-prey model. As the CRISPR incorporates genetic material, viruses are under pressure to evolve to escape recognition by CRISPR. When bacteria have a small rate of deleting spacers, a new parameter region in which bacteria and viruses can coexist arises, and it leads to a more complex coexistence patten for bacteria and viruses. For example, when the virus mutation rate is low, the virus extinction probability changes non-montonically with the bacterial exposure rate. The virus and bacteria coevolution not only alters the virus extinction probability, but also changes the bacterial population structure. Additionally, we show that recombination is a successful strategy for viruses to escape from CRISPR recognition when viruses have multiple proto-spacers, providing support for a recombination-mediated escape mechanism suggested experimentally. Finally, we suggest that the re-entrant phase diagram, in which phages can progress through three phases of extinction and two phases of abundance at low spacer deletion rates as a function of exposure rate to bacteria, is an experimentally testable phenomenon. © 2017 The Author(s).

Trans-Lesion DNA Polymerases May Be Involved in Yeast Meiosis

PubMed Central

Arbel-Eden, Ayelet; Joseph-Strauss, Daphna; Masika, Hagit; Printzental, Oxana; Rachi, Eléanor; Simchen, Giora

2013-01-01

Trans-lesion DNA polymerases (TLSPs) enable bypass of DNA lesions during replication and are also induced under stress conditions. Being only weakly dependent on their template during replication, TLSPs introduce mutations into DNA. The low processivity of these enzymes ensures that they fall off their template after a few bases are synthesized and are then replaced by the more accurate replicative polymerase. We find that the three TLSPs of budding yeast Saccharomyces cerevisiae Rev1, PolZeta (Rev3 and Rev7), and Rad30 are induced during meiosis at a time when DNA double-strand breaks (DSBs) are formed and homologous chromosomes recombine. Strains deleted for one or any combination of the three TLSPs undergo normal meiosis. However, in the triple-deletion mutant, there is a reduction in both allelic and ectopic recombination. We suggest that trans-lesion polymerases are involved in the processing of meiotic double-strand breaks that lead to mutations. In support of this notion, we report significant yeast two-hybrid (Y2H) associations in meiosis-arrested cells between the TLSPs and DSB proteins Rev1-Spo11, Rev1-Mei4, and Rev7-Rec114, as well as between Rev1 and Rad30. We suggest that the involvement of TLSPs in processing of meiotic DSBs could be responsible for the considerably higher frequency of mutations reported during meiosis compared with that found in mitotically dividing cells, and therefore may contribute to faster evolutionary divergence than previously assumed. PMID:23550131

Neuronal Deletion of Caspase 8 Protects against Brain Injury in Mouse Models of Controlled Cortical Impact and Kainic Acid-Induced Excitotoxicity

PubMed Central

Krajewska, Maryla; You, Zerong; Rong, Juan; Kress, Christina; Huang, Xianshu; Yang, Jinsheng; Kyoda, Tiffany; Leyva, Ricardo; Banares, Steven; Hu, Yue; Sze, Chia-Hung; Whalen, Michael J.; Salmena, Leonardo; Hakem, Razqallah; Head, Brian P.; Reed, John C.; Krajewski, Stan

2011-01-01

Background Acute brain injury is an important health problem. Given the critical position of caspase 8 at the crossroads of cell death pathways, we generated a new viable mouse line (Ncasp8 −/−), in which the gene encoding caspase 8 was selectively deleted in neurons by cre-lox system. Methodology/Principal Findings Caspase 8 deletion reduced rates of neuronal cell death in primary neuronal cultures and in whole brain organotypic coronal slice cultures prepared from 4 and 8 month old mice and cultivated up to 14 days in vitro. Treatments of cultures with recombinant murine TNFα (100 ng/ml) or TRAIL (250 ng/mL) plus cyclohexamide significantly protected neurons against cell death induced by these apoptosis-inducing ligands. A protective role of caspase 8 deletion in vivo was also demonstrated using a controlled cortical impact (CCI) model of traumatic brain injury (TBI) and seizure-induced brain injury caused by kainic acid (KA). Morphometric analyses were performed using digital imaging in conjunction with image analysis algorithms. By employing virtual images of hundreds of brain sections, we were able to perform quantitative morphometry of histological and immunohistochemical staining data in an unbiased manner. In the TBI model, homozygous deletion of caspase 8 resulted in reduced lesion volumes, improved post-injury motor performance, superior learning and memory retention, decreased apoptosis, diminished proteolytic processing of caspases and caspase substrates, and less neuronal degeneration, compared to wild type, homozygous cre, and caspase 8-floxed control mice. In the KA model, Ncasp8 −/− mice demonstrated superior survival, reduced seizure severity, less apoptosis, and reduced caspase 3 processing. Uninjured aged knockout mice showed improved learning and memory, implicating a possible role for caspase 8 in cognitive decline with aging. Conclusions Neuron-specific deletion of caspase 8 reduces brain damage and improves post-traumatic functional outcomes, suggesting an important role for this caspase in pathophysiology of acute brain trauma. PMID:21957448

A cytological-physical map of 22q11

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lindsay, E.A.; Rizzu, P.; Gaddini, L.

Our laboratory is involved in the construction of a cytological-physical map of 22q11 and isolation of expressed sequences from the region involved in DiGeorge syndrome (DGS) and Velo-Cardio-Facial syndrome (VCFS). One of the goals of the mapping is an understanding of the molecular mechanisms which generate the 22q11 microdeletions observed with high frequency in DGS and VCFS. Our of over 60 deleted patients studied in our laboratory, all but one were deleted for two loci approximately 1-2 Mb apart. There is evidence from patients with balanced and unbalanced translocations that deletion of the whole region is not necessary for determinationmore » of the clinical phenotype. Therefore, it is possible that deletion breakpoints occur as a consequence of structural characteristics of the DNA that predispose to rearrangements. A striking characteristic of the 22q11 region is the abundance of low copy repeat sequences. It is reasonable to think that recombination between these repeats may lead to microdeletions. However, a direct demonstration of such mechanism is not available yet. The presence of repeats makes standard physical mapping techniques based on hybridization or STS mapping often difficult to interpret. For example, we have found clones positive for the same STS that are located in different positions within 22q11. For this reason we have used high resolution cytological mapping as a supporting technique for map validation. We present the current status map which includes known polymorphic and non-polymorphic loci, newly isolated clones and chromosomal deletion breakpoints. The map extends from the loci D22S9/D22S24 to TOP1P2. Extended chromatin hybridization experiments visually demonstrate the presence of at least two repeat islands flanking (or at) the region where chromosomal breakpoints of the commonly deleted region occur.« less

African Swine Fever Virus Georgia Isolate Harboring Deletions of MGF360 and MGF505 Genes Is Attenuated in Swine and Confers Protection against Challenge with Virulent Parental Virus.

PubMed

O'Donnell, Vivian; Holinka, Lauren G; Gladue, Douglas P; Sanford, Brenton; Krug, Peter W; Lu, Xiqiang; Arzt, Jonathan; Reese, Bo; Carrillo, Consuelo; Risatti, Guillermo R; Borca, Manuel V

2015-06-01

African swine fever virus (ASFV) is the etiological agent of a contagious and often lethal disease of domestic pigs that has significant economic consequences for the swine industry. The control of African swine fever (ASF) has been hampered by the unavailability of vaccines. Experimental vaccines have been developed using genetically modified live attenuated ASFVs where viral genes involved in virus virulence were removed from the genome. Multigene family 360 (MGF360) and MGF505 represent a group of genes sharing partial sequence and structural identities that have been connected with ASFV host range specificity, blocking of the host innate response, and virus virulence. Here we report the construction of a recombinant virus (ASFV-G-ΔMGF) derived from the highly virulent ASFV Georgia 2007 isolate (ASFV-G) by specifically deleting six genes belonging to MGF360 or MGF505: MGF505-1R, MGF360-12L, MGF360-13L, MGF360-14L, MGF505-2R, and MGF505-3R. ASFV-G-ΔMGF replicates as efficiently in primary swine macrophage cell cultures as the parental virus. In vivo, ASFV-G-ΔMGF is completely attenuated in swine, since pigs inoculated intramuscularly (i.m.) with either 10(2) or 10(4) 50% hemadsorbing doses (HAD50) remained healthy, without signs of the disease. Importantly, when these animals were subsequently exposed to highly virulent parental ASFV-G, no signs of the disease were observed, although a proportion of these animals harbored the challenge virus. This is the first report demonstrating the role of MGF genes acting as independent determinants of ASFV virulence. Additionally, ASFV-G-ΔMGF is the first experimental vaccine reported to induce protection in pigs challenged with highly virulent and epidemiologically relevant ASFV-G. The main problem for controlling ASF is the lack of vaccines. Studies focusing on understanding ASFV virulence led to the production of genetically modified recombinant viruses that, while attenuated, are able to confer protection in pigs challenged with homologous viruses. Here we have produced an attenuated recombinant ASFV derived from highly virulent ASFV strain Georgia (ASFV-G) lacking only six of the multigene family 360 (MGF360) and MGF505 genes (ASFV-G-ΔMGF). It is demonstrated, by first time, that deleting specific MGF genes alone can completely attenuate a highly virulent field ASFV isolate. Recombinant virus ASFV-G-ΔMGF effectively confers protection in pigs against challenge with ASFV-G when delivered once via the intramuscular (i.m.) route. The protection against ASFV-G is highly effective by 28 days postvaccination. This is the first report of an experimental vaccine that induces solid protection against virulent ASFV-G. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Efficient gene editing in Corynebacterium glutamicum using the CRISPR/Cas9 system.

PubMed

Peng, Feng; Wang, Xinyue; Sun, Yang; Dong, Guibin; Yang, Yankun; Liu, Xiuxia; Bai, Zhonghu

2017-11-14

Corynebacterium glutamicum (C. glutamicum) has traditionally been used as a microbial cell factory for the industrial production of many amino acids and other industrially important commodities. C. glutamicum has recently been established as a host for recombinant protein expression; however, some intrinsic disadvantages could be improved by genetic modification. Gene editing techniques, such as deletion, insertion, or replacement, are important tools for modifying chromosomes. In this research, we report a CRISPR/Cas9 system in C. glutamicum for rapid and efficient genome editing, including gene deletion and insertion. The system consists of two plasmids: one containing a target-specific guide RNA and a homologous sequence to a target gene, the other expressing Cas9 protein. With high efficiency (up to 100%), this system was used to disrupt the porB, mepA, clpX and Ncgl0911 genes, which affect the ability to express proteins. The porB- and mepA-deletion strains had enhanced expression of green fluorescent protein, compared with the wild-type stain. This system can also be used to engineer point mutations and gene insertions. In this study, we adapted the CRISPR/Cas9 system from S. pyogens to gene deletion, point mutations and insertion in C. glutamicum. Compared with published genome modification methods, methods based on the CRISPR/Cas9 system can rapidly and efficiently achieve genome editing. Our research provides a powerful tool for facilitating the study of gene function, metabolic pathways, and enhanced productivity in C. glutamicum.

Selective deletion of leptin receptors in adult hippocampus induces depression-related behaviors

PubMed Central

Guo, Ming; Huang, Tung-Yi; Garza, Jacob C.; Chua, Streamson C.; Lu, Xin-Yun

2013-01-01

Previous studies have demonstrated that leptin and its receptors (LepRb) in the central nervous system play an important role in regulating depression- and anxiety-related behaviors. However, the physiological functions of LepRb in specific brain regions for mediating different emotional behaviors remain to be defined. In this study, we examined the behavioral effects of LepRb ablation in the adult hippocampus using a series of behavioral paradigms for assessing depression- and anxiety-related behaviors. Targeted deletion of LepRb was achieved using the Cre/loxP site-specific recombination system through bilateral stereotaxic delivery of an adeno-associated virus expressing Cre-recombinase (AAV-Cre) into the dentate gyrus of adult mice homozygous for a floxed leptin receptor allele. AAV-Cre-mediated deletion of the floxed region of LepRb was detected 2 weeks after injection. In accordance with this, leptin-stimulated phophorylation of Akt was attenuated in the hippocampus of AAV-Cre injected mice. Mice injected with AAV-Cre displayed normal locomotor activity and anxiety-like behavior, as determined in the elevated plus maze, light dark box and open field tests, but showed increased depression-like behaviors in the tail suspension, sucrose preference and learned helplessness tests. Taken together, this data suggests that deletion of LepRb in the adult hippocampus is sufficient to induce depression-like behaviors. Our results support the view that leptin signaling in the hippocampus may be essential for maintaining positive mood states and active coping to stress. PMID:22932068

Recombinant factor VIIa (eptacog alfa): a review of its use in congenital hemophilia with inhibitors, acquired hemophilia, and other congenital bleeding disorders.

PubMed

Croom, Katherine F; McCormack, Paul L

2008-01-01

Recombinant factor VIIa (NovoSeven; also known as recombinant activated factor VII or eptacog alfa) is structurally similar to human plasma-derived coagulation factor VIIa, but is manufactured using DNA biotechnology. Recombinant factor VIIa interacts with thrombin-activated platelets to produce a thrombin burst leading to accelerated fibrin clot formation localized to the site of vascular injury. It is approved in many countries for use as an intravenous hemostatic agent in patients with congenital hemophilia with inhibitors, and also for acquired hemophilia, factor VII deficiency, and Glanzmann thrombasthenia in some countries. Studies have shown it to be effective and generally well tolerated when used intravenously to treat bleeding episodes or provide hemostatic cover during surgery in patients with congenital hemophilia with inhibitors, acquired hemophilia, factor VII deficiency or Glanzmann thrombasthenia. Based on available data, its efficacy in terms of patient-assessed response may be similar to that of activated prothrombin complex concentrate (aPCC), but treatment with a single 270 microg/kg dose of recombinant factor VIIa might reduce the need for rescue therapy compared with aPCC. Recombinant factor VIIa is not immunogenic in patients with hemophilia, does not produce an anamnestic response in hemophilia patients with inhibitors, and has very low thrombogenicity. It is recommended in guidelines as the treatment of choice for bleeds in patients with hemophilia B with high-responding inhibitors and for patients with factor VII deficiency, and is also a first-line therapeutic option for high-responder hemophilia A patients with inhibitors and those with acquired hemophilia. Cost data from pharmacoeconomic analyses support its use in hemophilia patients with inhibitors. Thus, recombinant factor VIIa is a valuable treatment option for patients with these rare, but potentially serious, bleeding disorders.

Copy number variation is a fundamental aspect of the placental genome.

PubMed

Hannibal, Roberta L; Chuong, Edward B; Rivera-Mulia, Juan Carlos; Gilbert, David M; Valouev, Anton; Baker, Julie C

2014-05-01

Discovery of lineage-specific somatic copy number variation (CNV) in mammals has led to debate over whether CNVs are mutations that propagate disease or whether they are a normal, and even essential, aspect of cell biology. We show that 1,000 N polyploid trophoblast giant cells (TGCs) of the mouse placenta contain 47 regions, totaling 138 Megabases, where genomic copies are underrepresented (UR). UR domains originate from a subset of late-replicating heterochromatic regions containing gene deserts and genes involved in cell adhesion and neurogenesis. While lineage-specific CNVs have been identified in mammalian cells, classically in the immune system where V(D)J recombination occurs, we demonstrate that CNVs form during gestation in the placenta by an underreplication mechanism, not by recombination nor deletion. Our results reveal that large scale CNVs are a normal feature of the mammalian placental genome, which are regulated systematically during embryogenesis and are propagated by a mechanism of underreplication.

Synthetic biology approaches: Towards sustainable exploitation of marine bioactive molecules.

PubMed

Seghal Kiran, G; Ramasamy, Pasiyappazham; Sekar, Sivasankari; Ramu, Meenatchi; Hassan, Saqib; Ninawe, A S; Selvin, Joseph

2018-06-01

The discovery of genes responsible for the production of bioactive metabolites via metabolic pathways combined with the advances in synthetic biology tools, has allowed the establishment of numerous microbial cell factories, for instance the yeast cell factories, for the manufacture of highly useful metabolites from renewable biomass. Genome mining and metagenomics are two platforms provide base-line data for reconstruction of genomes and metabolomes which is based in the development of synthetic/semi-synthetic genomes for marine natural products discovery. Engineered biofilms are being innovated on synthetic biology platform using genetic circuits and cell signalling systems as represillators controlling biofilm formation. Recombineering is a process of homologous recombination mediated genetic engineering, includes insertion, deletion or modification of any sequence specifically. Although this discipline considered new to the scientific domain, this field has now developed as promising endeavor on the accomplishment of sustainable exploitation of marine natural products. Copyright © 2018 Elsevier B.V. All rights reserved.

Generation of astaxanthin mutants in Xanthophyllomyces dendrorhous using a double recombination method based on hygromycin resistance.

PubMed

Niklitschek, Mauricio; Baeza, Marcelo; Fernández-Lobato, María; Cifuentes, Víctor

2012-01-01

Generally two selection markers are required to obtain homozygous mutations in a diploid background, one for each gene copy that is interrupted. In this chapter is described a method that allows the double gene deletions of the two copies of a gene from a diploid organism, a wild-type strain of the Xanthophyllomyces dendrorhous yeast, using hygromycin B resistance as the only selection marker. To accomplish this, in a first step, a heterozygous hygromycin B-resistant strain is obtained by a single process of transformation (carrying the inserted hph gene). Following, the heterozygous mutant is grown in media with increasing concentrations of the antibiotic. In this way, the strains that became homozygous (by mitotic recombination) for the antibiotic marker would able to growth at higher concentration of the antibiotic than the heterozygous. The method can be potentially applied for obtaining double mutants of other diploid organisms.

The DNA damage checkpoint pathway promotes extensive resection and nucleotide synthesis to facilitate homologous recombination repair and genome stability in fission yeast.

PubMed

Blaikley, Elizabeth J; Tinline-Purvis, Helen; Kasparek, Torben R; Marguerat, Samuel; Sarkar, Sovan; Hulme, Lydia; Hussey, Sharon; Wee, Boon-Yu; Deegan, Rachel S; Walker, Carol A; Pai, Chen-Chun; Bähler, Jürg; Nakagawa, Takuro; Humphrey, Timothy C

2014-05-01

DNA double-strand breaks (DSBs) can cause chromosomal rearrangements and extensive loss of heterozygosity (LOH), hallmarks of cancer cells. Yet, how such events are normally suppressed is unclear. Here we identify roles for the DNA damage checkpoint pathway in facilitating homologous recombination (HR) repair and suppressing extensive LOH and chromosomal rearrangements in response to a DSB. Accordingly, deletion of Rad3(ATR), Rad26ATRIP, Crb2(53BP1) or Cdc25 overexpression leads to reduced HR and increased break-induced chromosome loss and rearrangements. We find the DNA damage checkpoint pathway facilitates HR, in part, by promoting break-induced Cdt2-dependent nucleotide synthesis. We also identify additional roles for Rad17, the 9-1-1 complex and Chk1 activation in facilitating break-induced extensive resection and chromosome loss, thereby suppressing extensive LOH. Loss of Rad17 or the 9-1-1 complex results in a striking increase in break-induced isochromosome formation and very low levels of chromosome loss, suggesting the 9-1-1 complex acts as a nuclease processivity factor to facilitate extensive resection. Further, our data suggest redundant roles for Rad3ATR and Exo1 in facilitating extensive resection. We propose that the DNA damage checkpoint pathway coordinates resection and nucleotide synthesis, thereby promoting efficient HR repair and genome stability. © The Author(s) 2014. Published by Oxford University Press.

Function of a Glutamine Synthetase-Like Protein in Bacterial Aniline Oxidation via γ-Glutamylanilide

PubMed Central

Ohara, Akira; Sakae, Shinji; Okamoto, Yasuhiro; Kitamura, Chitoshi; Kato, Dai-ichiro; Negoro, Seiji

2013-01-01

Acinetobacter sp. strain YAA has five genes (atdA1 to atdA5) involved in aniline oxidation as a part of the aniline degradation gene cluster. From sequence analysis, the five genes were expected to encode a glutamine synthetase (GS)-like protein (AtdA1), a glutamine amidotransferase-like protein (AtdA2), and an aromatic compound dioxygenase (AtdA3, AtdA4, and AtdA5) (M. Takeo, T. Fujii, and Y. Maeda, J. Ferment. Bioeng. 85:17-24, 1998). A recombinant Pseudomonas strain harboring these five genes quantitatively converted aniline into catechol, demonstrating that catechol is the major oxidation product from aniline. To elucidate the function of the GS-like protein AtdA1 in aniline oxidation, we purified it from recombinant Escherichia coli harboring atdA1. The purified AtdA1 protein produced gamma-glutamylanilide (γ-GA) quantitatively from aniline and l-glutamate in the presence of ATP and MgCl2. This reaction was identical to glutamine synthesis by GS, except for the use of aniline instead of ammonia as the substrate. Recombinant Pseudomonas strains harboring the dioxygenase genes (atdA3 to atdA5) were unable to degrade aniline but converted γ-GA into catechol, indicating that γ-GA is an intermediate to catechol and a direct substrate for the dioxygenase. Unexpectedly, a recombinant Pseudomonas strain harboring only atdA2 hydrolyzed γ-GA into aniline, reversing the γ-GA formation by AtdA1. Deletion of atdA2 from atdA1 to atdA5 caused γ-GA accumulation from aniline in recombinant Pseudomonas cells and inhibited the growth of a recombinant Acinetobacter strain on aniline, suggesting that AtdA2 prevents γ-GA accumulation that is harmful to the host cell. PMID:23893114

Function of a glutamine synthetase-like protein in bacterial aniline oxidation via γ-glutamylanilide.

PubMed

Takeo, Masahiro; Ohara, Akira; Sakae, Shinji; Okamoto, Yasuhiro; Kitamura, Chitoshi; Kato, Dai-ichiro; Negoro, Seiji

2013-10-01

Acinetobacter sp. strain YAA has five genes (atdA1 to atdA5) involved in aniline oxidation as a part of the aniline degradation gene cluster. From sequence analysis, the five genes were expected to encode a glutamine synthetase (GS)-like protein (AtdA1), a glutamine amidotransferase-like protein (AtdA2), and an aromatic compound dioxygenase (AtdA3, AtdA4, and AtdA5) (M. Takeo, T. Fujii, and Y. Maeda, J. Ferment. Bioeng. 85:17-24, 1998). A recombinant Pseudomonas strain harboring these five genes quantitatively converted aniline into catechol, demonstrating that catechol is the major oxidation product from aniline. To elucidate the function of the GS-like protein AtdA1 in aniline oxidation, we purified it from recombinant Escherichia coli harboring atdA1. The purified AtdA1 protein produced gamma-glutamylanilide (γ-GA) quantitatively from aniline and l-glutamate in the presence of ATP and MgCl2. This reaction was identical to glutamine synthesis by GS, except for the use of aniline instead of ammonia as the substrate. Recombinant Pseudomonas strains harboring the dioxygenase genes (atdA3 to atdA5) were unable to degrade aniline but converted γ-GA into catechol, indicating that γ-GA is an intermediate to catechol and a direct substrate for the dioxygenase. Unexpectedly, a recombinant Pseudomonas strain harboring only atdA2 hydrolyzed γ-GA into aniline, reversing the γ-GA formation by AtdA1. Deletion of atdA2 from atdA1 to atdA5 caused γ-GA accumulation from aniline in recombinant Pseudomonas cells and inhibited the growth of a recombinant Acinetobacter strain on aniline, suggesting that AtdA2 prevents γ-GA accumulation that is harmful to the host cell.

Mouse HFM1/Mer3 Is Required for Crossover Formation and Complete Synapsis of Homologous Chromosomes during Meiosis

PubMed Central

Guiraldelli, Michel F.; Eyster, Craig; Wilkerson, Joseph L.; Dresser, Michael E.; Pezza, Roberto J.

2013-01-01

Faithful chromosome segregation during meiosis requires that homologous chromosomes associate and recombine. Chiasmata, the cytological manifestation of recombination, provide the physical link that holds the homologs together as a pair, facilitating their orientation on the spindle at meiosis I. Formation of most crossover (CO) events requires the assistance of a group of proteins collectively known as ZMM. HFM1/Mer3 is in this group of proteins and is required for normal progression of homologous recombination and proper synapsis between homologous chromosomes in a number of model organisms. Our work is the first study in mammals showing the in vivo function of mouse HFM1. Cytological observations suggest that initial steps of recombination are largely normal in a majority of Hfm1−/− spermatocytes. Intermediate and late stages of recombination appear aberrant, as chromosomal localization of MSH4 is altered and formation of MLH1foci is drastically reduced. In agreement, chiasma formation is reduced, and cells arrest with subsequent apoptosis at diakinesis. Our results indicate that deletion of Hfm1 leads to the elimination of a major fraction but not all COs. Formation of chromosome axial elements and homologous pairing is apparently normal, and Hfm1−/− spermatocytes progress to the end of prophase I without apparent developmental delay or apoptosis. However, synapsis is altered with components of the central region of the synaptonemal complex frequently failing to extend the full length of the chromosome axes. We propose that initial steps of recombination are sufficient to support homology recognition, pairing, and initial chromosome synapsis and that HFM1 is required to form normal numbers of COs and to complete synapsis. PMID:23555294

Evaluation of two strains of Marek's disease virus serotype 1 for the development of recombinant vaccines against very virulent infectious bursal disease virus.

PubMed

Li, Kai; Liu, Yongzhen; Liu, Changjun; Gao, Li; Gao, Yulong; Zhang, Yanping; Cui, Hongyu; Qi, Xiaole; Zhong, Li; Wang, Xiaomei

2017-03-01

Attenuated strains of Marek's disease virus serotype 1 (MDV1), and the closely related herpesvirus of turkeys, are among the most potent vectors for development of recombinant vaccines for poultry. To investigate the effects of MDV1 strain characteristics on the protective efficacy of the recombinant vaccines, we developed two recombinant MDV1 vaccines for expressing the VP2 gene of infectious bursal disease virus (IBDV) based on two different MDV1 strains, the attenuated strain 814 and the Meq gene-deleted recombinant MDV1 strain rLMS△Meq, as the viral vectors. The r814-VP2 virus based on the 814 strain exhibited higher replication efficiency in cell culture while lower viral titers in chickens, compared to rLMS△Meq-VP2 derived from the rLMS△Meq strain. Further studies indicated that r814-VP2 produced higher levels of VP2 protein in cells and elicited stronger immune responses against IBDV in chickens than rLMS△Meq-VP2. After IBDV challenge, rLMS△Meq-VP2 provided 50% protection against mortality, and the birds that survived developed bursal atrophy and gross lesions. In contrast, r814-VP2 conferred complete protection not only against development of clinical signs and mortality, but also against the formation of bursal lesions. The results indicate that different MDV1 vector influences the protective efficacy of recombinant MDV1 vaccines. The r814-VP2 has the potential to serve as a bivalent vaccine against two important lethal pathogens of chickens. Copyright © 2016 Elsevier B.V. All rights reserved.

Large deletions play a minor but essential role in congenital coagulation factor VII and X deficiencies.

PubMed

Rath, M; Najm, J; Sirb, H; Kentouche, K; Dufke, A; Pauli, S; Hackmann, K; Liehr, T; Hübner, C A; Felbor, U

2015-01-01

Congenital factor VII (FVII) and factor X (FX) deficiencies belong to the group of rare bleeding disorders which may occur in separate or combined forms since both the F7 and F10 genes are located in close proximity on the distal long arm of chromosome 13 (13q34). We here present data of 192 consecutive index cases with FVII and/or FX deficiency. 10 novel and 53 recurrent sequence alterations were identified in the F7 gene and 5 novel as well as 11 recurrent in the F10 gene including one homozygous 4.35 kb deletion within F7 (c.64+430_131-6delinsTCGTAA) and three large heterozygous deletions involving both the F7 and F10 genes. One of the latter proved to be cytogenetically visible as a chromosome 13q34 deletion and associated with agenesis of the corpus callosum and psychomotor retardation. Large deletions play a minor but essential role in the mutational spectrum of the F7 and F10 genes. Copy number analyses (e. g. MLPA) should be considered if sequencing cannot clarify the underlying reason of an observed coagulopathy. Of note, in cases of combined FVII/FX deficiency, a deletion of the two contiguous genes might be part of a larger chromosomal rearrangement.

The Potential Role of Recombinant Activated Factor VIIa (rFVIIa) in Military Pre-Hospital Setting

DTIC Science & Technology

2004-09-01

coagulation factors and platelets by crystalloids, colloids, or blood products The severity of dilutional coagulopathy is determined by both volume and...RTO-MP-HFM-109 3 - 1 The Potential Role of Recombinant Activated Factor VIIa (rFVIIa) in Military Pre-Hospital Setting LTC (ret.) Uri...decrease mortality from exsanguinations. Recombinant factor VIIa (rFVIIa) has been shown to overcome a variety of coagulation and platelet disorders

Transcriptional activation of transforming growth factor alpha by estradiol: requirement for both a GC-rich site and an estrogen response element half-site.

PubMed

Vyhlidal, C; Samudio, I; Kladde, M P; Safe, S

2000-06-01

17beta-Estradiol (E2) induces transforming growth factor alpha (TGFalpha) gene expression in MCF-7 cells and previous studies have identified a 53 bp (-252 to -200) sequence containing two imperfect estrogen responsive elements (EREs) that contribute to E2 responsiveness. Deletion analysis of the TGFalpha gene promoter in this study identified a second upstream region of the promoter (-623 to -549) that is also E2 responsive. This sequence contains three GC-rich sites and an imperfect ERE half-site, and the specific cis-elements and trans-acting factors were determined by promoter analysis in transient transfection experiments, gel mobility shift assays and in vitro DNA footprinting. The results are consistent with an estrogen receptor alpha (ERalpha)/Sp1 complex interacting with an Sp1(N)(30) ERE half-site ((1/2)) motif in which both ERalpha and Sp1 bind promoter DNA. The ER/Sp1-DNA complex is formed using nuclear extracts from MCF-7 cells but not with recombinant human ERalpha or Sp1 proteins, suggesting that other nuclear factor(s) are required for complex stabilization. The E2-responsive Sp1(N)(x)ERE(1/2) motif identified in the TGFalpha gene promoter has also been characterized in the cathepsin D and heat shock protein 27 gene promoters; however, in the latter two promoters the numbers of intervening nucleotides are 23 and 10 respectively.