A screen to identify Drosophila genes required for integrin-mediated adhesion.

PubMed Central

Walsh, E P; Brown, N H

1998-01-01

Drosophila integrins have essential adhesive roles during development, including adhesion between the two wing surfaces. Most position-specific integrin mutations cause lethality, and clones of homozygous mutant cells in the wing do not adhere to the apposing surface, causing blisters. We have used FLP-FRT induced mitotic recombination to generate clones of randomly induced mutations in the F1 generation and screened for mutations that cause wing blisters. This phenotype is highly selective, since only 14 lethal complementation groups were identified in screens of the five major chromosome arms. Of the loci identified, 3 are PS integrin genes, 2 are blistered and bloated, and the remaining 9 appear to be newly characterized loci. All 11 nonintegrin loci are required on both sides of the wing, in contrast to integrin alpha subunit genes. Mutations in 8 loci only disrupt adhesion in the wing, similar to integrin mutations, while mutations in the 3 other loci cause additional wing defects. Mutations in 4 loci, like the strongest integrin mutations, cause a "tail-up" embryonic lethal phenotype, and mutant alleles of 1 of these loci strongly enhance an integrin mutation. Thus several of these loci are good candidates for genes encoding cytoplasmic proteins required for integrin function. PMID:9755209

RADIATION INDUCED VIABILITY MUTATIONS IN THE HONEY BEE

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

Lee, W.R.

The frequency of recessive detrimental mutations expressed in the haploid drone honey bee was investigated and compared with recessive and dominant lethal mutations detected in the haploid drone and diploid worker. A single queen was inseminated by a drone homozygous for three genetic markers. Viability of progeny was determined, and hybrid daughters bearing the genetic markers were stored in colonies. The spermatheca of the queen was then irradiated with 2600 r kvp x rays. Morphological defects and viability were studied in progeny and grand-progeny. A total of 92 pairs was tested during one season. Results showed that 60.8% of themore » sperm cells receiving radiation contained at least one or more dominant lethals. Correcting for the saturation effect on the assumption of independence of each dominant lethal, an average proportion of 0.94 dominant lethals were found per cell. The average reduction in embryonic viability was 28%. Forty per cent of the queens tested contained one or more recessive lethals. Corrections in procedure and plans for future work, as well as work in progress, are described. (H.M.G.)« less

Chitin synthase III: synthetic lethal mutants and "stress related" chitin synthesis that bypasses the CSD3/CHS6 localization pathway.

PubMed

Osmond, B C; Specht, C A; Robbins, P W

1999-09-28

We screened Saccharomyces strains for mutants that are synthetically lethal with deletion of the major chitin synthase gene CHS3. In addition to finding, not surprisingly, that mutations in major cell wall-related genes such as FKS1 (glucan synthase) and mutations in any of the Golgi glycosylation complex genes (MNN9 family) are lethal in combination with chs3Delta, we found that a mutation in Srv2p, a bifunctional regulatory gene, is notably lethal in the chs3 deletion. In extending studies of fks1-chitin synthase 3 interactions, we made the surprising discovery that deletion of CSD3/CHS6, a gene normally required for Chs3p delivery and activity in vivo, was not lethal with fks1 and, in fact, that lack of Csd3p/Chs6p did not decrease the high level of stress-related chitin made in the fks1 mutant. This finding suggests that "stress response" chitin synthesis proceeds through an alternate Chs3p targeting pathway.

Chitin synthase III: Synthetic lethal mutants and “stress related” chitin synthesis that bypasses the CSD3/CHS6 localization pathway

PubMed Central

Osmond, Barbara C.; Specht, Charles A.; Robbins, Phillips W.

1999-01-01

We screened Saccharomyces strains for mutants that are synthetically lethal with deletion of the major chitin synthase gene CHS3. In addition to finding, not surprisingly, that mutations in major cell wall-related genes such as FKS1 (glucan synthase) and mutations in any of the Golgi glycosylation complex genes (MNN9 family) are lethal in combination with chs3Δ, we found that a mutation in Srv2p, a bifunctional regulatory gene, is notably lethal in the chs3 deletion. In extending studies of fks1-chitin synthase 3 interactions, we made the surprising discovery that deletion of CSD3/CHS6, a gene normally required for Chs3p delivery and activity in vivo, was not lethal with fks1 and, in fact, that lack of Csd3p/Chs6p did not decrease the high level of stress-related chitin made in the fks1 mutant. This finding suggests that “stress response” chitin synthesis proceeds through an alternate Chs3p targeting pathway. PMID:10500155

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

Greenberg, C.R.; Taylor, C.D.; Haworth, J.C.

The authors have discovered a single homoallelic nucleotide substitution as the putative cause of the perinatal (lethal) form of hypophosphatasia in Canadian Mennonites. Previous linkage and haplotype analysis in this population suggested that a single mutational event was responsible for this autosomal recessive form of hypophosphatasia. The mutation is a guanosine-to-adenosine substitution at nucleotide position 1177 in exon 10 of the tissue nonspecific (liver/bone/kidney) alkaline phosphatase gene. This Gly[sup 317] [yields] Asp mutation segregates exclusively with the heterozygote phenotype previously assigned by biochemical testing (maximum combined lod score of 18.24 at [theta] = 0.00). This putative disease-causing mutation has notmore » been described in controls nor in other non-Mennonite probands with both lethal and nonlethal forms of hypophosphatasia studied to date. This Gly[sup 317] [yields] Asp mutation changes a polar glycine to an acidic aspartate at amino acid position 317 within the highly conserved active site region of the 507-amino-acid polypeptide. Carrier screening for this lethal mutation in a high-risk population is now feasible. 15 refs., 2 figs.« less

Mutations in the histone fold domain of the TAF12 gene show synthetic lethality with the TAF1 gene lacking the TAF N-terminal domain (TAND) by different mechanisms from those in the SPT15 gene encoding the TATA box-binding protein (TBP)

PubMed Central

Kobayashi, Akiko; Miyake, Tsuyoshi; Kawaichi, Masashi; Kokubo, Tetsuro

2003-01-01

The general transcription factor TFIID, composed of the TATA box-binding protein (TBP) and 14 TBP-associated factors (TAFs), is important for both basal and regulated transcription by RNA polymerase II. Although it is well known that the TAF N-terminal domain (TAND) at the amino-terminus of the TAF1 protein binds to TBP and thereby inhibits TBP function in vitro, the physiological role of this domain remains obscure. In our previous study, we screened for mutations that cause lethality when co-expressed with the TAF1 gene lacking TAND (taf1-ΔTAND) and identified two ΔTAND synthetic lethal (nsl) mutations as those in the SPT15 gene encoding TBP. In this study we isolated another nsl mutation in the same screen and identified it to be a mutation in the histone fold domain (HFD) of the TAF12 gene. Several other HFD mutations of this gene also exhibit nsl phenotypes, and all of them are more or less impaired in transcriptional activation in vivo. Interestingly, a set of genes affected in the taf1-ΔTAND mutant is similarly affected in the taf12 HFD mutants but not in the nsl mutants of TBP. Therefore, we discovered that the nsl mutations of these two genes cause lethality in the taf1-ΔTAND mutant by different mechanisms. PMID:12582246

Identification of a nonsense mutation in APAF1 that is likely causal for a decrease in reproductive efficiency in Holstein dairy cattle

USDA-ARS?s Scientific Manuscript database

A haplotype on cattle chromosome 5 carrying a recessive lethal allele was found to originate in a Holstein-Friesian foundation sire. Resequencing led to the identification of a stop-gain mutation in exon 11 of APAF1, a gene known to cause embryonic lethality and neurodevelopmental abnormalities in ...

Use of the Polymerase Chain Reaction and Complementary DNA Probes in the Detection of Duchenne Muscular Dystrophy Carriers

DTIC Science & Technology

1990-01-01

dominant or X-linked mutations, for example DMD and lethal osteogenesis imperfecta (1, 97). This phenomenon is the result of a dual population of...of the mutations. Am J Hum Genet 1988; 43: 620-29. 97. Cohn DH, Starman B, Blumberg B, Byers PH. Recurrence of lethal osteogenesis imperfecta due to

A Strong Loss-of-Function Mutation in RAN1 Results in Constitutive Activation of the Ethylene Response Pathway as Well as a Rosette-Lethal Phenotype

PubMed Central

Woeste, Keith E.; Kieber, Joseph J.

2000-01-01

A recessive mutation was identified that constitutively activated the ethylene response pathway in Arabidopsis and resulted in a rosette-lethal phenotype. Positional cloning of the gene corresponding to this mutation revealed that it was allelic to responsive to antagonist1 (ran1), a mutation that causes seedlings to respond in a positive manner to what is normally a competitive inhibitor of ethylene binding. In contrast to the previously identified ran1-1 and ran1-2 alleles that are morphologically indistinguishable from wild-type plants, this ran1-3 allele results in a rosette-lethal phenotype. The predicted protein encoded by the RAN1 gene is similar to the Wilson and Menkes disease proteins and yeast Ccc2 protein, which are integral membrane cation-transporting P-type ATPases involved in copper trafficking. Genetic epistasis analysis indicated that RAN1 acts upstream of mutations in the ethylene receptor gene family. However, the rosette-lethal phenotype of ran1-3 was not suppressed by ethylene-insensitive mutants, suggesting that this mutation also affects a non-ethylene-dependent pathway regulating cell expansion. The phenotype of ran1-3 mutants is similar to loss-of-function ethylene receptor mutants, suggesting that RAN1 may be required to form functional ethylene receptors. Furthermore, these results suggest that copper is required not only for ethylene binding but also for the signaling function of the ethylene receptors. PMID:10715329

A strong loss-of-function mutation in RAN1 results in constitutive activation of the ethylene response pathway as well as a rosette-lethal phenotype

NASA Technical Reports Server (NTRS)

Woeste, K. E.; Kieber, J. J.; Evans, M. L. (Principal Investigator)

2000-01-01

A recessive mutation was identified that constitutively activated the ethylene response pathway in Arabidopsis and resulted in a rosette-lethal phenotype. Positional cloning of the gene corresponding to this mutation revealed that it was allelic to responsive to antagonist1 (ran1), a mutation that causes seedlings to respond in a positive manner to what is normally a competitive inhibitor of ethylene binding. In contrast to the previously identified ran1-1 and ran1-2 alleles that are morphologically indistinguishable from wild-type plants, this ran1-3 allele results in a rosette-lethal phenotype. The predicted protein encoded by the RAN1 gene is similar to the Wilson and Menkes disease proteins and yeast Ccc2 protein, which are integral membrane cation-transporting P-type ATPases involved in copper trafficking. Genetic epistasis analysis indicated that RAN1 acts upstream of mutations in the ethylene receptor gene family. However, the rosette-lethal phenotype of ran1-3 was not suppressed by ethylene-insensitive mutants, suggesting that this mutation also affects a non-ethylene-dependent pathway regulating cell expansion. The phenotype of ran1-3 mutants is similar to loss-of-function ethylene receptor mutants, suggesting that RAN1 may be required to form functional ethylene receptors. Furthermore, these results suggest that copper is required not only for ethylene binding but also for the signaling function of the ethylene receptors.

Inferring the distribution of mutational effects on fitness in Drosophila.

PubMed

Loewe, Laurence; Charlesworth, Brian

2006-09-22

The properties of the distribution of deleterious mutational effects on fitness (DDME) are of fundamental importance for evolutionary genetics. Since it is extremely difficult to determine the nature of this distribution, several methods using various assumptions about the DDME have been developed, for the purpose of parameter estimation. We apply a newly developed method to DNA sequence polymorphism data from two Drosophila species and compare estimates of the parameters of the distribution of the heterozygous fitness effects of amino acid mutations for several different distribution functions. The results exclude normal and gamma distributions, since these predict too few effectively lethal mutations and power-law distributions as a result of predicting too many lethals. Only the lognormal distribution appears to fit both the diversity data and the frequency of lethals. This DDME arises naturally in complex systems when independent factors contribute multiplicatively to an increase in fitness-reducing damage. Several important parameters, such as the fraction of effectively neutral non-synonymous mutations and the harmonic mean of non-neutral selection coefficients, are robust to the form of the DDME. Our results suggest that the majority of non-synonymous mutations in Drosophila are under effective purifying selection.

GENETIC EFFECTS OF SPACEFLIGHT FACTORS

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

Glembotskii, Ya.L.; Parfenov, G.P.

1962-12-01

With the object of investigating effects of spaceflight factors on heredity, Drosophila melanogaster was carried on the second, fourth, and fifth orbital spaceships and on Vostok-1 and Vostok-2. Four different spaceflight effects were investigated. Nondisjunction of chromosomes was investigated by exposing unfertilized white-eyed Drosophila females on Vostoks 1 and 2 and mating them on their return with red-eyed males. Primary nondisjunction of chromosomes resulted in the appearance of four times as many unusual genotypes (XXY females and XO males) among the progeny of the exposed group as among offspring of the controls. However, the increase in nondisjunction cannot be ascribedmore » to radiation effects. Induced crossovers were investigated by exposing heterozygotic males (having normal phenotypes but three recessive genes in the second chromosome) on the fifth orbital spaceship and on Vostoks 1 and 2. Upon return they were mated with homozygotic females displaying the three recessive characteristics (black body, cinnabar eyes, and vestigial wings). Drosophila carried in the fifth orbital spaceship with no protection against low-frequency vibrations showed crossover incidence of 0.50 450 deg C in a 0.12%, compared to an incidence of 0.05 450 deg C in a 0.05% or none at all on Vostok spaceships, where the insect containers were cushioned against vibration. Dominant lethal mutations were investigated by exposing two strains of Drosophila melanogaster (D- 18 with a high rate of spontaneous lethal mutations, and D-32 with a low rate for the same mutations) of the five spacecraft. The number of dominant lethal mutations was found to increase somewhat in all groups exposed to space flight. Sex-linked recessive lethal mutations were investigated by exposing young males of the D-18 and D-32 strains of Drosophila melanogaster on all five vehicles. Exposure on the second and fourth orbital spaceships and on Vostok-1 resulted in statistically significant numbers of sex-linked recessive lethal mutations for spermatozoa and spermatids of both strains. However, no increase in mutations was observed following exposure on the fifth orbital spaceship and on Vostok-2. (TCO)« less

Suppression Analysis Reveals a Functional Difference between the Serines in Positions Two and Five in the Consensus Sequence of the C-Terminal Domain of Yeast RNA Polymerase II

PubMed Central

Yuryev, A.; Corden, J. L.

1996-01-01

The largest subunit of RNA polymerase II contains a repetitive C-terminal domain (CTD) consisting of tandem repeats of the consensus sequence Tyr(1)Ser(2)Pro(3)Thr(4) Ser(5)Pro(6) Ser(7). Substitution of nonphosphorylatable amino acids at positions two or five of the Saccharomyces cerevisiae CTD is lethal. We developed a selection ssytem for isolating suppressors of this lethal phenotype and cloned a gene, SCA1 (suppressor of CTD alanine), which complements recessive suppressors of lethal multiple-substitution mutations. A partial deletion of SCA1 (sca1Δ::hisG) suppresses alanine or glutamate substitutions at position two of the consensus CTD sequence, and a lethal CTD truncation mutation, but SCA1 deletion does not suppress alanine or glutamate substitutions at position five. SCA1 is identical to SRB9, a suppressor of a cold-sensitive CTD truncation mutation. Strains carrying dominant SRB mutations have the same suppression properties as a sca1Δ::hisG strain. These results reveal a functional difference between positions two and five of the consensus CTD heptapeptide repeat. The ability of SCA1 and SRB mutant alleles to suppress CTD truncation mutations suggest that substitutions at position two, but not at position five, cause a defect in RNA polymerase II function similar to that introduced by CTD truncation. PMID:8725217

Mutations in new cell cycle genes that fail to complement a multiply mutant third chromosome of Drosophila.

PubMed

White-Cooper, H; Carmena, M; Gonzalez, C; Glover, D M

1996-11-01

We have simultaneously screened for new alleles and second site mutations that fail to complement five cell cycle mutations of Drosphila carried on a single third chromosome (gnu, polo, mgr, asp, stg). Females that are either transheterozygous for scott of the antartic (scant) and polo, or homozygous for scant produce embryos that show mitotic defects. A maternal effect upon embryonic mitoses is also seen in embryos derived from females transheterozygous with helter skelter (hsk) and either mgr or asp. cleopatra (cleo), fails to complement asp but is not uncovered by a deficiency for asp. The mitotic phenotype of larvae heterozygous for cleo and the multiple mutant chromosome is similar to weak alleles of asp, but there are no defects in male meiosis. Mutations that failed to complement stg fell into two complementation groups corresponding to stg and a new gene noose. Three of the new stg alleles are early zygotic lethals, whereas the fourth is a pharate adult lethal allele that affects both mitosis and meiosis. Mutations in noose fully complement a small deficiency that removes stg, but when placed in trans to certain stg alleles, result in late lethality and mitotic abnormalities in larval brains.

[Mutagenic and antimutagenic properties of bemitil].

PubMed

Seredenin, S B; Bobkov, Iu G; Durnev, A D; Dubovskaia, O Iu

1986-07-01

Complex research of the genetic activity of a new 2-mercaptobenzimidazole derivative bemythyl has shown that the drug failed to induce recessive, age-related lethal mutations in drosophila, dominant lethal mutations in germ mammalian cells and chromosomal damage in murine bone marrow cells and human peripheral blood cell cultures. The experiments on mice have demonstrated that therapeutic bemythyl doses caused a two-fold decrease in the level of aberrant cells induced by alkylating agents--fotrin and fopurin.

Empirical complexities in the genetic foundations of lethal mutagenesis.

PubMed

Bull, James J; Joyce, Paul; Gladstone, Eric; Molineux, Ian J

2013-10-01

From population genetics theory, elevating the mutation rate of a large population should progressively reduce average fitness. If the fitness decline is large enough, the population will go extinct in a process known as lethal mutagenesis. Lethal mutagenesis has been endorsed in the virology literature as a promising approach to viral treatment, and several in vitro studies have forced viral extinction with high doses of mutagenic drugs. Yet only one empirical study has tested the genetic models underlying lethal mutagenesis, and the theory failed on even a qualitative level. Here we provide a new level of analysis of lethal mutagenesis by developing and evaluating models specifically tailored to empirical systems that may be used to test the theory. We first quantify a bias in the estimation of a critical parameter and consider whether that bias underlies the previously observed lack of concordance between theory and experiment. We then consider a seemingly ideal protocol that avoids this bias-mutagenesis of virions-but find that it is hampered by other problems. Finally, results that reveal difficulties in the mere interpretation of mutations assayed from double-strand genomes are derived. Our analyses expose unanticipated complexities in testing the theory. Nevertheless, the previous failure of the theory to predict experimental outcomes appears to reside in evolutionary mechanisms neglected by the theory (e.g., beneficial mutations) rather than from a mismatch between the empirical setup and model assumptions. This interpretation raises the specter that naive attempts at lethal mutagenesis may augment adaptation rather than retard it.

Lethal and mutagenic effects of ion beams and γ-rays in Aspergillus oryzae.

PubMed

Toyoshima, Yoshiyuki; Takahashi, Akemi; Tanaka, Hisaki; Watanabe, Jun; Mogi, Yoshinobu; Yamazaki, Tatsuo; Hamada, Ryoko; Iwashita, Kazuhiro; Satoh, Katsuya; Narumi, Issay

2012-12-01

Aspergillus oryzae is a fungus that is used widely in traditional Japanese fermentation industries. In this study, the lethal and mutagenic effects of different linear energy transfer (LET) radiation in freeze-dried conidia of A. oryzae were investigated. The lethal effect, which was evaluated by a 90% lethal dose, was dependent on the LET value of the ionizing radiation. The most lethal ionizing radiation among that tested was (12)C(5+) ion beams with an LET of 121keV/μm. The (12)C(5+) ion beams had a 3.6-times higher lethal effect than low-LET (0.2keV/μm) γ-rays. The mutagenic effect was evaluated by the frequency of selenate resistant mutants. (12)C(6+) ion beams with an LET of 86keV/μm were the most effective in inducing selenate resistance. The mutant frequency following exposure to (12)C(6+) ion beams increased with an increase in dose and reached 3.47×10(-3) at 700Gy. In the dose range from 0 to 700Gy, (12)C(5+) ion beams were the second most effective in inducing selenate resistance, the mutant frequency of which reached a maximum peak (1.67×10(-3)) at 400Gy. To elucidate the characteristics of mutation induced by ionizing radiation, mutations in the sulphate permease gene (sB) and ATP sulfurylase gene (sC) loci, the loss of function of which results in a selenate resistant phenotype, were compared between (12)C(5+) ion beams and γ-rays. We detected all types of transversions and transitions. For frameshifts, the frequency of a +1 frameshift was the highest in all cases. Although the incidence of deletions >2bp was generally low, deletions >20bp were characteristic for (12)C(5+) ion beams. γ-rays had a tendency to generate mutants carrying a multitude of mutations in the same locus. Both forms of radiation also induced genome-wide large-scale mutations including chromosome rearrangements and large deletions. These results provide new basic insights into the mutation breeding of A. oryzae using ionizing radiation. Crown Copyright © 2012. Published by Elsevier B.V. All rights reserved.

A recurrent mutation causing Melnick-Needles syndrome in females confers a severe, lethal phenotype in males.

PubMed

Spencer, Careni; Lombaard, Hendrik; Wise, Amy; Krause, Amanda; Robertson, Stephen P

2018-04-01

Melnick-Needles syndrome (MNS; MIM 309350) is an X-linked skeletal dysplasia caused by mutations in FLNA. Females with the condition present with characteristic facial features, short stature, skeletal anomalies, including poorly modeled and sclerotic bones, and structural abnormalities such as cardiac and urological defects. Previously males were thought to present with either a mild phenotype compatible with life or a severe lethal presentation depending on the maternal phenotype. The discovery of a limited number of mutations in FLNA as the cause of the condition has clarified the molecular basis of the disorder, but only a very small number of severely affected males have been reported with MNS. Furthermore, no mildly affected males have been described with a molecular confirmation of the condition. In this report, we describe the clinical and molecular findings of a mildly affected mother with MNS and her severely affected son. They shared a well-documented disease-causing variant in FLNA, p.(Ala1188Thr), one of two highly recurrent mutations leading to the disorder. This is only the fourth report of a male with perinatal lethal MNS and a molecular confirmation; it is the first description of this specific mutation in a male. © 2018 Wiley Periodicals, Inc.

Mutations in KIAA0586 Cause Lethal Ciliopathies Ranging from a Hydrolethalus Phenotype to Short-Rib Polydactyly Syndrome

PubMed Central

Alby, Caroline; Piquand, Kevin; Huber, Céline; Megarbané, André; Ichkou, Amale; Legendre, Marine; Pelluard, Fanny; Encha-Ravazi, Ferechté; Abi-Tayeh, Georges; Bessières, Bettina; El Chehadeh-Djebbar, Salima; Laurent, Nicole; Faivre, Laurence; Sztriha, László; Zombor, Melinda; Szabó, Hajnalka; Failler, Marion; Garfa-Traore, Meriem; Bole, Christine; Nitschké, Patrick; Nizon, Mathilde; Elkhartoufi, Nadia; Clerget-Darpoux, Françoise; Munnich, Arnold; Lyonnet, Stanislas; Vekemans, Michel; Saunier, Sophie; Cormier-Daire, Valérie; Attié-Bitach, Tania; Thomas, Sophie

2015-01-01

KIAA0586, the human ortholog of chicken TALPID3, is a centrosomal protein that is essential for primary ciliogenesis. Its disruption in animal models causes defects attributed to abnormal hedgehog signaling; these defects include polydactyly and abnormal dorsoventral patterning of the neural tube. Here, we report homozygous mutations of KIAA0586 in four families affected by lethal ciliopathies ranging from a hydrolethalus phenotype to short-rib polydactyly. We show defective ciliogenesis, as well as abnormal response to SHH-signaling activation in cells derived from affected individuals, consistent with a role of KIAA0586 in primary cilia biogenesis. Whereas centriolar maturation seemed unaffected in mutant cells, we observed an abnormal extended pattern of CEP290, a centriolar satellite protein previously associated with ciliopathies. Our data show the crucial role of KIAA0586 in human primary ciliogenesis and subsequent abnormal hedgehog signaling through abnormal GLI3 processing. Our results thus establish that KIAA0586 mutations cause lethal ciliopathies. PMID:26166481

X-ray induced dominant lethal mutations in mature and immature oocytes of guinea-pigs and golden hamsters.

PubMed

Cox, B D; Lyon, M F

1975-06-01

The induction of dominant lethal mutations by doses of 100-400 rad X-rays in oocytes of the guinea-pig and golden hamster was studied using criteria of embryonic mortality. For both species higher yields were obtained from mature than from immature oocytes, in contrast to results for the mouse. Data on fertility indicated that in the golden hamster, as in the mouse, immature oocytes were more sensitive to killing by X-rays than mature oocytes but that the converse was true in the guinea-pig. The dose-response relationship for mutation to dominant lethals in pre-ovulatory oocytes of guinea-pig and golden hamsters was linear, both when based on pre- and post-implantation loss and when on post-implantation loss only. The rate per unit dose was higher for the golden hamster, and the old golden hamsters were possibly slightly more sensitive than young ones. The mutation rate data for mature oocytes of the mouse, using post-implantation loss alone, also fitted a linear dose-response relationship, except that the rate per unit dose was lower than for the other two species.

Novel TMEM67 Mutations and Genotype-phenotype Correlates in Meckelin-related Ciliopathies

PubMed Central

Iannicelli, Miriam; Brancati, Francesco; Mougou-Zerelli, Soumaya; Mazzotta, Annalisa; Thomas, Sophie; Elkhartoufi, Nadia; Travaglini, Lorena; Gomes, Céline; Ardissino, Gian Luigi; Bertini, Enrico; Boltshauser, Eugen; Castorina, Pierangela; D'Arrigo, Stefano; Fischetto, Rita; Leroy, Brigitte; Loget, Philippe; Bonnière, Maryse; Starck, Lena; Tantau, Julia; Gentilin, Barbara; Majore, Silvia; Swistun, Dominika; Flori, Elizabeth; Lalatta, Faustina; Pantaleoni, Chiara; Johannes.Penzien; Grammatico, Paola; Dallapiccola, Bruno; Gleeson, Joseph G.; Attie-Bitach, Tania; Valente, Enza Maria

2010-01-01

Human ciliopathies are hereditary conditions caused by defects of proteins expressed at the primary cilium. Among ciliopathies, Joubert syndrome and related disorders (JSRD), Meckel syndrome (MKS) and nephronophthisis (NPH) present clinical and genetic overlap, being allelic at several loci. One of the most interesting gene is TMEM67, encoding the transmembrane protein meckelin. We performed mutation analysis of TMEM67 in 341 probands, including 265 JSRD representative of all clinical subgroups and 76 MKS fetuses. We identified 33 distinct mutations, of which 20 were novel, in 8/10 (80%) JS with liver involvement (COACH phenotype) and 12/76 (16%) MKS fetuses. No mutations were found in other JSRD subtypes, confirming the strong association between TMEM67 mutations and liver involvement. Literature review of all published TMEM67 mutated cases was performed to delineate genotype-phenotype correlates. In particular, comparison of the types of mutations and their distribution along the gene in lethal versus non lethal phenotypes showed in MKS patients a significant enrichment of missense mutations falling in TMEM67 exons 8 to 15, especially when in combination with a truncating mutation. These exons encode for a region of unknown function in the extracellular domain of meckelin. PMID:20232449

Loss of ATM kinase activity leads to embryonic lethality in mice.

PubMed

Daniel, Jeremy A; Pellegrini, Manuela; Lee, Baeck-Seung; Guo, Zhi; Filsuf, Darius; Belkina, Natalya V; You, Zhongsheng; Paull, Tanya T; Sleckman, Barry P; Feigenbaum, Lionel; Nussenzweig, André

2012-08-06

Ataxia telangiectasia (A-T) mutated (ATM) is a key deoxyribonucleic acid (DNA) damage signaling kinase that regulates DNA repair, cell cycle checkpoints, and apoptosis. The majority of patients with A-T, a cancer-prone neurodegenerative disease, present with null mutations in Atm. To determine whether the functions of ATM are mediated solely by its kinase activity, we generated two mouse models containing single, catalytically inactivating point mutations in Atm. In this paper, we show that, in contrast to Atm-null mice, both D2899A and Q2740P mutations cause early embryonic lethality in mice, without displaying dominant-negative interfering activity. Using conditional deletion, we find that the D2899A mutation in adult mice behaves largely similar to Atm-null cells but shows greater deficiency in homologous recombination (HR) as measured by hypersensitivity to poly (adenosine diphosphate-ribose) polymerase inhibition and increased genomic instability. These results may explain why missense mutations with no detectable kinase activity are rarely found in patients with classical A-T. We propose that ATM kinase-inactive missense mutations, unless otherwise compensated for, interfere with HR during embryogenesis.

Pancreatic SEC23B deficiency is sufficient to explain the perinatal lethality of germline SEC23B deficiency in mice

PubMed Central

Khoriaty, Rami; Everett, Lesley; Chase, Jennifer; Zhu, Guojing; Hoenerhoff, Mark; McKnight, Brooke; Vasievich, Matthew P.; Zhang, Bin; Tomberg, Kärt; Williams, John; Maillard, Ivan; Ginsburg, David

2016-01-01

In humans, loss of function mutations in SEC23B result in Congenital Dyserythropoietic Anemia type II (CDAII), a disease limited to defective erythroid development. Patients with two nonsense SEC23B mutations have not been reported, suggesting that complete SEC23B deficiency might be lethal. We previously reported that SEC23B-deficient mice die perinatally, exhibiting massive pancreatic degeneration and that mice with hematopoietic SEC23B deficiency do not exhibit CDAII. We now show that SEC23B deficiency restricted to the pancreas is sufficient to explain the lethality observed in mice with global SEC23B-deficiency. Immunohistochemical stains demonstrate an acinar cell defect but normal islet cells. Mammalian genomes contain two Sec23 paralogs, Sec23A and Sec23B. The encoded proteins share ~85% amino acid sequence identity. We generate mice with pancreatic SEC23A deficiency and demonstrate that these mice survive normally, exhibiting normal pancreatic weights and histology. Taken together, these data demonstrate that SEC23B but not SEC23A is essential for murine pancreatic development. We also demonstrate that two BAC transgenes spanning Sec23b rescue the lethality of mice homozygous for a Sec23b gene trap allele, excluding a passenger gene mutation as the cause of the pancreatic lethality, and indicating that the regulatory elements critical for Sec23b pancreatic function reside within the BAC transgenes. PMID:27297878

Drugging the Cancers Addicted to DNA Repair.

PubMed

Nickoloff, Jac A; Jones, Dennie; Lee, Suk-Hee; Williamson, Elizabeth A; Hromas, Robert

2017-11-01

Defects in DNA repair can result in oncogenic genomic instability. Cancers occurring from DNA repair defects were once thought to be limited to rare inherited mutations (such as BRCA1 or 2). It now appears that a clinically significant fraction of cancers have acquired DNA repair defects. DNA repair pathways operate in related networks, and cancers arising from loss of one DNA repair component typically become addicted to other repair pathways to survive and proliferate. Drug inhibition of the rescue repair pathway prevents the repair-deficient cancer cell from replicating, causing apoptosis (termed synthetic lethality). However, the selective pressure of inhibiting the rescue repair pathway can generate further mutations that confer resistance to the synthetic lethal drugs. Many such drugs currently in clinical use inhibit PARP1, a repair component to which cancers arising from inherited BRCA1 or 2 mutations become addicted. It is now clear that drugs inducing synthetic lethality may also be therapeutic in cancers with acquired DNA repair defects, which would markedly broaden their applicability beyond treatment of cancers with inherited DNA repair defects. Here we review how each DNA repair pathway can be attacked therapeutically and evaluate DNA repair components as potential drug targets to induce synthetic lethality. Clinical use of drugs targeting DNA repair will markedly increase when functional and genetic loss of repair components are consistently identified. In addition, future therapies will exploit artificial synthetic lethality, where complementary DNA repair pathways are targeted simultaneously in cancers without DNA repair defects. © The Author 2017. Published by Oxford University Press.

The maternally expressed WRKY transcription factor TTG2 controls lethality in interploidy crosses of Arabidopsis.

PubMed

Dilkes, Brian P; Spielman, Melissa; Weizbauer, Renate; Watson, Brian; Burkart-Waco, Diana; Scott, Rod J; Comai, Luca

2008-12-09

The molecular mechanisms underlying lethality of F1 hybrids between diverged parents are one target of speciation research. Crosses between diploid and tetraploid individuals of the same genotype can result in F1 lethality, and this dosage-sensitive incompatibility plays a role in polyploid speciation. We have identified variation in F1 lethality in interploidy crosses of Arabidopsis thaliana and determined the genetic architecture of the maternally expressed variation via QTL mapping. A single large-effect QTL, DR. STRANGELOVE 1 (DSL1), was identified as well as two QTL with epistatic relationships to DSL1. DSL1 affects the rate of postzygotic lethality via expression in the maternal sporophyte. Fine mapping placed DSL1 in an interval encoding the maternal effect transcription factor TTG2. Maternal parents carrying loss-of-function mutations in TTG2 suppressed the F1 lethality caused by paternal excess interploidy crosses. The frequency of cellularization in the endosperm was similarly affected by both natural variation and ttg2 loss-of-function mutants. The simple genetic basis of the natural variation and effects of single-gene mutations suggests that F1 lethality in polyploids could evolve rapidly. Furthermore, the role of the sporophytically active TTG2 gene in interploidy crosses indicates that the developmental programming of the mother regulates the viability of interploidy hybrid offspring.

The Generation and Genetic Analysis of Suppressors of Lethal Mutations in the Caenorhabditis Elegans Rol-3(v) Gene

PubMed Central

Barbazuk, W. B.; Johnsen, R. C.; Baillie, D. L.

1994-01-01

The Caenorhabditis elegans rol-3(e754) mutation is a member of a general glass of mutations affecting gross morphology, presumably through disruption of the nematode cuticle. Adult worms homozygous for rol-3(e754) exhibit rotation about their long axis associated with a left-hand twisted cuticle, musculature, gut and ventral nerve cord. Our laboratory previously isolated 12 recessive lethal alleles of rol-3. All these lethal alleles cause an arrest in development at either early or mid-larval stages, suggesting that the rol-3 gene product performs an essential developmental function. Furthermore, through the use of the heterochronic mutants lin-14 and lin-29, we have established that the expression of rol-3(e754)'s adult specific visible function is not dependent on the presence of an adult cuticle. In an attempt to understand rol-3's developmental role we sought to identify other genes whose products interact with that of rol-3. Toward this end, we generated eight EMS induced and two gamma irradiation-induced recessive suppressors of the temperature sensitive (ts) mid-larval lethal phenotype of rol-3(s1040ts). These suppressors define two complementation groups srl-1 II and srl-2 III; and, while they suppress the rol-3(s1040) lethality, they do not suppress the adult specific visible rolling phenotype. Furthermore, there is a complex genetic interaction between srl-2 and srl-1 such that srl-2(s2506) fails to complement all srl alleles tested. These results suggest that srl-1 and srl-2 may share a common function and, thus, possibly constitute members of the same gene family. Mutations in both srl-1 and srl-2 produce no obvious hermaphrodite phenotypes in the absence of rol-3(s1040ts); however, males homozygous for either srl-1 or srl-2 display aberrant tail morphology. We present evidence suggesting that the members of srl-2 are not allele specific with respect to their suppression of rol-3 lethality, and that rol-3 may act in some way to influence proper posterior morphogenesis. Finally, based on our genetic analysis of rol-3 and the srl mutations, we present a model whereby the wild-type products of the srl loci act in a concerted manner to negatively regulate the rol-3 gene. PMID:8138151

Mutation in fission yeast phosphatidylinositol 4-kinase Pik1 is synthetically lethal with defect in telomere protection protein Pot1.

PubMed

Sugihara, Asami; Nguyen, Luan Cao; Shamim, Hossain Mohammad; Iida, Tetsushi; Nakase, Mai; Takegawa, Kaoru; Senda, Mitsuhisa; Jida, Shohei; Ueno, Masaru

2018-02-19

Fission yeast Pik1p is one of three phosphatidylinositol 4-kinases associated with the Golgi complex, but its function is not fully understood. Deletion of pot1 + causes telomere degradation and chromosome circularization. We searched for the gene which becomes synthetically lethal with pot1Δ. We obtained a novel pik1 mutant, pik1-1, which is synthetically lethal with pot1Δ. We found phosphoinositol 4-phosphate in the Golgi was reduced in pik1-1. To investigate the mechanism of the lethality of the pot1Δ pik1-1 double mutant, we constructed the nmt-pot1-aid pik1-1 strain, where Pot1 function becomes low by drugs, which leads to telomere loss and chromosome circularization, and found pik1-1 mutation does not affect telomere resection and chromosome circularization. Thus, our results suggest that pik1 + is required for the maintenance of circular chromosomes. Copyright © 2018 Elsevier Inc. All rights reserved.

The induction of mutation and recombination following UV irradiation during meiosis in Saccharomyces cerevisiae.

PubMed

Kelly, S L; Parry, J M

1983-03-01

Irradiation of yeast cultures with ultraviolet light at discrete stages during meiosis produces cyclic variations in sensitivity, i.e. cells are more sensitive to the lethal effects of UV light prior to entry into the meiotic DNA synthesis, and this corresponds to a peak of induction of point mutation. Cells become more resistant to both induced point mutation and lethality as they enter meiotic DNA synthesis, but become more sensitive again during spore formation. The induced level of intragenic recombination rises during the period of commitment to recombination to a level indistinguishable from the full meiotic level of spontaneous intragenic recombination. Induced reciprocal recombination remains above the spontaneous level up to the point of commitment to sporulation.

Evolution of high-level resistance during low-level antibiotic exposure.

PubMed

Wistrand-Yuen, Erik; Knopp, Michael; Hjort, Karin; Koskiniemi, Sanna; Berg, Otto G; Andersson, Dan I

2018-04-23

It has become increasingly clear that low levels of antibiotics present in many environments can select for resistant bacteria, yet the evolutionary pathways for resistance development during exposure to low amounts of antibiotics remain poorly defined. Here we show that Salmonella enterica exposed to sub-MIC levels of streptomycin evolved high-level resistance via novel mechanisms that are different from those observed during lethal selections. During lethal selection only rpsL mutations are found, whereas at sub-MIC selection resistance is generated by several small-effect resistance mutations that combined confer high-level resistance via three different mechanisms: (i) alteration of the ribosomal RNA target (gidB mutations), (ii) reduction in aminoglycoside uptake (cyoB, nuoG, and trkH mutations), and (iii) induction of the aminoglycoside-modifying enzyme AadA (znuA mutations). These results demonstrate how the strength of the selective pressure influences evolutionary trajectories and that even weak selective pressures can cause evolution of high-level resistance.

Embryonic lethality is not sufficient to explain hourglass-like conservation of vertebrate embryos.

PubMed

Uchida, Yui; Uesaka, Masahiro; Yamamoto, Takayoshi; Takeda, Hiroyuki; Irie, Naoki

2018-01-01

Understanding the general trends in developmental changes during animal evolution, which are often associated with morphological diversification, has long been a central issue in evolutionary developmental biology. Recent comparative transcriptomic studies revealed that gene expression profiles of mid-embryonic period tend to be more evolutionarily conserved than those in earlier or later periods. While the hourglass-like divergence of developmental processes has been demonstrated in a variety of animal groups such as vertebrates, arthropods, and nematodes, the exact mechanism leading to this mid-embryonic conservation remains to be clarified. One possibility is that the mid-embryonic period (pharyngula period in vertebrates) is highly prone to embryonic lethality, and the resulting negative selections lead to evolutionary conservation of this phase. Here, we tested this "mid-embryonic lethality hypothesis" by measuring the rate of lethal phenotypes of three different species of vertebrate embryos subjected to two kinds of perturbations: transient perturbations and genetic mutations. By subjecting zebrafish ( Danio rerio ), African clawed frog ( Xenopus laevis ), and chicken ( Gallus gallus ) embryos to transient perturbations, namely heat shock and inhibitor treatments during three developmental periods [early (represented by blastula and gastrula), pharyngula, and late], we found that the early stages showed the highest rate of lethal phenotypes in all three species. This result was corroborated by perturbation with genetic mutations. By tracking the survival rate of wild-type embryos and embryos with genetic mutations induced by UV irradiation in zebrafish and African clawed frogs, we found that the highest decrease in survival rate was at the early stages particularly around gastrulation in both these species. In opposition to the "mid-embryonic lethality hypothesis," our results consistently showed that the stage with the highest lethality was not around the conserved pharyngula period, but rather around the early period in all the vertebrate species tested. These results suggest that negative selection by embryonic lethality could not explain hourglass-like conservation of animal embryos. This highlights the potential contribution of alternative mechanisms such as the diversifying effect of positive selections against earlier and later stages, and developmental constraints which lead to conservation of mid-embryonic stages.

Inviability of a DNA2 deletion mutant is due to the DNA damage checkpoint.

PubMed

Budd, Martin E; Antoshechkin, Igor A; Reis, Clara; Wold, Barbara J; Campbell, Judith L

2011-05-15

Dna2 is a dual polarity exo/endonuclease, and 5' to 3' DNA helicase involved in Okazaki Fragment Processing (OFP) and Double-Strand Break (DSB) Repair. In yeast, DNA2 is an essential gene, as expected for a DNA replication protein. Suppression of the lethality of dna2Δ mutants has been found to occur by two mechanisms: overexpression of RAD27 (scFEN1) , encoding a 5' to 3' exo/endo nuclease that processes Okazaki fragments (OFs) for ligation, or deletion of PIF1, a 5' to 3' helicase involved in mitochondrial recombination, telomerase inhibition and OFP. Mapping of a novel, spontaneously arising suppressor of dna2Δ now reveals that mutation of rad9 and double mutation of rad9 mrc1 can also suppress the lethality of dna2Δ mutants. Interaction of dna2Δ and DNA damage checkpoint mutations provides insight as to why dna2Δ is lethal but rad27Δ is not, even though evidence shows that Rad27 (ScFEN1) processes most of the Okazaki fragments, while Dna2 processes only a subset.

Inactivating mutations in MFSD2A, required for omega-3 fatty acid transport in brain, cause a lethal microcephaly syndrome.

PubMed

Guemez-Gamboa, Alicia; Nguyen, Long N; Yang, Hongbo; Zaki, Maha S; Kara, Majdi; Ben-Omran, Tawfeg; Akizu, Naiara; Rosti, Rasim Ozgur; Rosti, Basak; Scott, Eric; Schroth, Jana; Copeland, Brett; Vaux, Keith K; Cazenave-Gassiot, Amaury; Quek, Debra Q Y; Wong, Bernice H; Tan, Bryan C; Wenk, Markus R; Gunel, Murat; Gabriel, Stacey; Chi, Neil C; Silver, David L; Gleeson, Joseph G

2015-07-01

Docosahexanoic acid (DHA) is the most abundant omega-3 fatty acid in brain, and, although it is considered essential, deficiency has not been linked to disease. Despite the large mass of DHA in phospholipids, the brain does not synthesize it. DHA is imported across the blood-brain barrier (BBB) through the major facilitator superfamily domain-containing 2a (MFSD2A) protein. MFSD2A transports DHA as well as other fatty acids in the form of lysophosphatidylcholine (LPC). We identify two families displaying MFSD2A mutations in conserved residues. Affected individuals exhibited a lethal microcephaly syndrome linked to inadequate uptake of LPC lipids. The MFSD2A mutations impaired transport activity in a cell-based assay. Moreover, when expressed in mfsd2aa-morphant zebrafish, mutants failed to rescue microcephaly, BBB breakdown and lethality. Our results establish a link between transport of DHA and LPCs by MFSD2A and human brain growth and function, presenting the first evidence of monogenic disease related to transport of DHA in humans.

Mutations in KIAA0586 Cause Lethal Ciliopathies Ranging from a Hydrolethalus Phenotype to Short-Rib Polydactyly Syndrome.

PubMed

Alby, Caroline; Piquand, Kevin; Huber, Céline; Megarbané, André; Ichkou, Amale; Legendre, Marine; Pelluard, Fanny; Encha-Ravazi, Ferechté; Abi-Tayeh, Georges; Bessières, Bettina; El Chehadeh-Djebbar, Salima; Laurent, Nicole; Faivre, Laurence; Sztriha, László; Zombor, Melinda; Szabó, Hajnalka; Failler, Marion; Garfa-Traore, Meriem; Bole, Christine; Nitschké, Patrick; Nizon, Mathilde; Elkhartoufi, Nadia; Clerget-Darpoux, Françoise; Munnich, Arnold; Lyonnet, Stanislas; Vekemans, Michel; Saunier, Sophie; Cormier-Daire, Valérie; Attié-Bitach, Tania; Thomas, Sophie

2015-08-06

KIAA0586, the human ortholog of chicken TALPID3, is a centrosomal protein that is essential for primary ciliogenesis. Its disruption in animal models causes defects attributed to abnormal hedgehog signaling; these defects include polydactyly and abnormal dorsoventral patterning of the neural tube. Here, we report homozygous mutations of KIAA0586 in four families affected by lethal ciliopathies ranging from a hydrolethalus phenotype to short-rib polydactyly. We show defective ciliogenesis, as well as abnormal response to SHH-signaling activation in cells derived from affected individuals, consistent with a role of KIAA0586 in primary cilia biogenesis. Whereas centriolar maturation seemed unaffected in mutant cells, we observed an abnormal extended pattern of CEP290, a centriolar satellite protein previously associated with ciliopathies. Our data show the crucial role of KIAA0586 in human primary ciliogenesis and subsequent abnormal hedgehog signaling through abnormal GLI3 processing. Our results thus establish that KIAA0586 mutations cause lethal ciliopathies. Copyright © 2015 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

New insights into genotype–phenotype correlation for GLI3 mutations

PubMed Central

Démurger, Florence; Ichkou, Amale; Mougou-Zerelli, Soumaya; Le Merrer, Martine; Goudefroye, Géraldine; Delezoide, Anne-Lise; Quélin, Chloé; Manouvrier, Sylvie; Baujat, Geneviève; Fradin, Mélanie; Pasquier, Laurent; Megarbané, André; Faivre, Laurence; Baumann, Clarisse; Nampoothiri, Sheela; Roume, Joëlle; Isidor, Bertrand; Lacombe, Didier; Delrue, Marie-Ange; Mercier, Sandra; Philip, Nicole; Schaefer, Elise; Holder, Muriel; Krause, Amanda; Laffargue, Fanny; Sinico, Martine; Amram, Daniel; André, Gwenaelle; Liquier, Alain; Rossi, Massimiliano; Amiel, Jeanne; Giuliano, Fabienne; Boute, Odile; Dieux-Coeslier, Anne; Jacquemont, Marie-Line; Afenjar, Alexandra; Van Maldergem, Lionel; Lackmy-Port-Lis, Marylin; Vincent- Delorme, Catherine; Chauvet, Marie-Liesse; Cormier-Daire, Valérie; Devisme, Louise; Geneviève, David; Munnich, Arnold; Viot, Géraldine; Raoul, Odile; Romana, Serge; Gonzales, Marie; Encha-Razavi, Ferechte; Odent, Sylvie; Vekemans, Michel; Attie-Bitach, Tania

2015-01-01

The phenotypic spectrum of GLI3 mutations includes autosomal dominant Greig cephalopolysyndactyly syndrome (GCPS) and Pallister–Hall syndrome (PHS). PHS was first described as a lethal condition associating hypothalamic hamartoma, postaxial or central polydactyly, anal atresia and bifid epiglottis. Typical GCPS combines polysyndactyly of hands and feet and craniofacial features. Genotype–phenotype correlations have been found both for the location and the nature of GLI3 mutations, highlighting the bifunctional nature of GLI3 during development. Here we report on the molecular and clinical study of 76 cases from 55 families with either a GLI3 mutation (49 GCPS and 21 PHS), or a large deletion encompassing the GLI3 gene (6 GCPS cases). Most of mutations are novel and consistent with the previously reported genotype–phenotype correlation. Our results also show a correlation between the location of the mutation and abnormal corpus callosum observed in some patients with GCPS. Fetal PHS observations emphasize on the possible lethality of GLI3 mutations and extend the phenotypic spectrum of malformations such as agnathia and reductional limbs defects. GLI3 expression studied by in situ hybridization during human development confirms its early expression in target tissues. PMID:24736735

New insights into genotype-phenotype correlation for GLI3 mutations.

PubMed

Démurger, Florence; Ichkou, Amale; Mougou-Zerelli, Soumaya; Le Merrer, Martine; Goudefroye, Géraldine; Delezoide, Anne-Lise; Quélin, Chloé; Manouvrier, Sylvie; Baujat, Geneviève; Fradin, Mélanie; Pasquier, Laurent; Megarbané, André; Faivre, Laurence; Baumann, Clarisse; Nampoothiri, Sheela; Roume, Joëlle; Isidor, Bertrand; Lacombe, Didier; Delrue, Marie-Ange; Mercier, Sandra; Philip, Nicole; Schaefer, Elise; Holder, Muriel; Krause, Amanda; Laffargue, Fanny; Sinico, Martine; Amram, Daniel; André, Gwenaelle; Liquier, Alain; Rossi, Massimiliano; Amiel, Jeanne; Giuliano, Fabienne; Boute, Odile; Dieux-Coeslier, Anne; Jacquemont, Marie-Line; Afenjar, Alexandra; Van Maldergem, Lionel; Lackmy-Port-Lis, Marylin; Vincent-Delorme, Catherine; Chauvet, Marie-Liesse; Cormier-Daire, Valérie; Devisme, Louise; Geneviève, David; Munnich, Arnold; Viot, Géraldine; Raoul, Odile; Romana, Serge; Gonzales, Marie; Encha-Razavi, Ferechte; Odent, Sylvie; Vekemans, Michel; Attie-Bitach, Tania

2015-01-01

The phenotypic spectrum of GLI3 mutations includes autosomal dominant Greig cephalopolysyndactyly syndrome (GCPS) and Pallister-Hall syndrome (PHS). PHS was first described as a lethal condition associating hypothalamic hamartoma, postaxial or central polydactyly, anal atresia and bifid epiglottis. Typical GCPS combines polysyndactyly of hands and feet and craniofacial features. Genotype-phenotype correlations have been found both for the location and the nature of GLI3 mutations, highlighting the bifunctional nature of GLI3 during development. Here we report on the molecular and clinical study of 76 cases from 55 families with either a GLI3 mutation (49 GCPS and 21 PHS), or a large deletion encompassing the GLI3 gene (6 GCPS cases). Most of mutations are novel and consistent with the previously reported genotype-phenotype correlation. Our results also show a correlation between the location of the mutation and abnormal corpus callosum observed in some patients with GCPS. Fetal PHS observations emphasize on the possible lethality of GLI3 mutations and extend the phenotypic spectrum of malformations such as agnathia and reductional limbs defects. GLI3 expression studied by in situ hybridization during human development confirms its early expression in target tissues.

Effect of lethality on the extinction and on the error threshold of quasispecies.

PubMed

Tejero, Hector; Marín, Arturo; Montero, Francisco

2010-02-21

In this paper the effect of lethality on error threshold and extinction has been studied in a population of error-prone self-replicating molecules. For given lethality and a simple fitness landscape, three dynamic regimes can be obtained: quasispecies, error catastrophe, and extinction. Using a simple model in which molecules are classified as master, lethal and non-lethal mutants, it is possible to obtain the mutation rates of the transitions between the three regimes analytically. The numerical resolution of the extended model, in which molecules are classified depending on their Hamming distance to the master sequence, confirms the results obtained in the simple model and shows how an error catastrophe regime changes when lethality is taken in account. (c) 2009 Elsevier Ltd. All rights reserved.

CDK1 Is a Synthetic Lethal Target for KRAS Mutant Tumours

PubMed Central

Costa-Cabral, Sara; Brough, Rachel; Konde, Asha; Aarts, Marieke; Campbell, James; Marinari, Eliana; Riffell, Jenna; Bardelli, Alberto; Torrance, Christopher; Lord, Christopher J.; Ashworth, Alan

2016-01-01

Activating KRAS mutations are found in approximately 20% of human cancers but no RAS-directed therapies are currently available. Here we describe a novel, robust, KRAS synthetic lethal interaction with the cyclin dependent kinase, CDK1. This was discovered using parallel siRNA screens in KRAS mutant and wild type colorectal isogenic tumour cells and subsequently validated in a genetically diverse panel of 26 colorectal and pancreatic tumour cell models. This established that the KRAS/CDK1 synthetic lethality applies in tumour cells with either amino acid position 12 (p.G12V, pG12D, p.G12S) or amino acid position 13 (p.G13D) KRAS mutations and can also be replicated in vivo in a xenograft model using a small molecule CDK1 inhibitor. Mechanistically, CDK1 inhibition caused a reduction in the S-phase fraction of KRAS mutant cells, an effect also characterised by modulation of Rb, a master control of the G1/S checkpoint. Taken together, these observations suggest that the KRAS/CDK1 interaction is a robust synthetic lethal effect worthy of further investigation. PMID:26881434

Dominant Drop mutants are gain-of-function alleles of the muscle segment homeobox gene (msh) whose overexpression leads to the arrest of eye development.

PubMed

Mozer, B A

2001-05-15

Dominant Drop (Dr) mutations are nearly eyeless and have additional recessive phenotypes including lethality and patterning defects in eye and sensory bristles due to cis-regulatory lesions in the cell cycle regulator string (stg). Genetic analysis demonstrates that the dominant small eye phenotype is the result of separate gain-of-function mutations in the closely linked muscle segment homeobox (msh) gene, encoding a homeodomain transcription factor required for patterning of muscle and nervous system. Reversion of the Dr(Mio) allele was coincident with the generation of lethal loss-of-function mutations in msh in cis, suggesting that the dominant eye phenotype is the result of ectopic expression. Molecular genetic analysis revealed that two dominant Dr alleles contain lesions upstream of the msh transcription start site. In the Dr(Mio) mutant, a 3S18 retrotransposon insertion is the target of second-site mutations (P-element insertions or deletions) which suppress the dominant eye phenotype following reversion. The pattern of 3S18 expression and the absence of msh in eye imaginal discs suggest that transcriptional activation of the msh promoter accounts for ectopic expression. Dr dominant mutations arrest eye development by blocking the progression of the morphogenetic furrow leading to photoreceptor cell loss via apoptosis. Gal4-mediated ubiquitous expression of msh in third-instar larvae was sufficient to arrest the morphogenetic furrow in the eye imaginal disc and resulted in lethality prior to eclosion. Dominant mutations in the human msx2 gene, one of the vertebrate homologs of msh, are associated with craniosynostosis, a disease affecting cranial development. The Dr mutations are the first example of gain-of-function mutations in the msh/msx gene family identified in a genetically tractible model organism and may serve as a useful tool to identify additional genes that regulate this class of homeodomain proteins. Copyright 2001 Academic Press.

Functional genomics reveals the induction of inflammatory response and metalloproteinase gene expression during lethal Ebola virus infection.

PubMed

Cilloniz, Cristian; Ebihara, Hideki; Ni, Chester; Neumann, Gabriele; Korth, Marcus J; Kelly, Sara M; Kawaoka, Yoshihiro; Feldmann, Heinz; Katze, Michael G

2011-09-01

Ebola virus is the etiologic agent of a lethal hemorrhagic fever in humans and nonhuman primates with mortality rates of up to 90%. Previous studies with Zaire Ebola virus (ZEBOV), mouse-adapted virus (MA-ZEBOV), and mutant viruses (ZEBOV-NP(ma), ZEBOV-VP24(ma), and ZEBOV-NP/VP24(ma)) allowed us to identify the mutations in viral protein 24 (VP24) and nucleoprotein (NP) responsible for acquisition of high virulence in mice. To elucidate specific molecular signatures associated with lethality, we compared global gene expression profiles in spleen samples from mice infected with these viruses and performed an extensive functional analysis. Our analysis showed that the lethal viruses (MA-ZEBOV and ZEBOV-NP/VP24(ma)) elicited a strong expression of genes 72 h after infection. In addition, we found that although the host transcriptional response to ZEBOV-VP24(ma) was nearly the same as that to ZEBOV-NP/VP24(ma), the contribution of a mutation in the NP gene was required for a lethal phenotype. Further analysis indicated that one of the most relevant biological functions differentially regulated by the lethal viruses was the inflammatory response, as was the induction of specific metalloproteinases, which were present in our newly identify functional network that was associated with Ebola virus lethality. Our results suggest that this dysregulated proinflammatory response increased the severity of disease. Consequently, the newly discovered molecular signature could be used as the starting point for the development of new drugs and therapeutics. To our knowledge, this is the first study that clearly defines unique molecular signatures associated with Ebola virus lethality.

Molecular and Genetic Characterization of the Drosophila Melanogaster 87e Actin Gene Region

PubMed Central

Manseau, L. J.; Ganetzky, B.; Craig, E. A.

1988-01-01

A combined molecular and genetic analysis of the 87E actin gene (Act87E) in Drosophila melanogaster was undertaken. A clone of Act87E was isolated and characterized. The Act87E transcription unit is 1.57 kb and includes a 556-base intervening sequence in the 5' leader of the gene. The protein-coding region is contiguous and encodes a protein that is >93% identical to the other Drosophila actins. By in situ hybridization with a series of deficiencies that break in 87E, Act87E was localized to a region encompassing one to three faint, polytene chromosome bands. The region between the deficiency endpoints that flank the actin gene was isolated and measures approximately 24-30 kb. The closest proximal deficiency endpoint lies 8-10 kb 5' to the actin gene; the closest distal deficiency endpoint lies 16-20 kb 3' to the actin gene. A single, recessive lethal complementation group lies between the deficiency endpoints that flank the actin gene. An EMS mutagenesis screen produced four additional members of this recessive lethal complementation group. Molecular analysis of the members of this complementation group indicated that two of the newly induced mutations have deletions of approximately 1 kb in a transcribed region 4-5 kb 3' (distal) to the actin gene. This result suggests that the recessive lethal complementation group represents a gene separate from and distal to the actin gene. The mutagenesis screen failed to identify additional recessive lethal complementation groups in the actin gene-containing region. The implications of the failure to identify recessive lethal mutations in the actin gene are discussed in reference to studies of other conserved multigene families and other muscle protein mutations. PMID:2840338

The timing of UV mutagenesis in yeast: a pedigree analysis of induced recessive mutation.

PubMed

James, A P; Kilbey, B J

1977-10-01

The mechanism of UV-induced mutation in eukaryotes was studied in individual yeast cells by a procedure that combined pedigree analysis and tetrad analysis. The technique involved the induction of recessive lethals and semilethals in G1 diploid cells. Induced frequencies were 25 and 61 percent at survival levels of 90 and 77 percent, respectively. No evidence of gross chromosome aberrations was detected. Recessive mutations that affect only one strand or that affect both strands of the DNA molecule are induced much at random among a population of cells, and both types can occur within the same cell. However, the data confirm that two-strand mutations are in the majority after a low level of irradiation. The simplest explanation involves a mechanism whereby most mutations are fixed in both strands prior to the first round of post-irradiation DNA replication. The recessive mutational consequences of irradiation are exhausted at the conclusion of the first post-irradiation cell division, although dominant-lethal sectoring continues at a high level through the second post-irradiation division. It is concluded that pyrimidine dimers that persist to the second round of DNA replication are rare or ineffective.

Biological responses of Habrobracon to spaceflight.

PubMed

von Borstel, R C; Smith, R H; Whiting, A R; Grosch, D S

1970-01-01

Since the interaction of the parasitic wasp Habrobracon with the space environment could not be prejudged, we decided to test approximately 30 different parameters of a genetic, mutational, biochemical, behavioral, and physiological character in the one spaceflight we had at our disposal. These parameters were examined at six different exposures of gamma-radiation (including 0 dose) in flight, resulting in about 180 different endpoints in all. The most profound effects of spaceflight in conjunction with radiation were decreased hatchability and enhanced fecundity of eggs exposed to spaceflight at different stages of oogenesis. The interpretation we favor is that these two endpoints are reflections of chromosomal non-disjunction in the former case and inhibition of cell division in the latter. Our most comprehensive study of mutagenesis was on sperm, where dominant lethality, recessive lethality, translocations, and visible mutations were assayed; the only effect found was a threefold enhancement of the recessive lethal mutation frequency in the non-irradiated sperm in the orbited Habrobracon males. Behavioral and biochemical differences were found. Mating activity of orbited males was severely disrupted and xanthine dehydrogenase activity was sharply decreased in the irradiated flight animals, an unexpected observation. Postflight experiments were like the ground-based control experiments in all aspects but one. Under conditions of vibration similar to those encountered during the launch and re-entry, the mutation frequency in the sperm increased by a factor of three over that of the non-vibrated control.

RNase H2 catalytic core Aicardi-Goutières syndrome–related mutant invokes cGAS–STING innate immune-sensing pathway in mice

PubMed Central

Pokatayev, Vladislav; Hasin, Naushaba; Chon, Hyongi; Cerritelli, Susana M.; Sakhuja, Kiran; Ward, Jerrold M.; Morris, H. Douglas; Yan, Nan

2016-01-01

The neuroinflammatory autoimmune disease Aicardi-Goutières syndrome (AGS) develops from mutations in genes encoding several nucleotide-processing proteins, including RNase H2. Defective RNase H2 may induce accumulation of self-nucleic acid species that trigger chronic type I interferon and inflammatory responses, leading to AGS pathology. We created a knock-in mouse model with an RNase H2 AGS mutation in a highly conserved residue of the catalytic subunit, Rnaseh2aG37S/G37S (G37S), to understand disease pathology. G37S homozygotes are perinatal lethal, in contrast to the early embryonic lethality previously reported for Rnaseh2b- or Rnaseh2c-null mice. Importantly, we found that the G37S mutation led to increased expression of interferon-stimulated genes dependent on the cGAS–STING signaling pathway. Ablation of STING in the G37S mice results in partial rescue of the perinatal lethality, with viable mice exhibiting white spotting on their ventral surface. We believe that the G37S knock-in mouse provides an excellent animal model for studying RNASEH2-associated autoimmune diseases. PMID:26880576

Leapfrogging: primordial germ cell transplantation permits recovery of CRISPR/Cas9-induced mutations in essential genes

PubMed Central

Fish, Margaret B.; Cho, Ken W. Y.

2016-01-01

CRISPR/Cas9 genome editing is revolutionizing genetic loss-of-function analysis but technical limitations remain that slow progress when creating mutant lines. First, in conventional genetic breeding schemes, mosaic founder animals carrying mutant alleles are outcrossed to produce F1 heterozygotes. Phenotypic analysis occurs in the F2 generation following F1 intercrosses. Thus, mutant analyses will require multi-generational studies. Second, when targeting essential genes, efficient mutagenesis of founders is often lethal, preventing the acquisition of mature animals. Reducing mutagenesis levels may improve founder survival, but results in lower, more variable rates of germline transmission. Therefore, an efficient approach to study lethal mutations would be useful. To overcome these shortfalls, we introduce ‘leapfrogging’, a method combining efficient CRISPR mutagenesis with transplantation of mutated primordial germ cells into a wild-type host. Tested using Xenopus tropicalis, we show that founders containing transplants transmit mutant alleles with high efficiency. F1 offspring from intercrosses between F0 animals that carry embryonic lethal alleles recapitulate loss-of-function phenotypes, circumventing an entire generation of breeding. We anticipate that leapfrogging will be transferable to other species. PMID:27385011

Evasion of adaptive immunity by HIV through the action of host APOBEC3G/F enzymes.

PubMed

Grant, Michael; Larijani, Mani

2017-09-12

APOBEC3G (A3G) and APOBEC3F (A3F) are DNA-mutating enzymes expressed in T cells, dendritic cells and macrophages. A3G/F have been considered innate immune host factors, based on reports that they lethally mutate the HIV genome in vitro. In vivo, A3G/F effectiveness is limited by viral proteins, entrapment in inactive complexes and filtration of mutations during viral life cycle. We hypothesized that the impact of sub-lethal A3G/F action could extend beyond the realm of innate immunity confined to the cytoplasm of infected cells. We measured recognition of wild type and A3G/F-mutated epitopes by cytotoxic T lymphocytes (CTL) from HIV-infected individuals and found that A3G/F-induced mutations overwhelmingly diminished CTL recognition of HIV peptides, in a human histocompatibility-linked leukocyte antigen (HLA)-dependent manner. Furthermore, we found corresponding enrichment of A3G/F-favored motifs in CTL epitope-encoding sequences within the HIV genome. These findings illustrate that A3G/F-mediated mutations mediate immune evasion by HIV in vivo. Therefore, we suggest that vaccine strategies target T cell or antibody epitopes that are not poised for mutation into escape variants by A3G/F action.

Survival and dominant transmission of "lethal" platyspondylic dwarfism of the "West coast" types.

PubMed

Omran, H; Uhl, M; Brandis, M; Wolff, G

2000-03-01

Torrance, San Diego, and Luton types ("West coast" types) of neonatal platyspondylic short-limbed dwarfism are suspected to be caused by dominant mutations that are obligatorily lethal. We report on an affected mother, who passed the disease to her daughter, confirming dominant disease transmission. Survival of the mother indicates a wider phenotypic spectrum.

Topology of evolving, mutagenized viral populations: quasispecies expansion, compression, and operation of negative selection.

PubMed

Ojosnegros, Samuel; Agudo, Rubén; Sierra, Macarena; Briones, Carlos; Sierra, Saleta; González-López, Claudia; Domingo, Esteban; Cristina, Juan

2008-07-17

The molecular events and evolutionary forces underlying lethal mutagenesis of virus (or virus extinction through an excess of mutations) are not well understood. Here we apply for the first time phylogenetic methods and Partition Analysis of Quasispecies (PAQ) to monitor genetic distances and intra-population structures of mutant spectra of foot-and-mouth disease virus (FMDV) quasispecies subjected to mutagenesis by base and nucleoside analogues. Phylogenetic and PAQ analyses have revealed a highly dynamic variation of intrapopulation diversity of FMDV quasispecies. The population diversity first suffers striking expansions in the presence of mutagens and then compressions either when the presence of the mutagenic analogue was discontinued or when a mutation that decreased sensitivity to a mutagen was selected. The pattern of mutations found in the populations was in agreement with the behavior of the corresponding nucleotide analogues with FMDV in vitro. Mutations accumulated at preferred genomic sites, and dn/ds ratios indicate the operation of negative (or purifying) selection in populations subjected to mutagenesis. No evidence of unusually elevated genetic distances has been obtained for FMDV populations approaching extinction. Phylogenetic and PAQ analysis provide adequate procedures to describe the evolution of viral sequences subjected to lethal mutagenesis. These methods define the changes of intra-population structure more precisely than mutation frequencies and Shannon entropies. PAQ is very sensitive to variations of intrapopulation genetic distances. Strong negative (or purifying) selection operates in FMDV populations subjected to enhanced mutagenesis. The quantifications provide evidence that extinction does not imply unusual increases of intrapopulation complexity, in support of the lethal defection model of virus extinction.

Narrowing the wingless-2 mutation to a 227 Kb candidate region on chicken chromosome 12

USDA-ARS?s Scientific Manuscript database

Wingless-2 (wg-2) is an autosomal recessive mutation in chicken that results in an embryonic lethal condition. Affected individuals exhibit a multisystem syndrome characterized by absent wings, truncated legs, and craniofacial, kidney, and feather malformations. Previously, work focused on phenotype...

THE EFFECTS OF CHLORAMPHENICOL, STREPTOMYCIN, AND PENICILLIN ON THE INDUCTION OF MUTATIONS BY X-RAYS IN DROSOPHILA MELANOGASTER

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

Clark, A.M.

The injection of chloramphenicol, streptomycin, or penicillin into Drosophila males just before exposure to x irradiation caused a reduction in the yield of sex linked recessive lethal mutations. The effect appears to be primarily on spermatids and possibly spermatocytes. (auth)

FIRST RESULTS ON X-RAY-INDUCED GENETIC DAMAGE IN ARTEMIA SALINA LEACH

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

Metalli, P.; Ballardin, E.

1962-01-01

Prophase oocytes of diploid and tetraploid pantenogenetic Antemia salina were x irradiated with 1000 r and damage was scored as oocyte or embryo lethal mutations at the first (X/sub 1/) and at the second (X/sub 2/) generations after irradiation. Dominant lethality shown at X/sub 1/ was much greater for the diploid strain than for the tetraploid; lethality observed at X/sub 2/ was increased with respect to X/sub 1/ in the diploid strain, while in the tetraploid it remained unmodified. (auth)

Mutational analysis of varicella-zoster virus (VZV) immediate early protein (IE62) subdomains and their importance in viral replication

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

Khalil, Mohamed I., E-mail: mkhalil2@stanford.edu; Department of Molecular Biology, National Research Centre, El-Buhouth St., Cairo; Che, Xibing

VZV IE62 is an essential, immediate-early, tegument protein and consists of five domains. We generated recombinant viruses carrying mutations in the first three IE62 domains and tested their influence on VZV replication kinetics. The mutations in domain I did not affect replication kinetics while domain II mutations, disrupting the DNA binding and dimerization domain (DBD), were lethal for VZV replication. Mutations in domain III of the nuclear localization signal (NLS) and the two phosphorylation sites S686A/S722A resulted in slower growth in early and late infection respectively and were associated with IE62 accumulation in the cytoplasm and nucleus respectively. This studymore » mapped the functional domains of IE62 in context of viral infection, indicating that DNA binding and dimerization domain is essential for VZV replication. In addition, the correct localization of IE62, whether nuclear or cytoplasmic, at different points in the viral life cycle, is important for normal progression of VZV replication. - Highlights: • Mutation of IE62 domain I did not affect VZV replication in melanoma cells. • IE62 domain II and III are important for VZV replication in melanoma cells. • Mutations of IE62 domain II (DBD) were lethal for virus replication. • Mutations of IE62 NLS and phosphorylation sites inhibited VZV replication. • NLS and S686A/S722A mutations altered localization of IE62 during early and late infection.« less

Drosophila Lin-52 Acts in Opposition to Repressive Components of the Myb-MuvB/dREAM Complex

PubMed Central

Lewis, Peter W.; Sahoo, Debashis; Geng, Cuiyun; Bell, Maren

2012-01-01

The Drosophila melanogaster Myb-MuvB/dREAM complex (MMB/dREAM) participates in both the activation and repression of developmentally regulated genes and origins of DNA replication. Mutants in MMB subunits exhibit diverse phenotypes, including lethality, eye defects, reduced fecundity, and sterility. Here, we used P-element excision to generate mutations in lin-52, which encodes the smallest subunit of the MMB/dREAM complex. lin-52 is required for viability, as null mutants die prior to pupariation. The generation of somatic and germ line mutant clones indicates that lin-52 is required for adult eye development and for early embryogenesis via maternal effects. Interestingly, the maternal-effect embryonic lethality, larval lethality, and adult eye defects could be suppressed by mutations in other subunits of the MMB/dREAM complex. These results suggest that a partial MMB/dREAM complex is responsible for the lethality and eye defects of lin-52 mutants. Furthermore, these findings support a model in which the Lin-52 and Myb proteins counteract the repressive activities of the other members of the MMB/dREAM complex at specific genomic loci in a developmentally controlled manner. PMID:22688510

Complementation Studies of Bacteriophage λ O Amber Mutants by Allelic Forms of O Expressed from Plasmid, and O-P Interaction Phenotypes.

PubMed

Hayes, Sidney; Rajamanickam, Karthic; Hayes, Connie

2018-04-05

λ genes O and P are required for replication initiation from the bacteriophage λ origin site, ori λ, located within gene O . Questions have persisted for years about whether O-defects can indeed be complemented in trans . We show the effect of original null mutations in O and the influence of four origin mutations (three are in-frame deletions and one is a point mutation) on complementation. This is the first demonstration that O proteins with internal deletions can complement for O activity, and that expression of the N-terminal portion of gene P can completely prevent O complementation. We show that O-P co-expression can limit the lethal effect of P on cell growth. We explore the influence of the contiguous small RNA OOP on O complementation and P-lethality.

Synthetic Lethality Reveals Mechanisms of Mycobacterium tuberculosis Resistance to β-Lactams

PubMed Central

Lun, Shichun; Miranda, David; Kubler, Andre; Guo, Haidan; Maiga, Mariama C.; Winglee, Kathryn; Pelly, Shaaretha

2014-01-01

ABSTRACT Most β-lactam antibiotics are ineffective against Mycobacterium tuberculosis due to the microbe’s innate resistance. The emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains has prompted interest to repurpose this class of drugs. To identify the genetic determinants of innate β-lactam resistance, we carried out a synthetic lethality screen on a transposon mutant library for susceptibility to imipenem, a carbapenem β-lactam antibiotic. Mutations in 74 unique genes demonstrated synthetic lethality. The majority of mutations were in genes associated with cell wall biosynthesis. A second quantitative real-time PCR (qPCR)-based synthetic lethality screen of randomly selected mutants confirmed the role of cell wall biosynthesis in β-lactam resistance. The global transcriptional response of the bacterium to β-lactams was investigated, and changes in levels of expression of cell wall biosynthetic genes were identified. Finally, we validated these screens in vivo using the MT1616 transposon mutant, which lacks a functional acyl-transferase gene. Mice infected with the mutant responded to β-lactam treatment with a 100-fold decrease in bacillary lung burden over 4 weeks, while the numbers of organisms in the lungs of mice infected with wild-type bacilli proliferated. These findings reveal a road map of genes required for β-lactam resistance and validate synthetic lethality screening as a promising tool for repurposing existing classes of licensed, safe, well-characterized antimicrobials against tuberculosis. PMID:25227469

A Functional Genomics Approach to Identify Novel Breast Cancer Gene Targets in Yeast

DTIC Science & Technology

2005-05-01

Chaleff DT, Valent B, Fink GR. Mutations affecting Ty-mediated expression of the HIS4 gene of Saccharomyces cerevisiae. Genetics 1984; 107(2): 179-97... mutations , and are synthetically lethal with rotl mutations ROX3 YBL093C Repressor Of hypoXic genes : RNA polymerase I1 holcenzyme component 3,3 SSS...mitochondrial gene products; mutation causes an elevated rate of mitochondrial turnover; 3 MOD after 60 generations, MOD on NaCI YNDI YER005W Yeast Nucleoside

Lethal mitochondrial cardiomyopathy in a hypomorphic Med30 mouse mutant is ameliorated by ketogenic diet

PubMed Central

Krebs, Philippe; Fan, Weiwei; Chen, Yen-Hui; Tobita, Kimimasa; Downes, Michael R.; Wood, Malcolm R.; Sun, Lei; Xia, Yu; Ding, Ning; Spaeth, Jason M.; Moresco, Eva Marie Y.; Boyer, Thomas G.; Lo, Cecilia Wen Ya; Yen, Jeffrey; Evans, Ronald M.; Beutler, Bruce

2011-01-01

Deficiencies of subunits of the transcriptional regulatory complex Mediator generally result in embryonic lethality, precluding study of its physiological function. Here we describe a missense mutation in Med30 causing progressive cardiomyopathy in homozygous mice that, although viable during lactation, show precipitous lethality 2–3 wk after weaning. Expression profiling reveals pleiotropic changes in transcription of cardiac genes required for oxidative phosphorylation and mitochondrial integrity. Weaning mice to a ketogenic diet extends viability to 8.5 wk. Thus, we establish a mechanistic connection between Mediator and induction of a metabolic program for oxidative phosphorylation and fatty acid oxidation, in which lethal cardiomyopathy is mitigated by dietary intervention. PMID:22106289

Truncating Mutations of MAGEL2, a Gene within the Prader-Willi Locus, Are Responsible for Severe Arthrogryposis

PubMed Central

Mejlachowicz, Dan; Nolent, Flora; Maluenda, Jérome; Ranjatoelina-Randrianaivo, Hanitra; Giuliano, Fabienne; Gut, Ivo; Sternberg, Damien; Laquerrière, Annie; Melki, Judith

2015-01-01

Arthrogryposis multiplex congenita (AMC) is characterized by the presence of multiple joint contractures resulting from reduced or absent fetal movement. Here, we report two unrelated families affected by lethal AMC. By genetic mapping and whole-exome sequencing in a multiplex family, a heterozygous truncating MAGEL2 mutation leading to frameshift and a premature stop codon (c.1996delC, p.Gln666Serfs∗36) and inherited from the father was identified in the probands. In another family, a distinct heterozygous truncating mutation leading to frameshift (c.2118delT, p.Leu708Trpfs∗7) and occurring de novo on the paternal allele of MAGEL2 was identified in the affected individual. In both families, RNA analysis identified the mutated paternal MAGEL2 transcripts only in affected individuals. MAGEL2 is one of the paternally expressed genes within the Prader-Willi syndrome (PWS) locus. PWS is associated with, to varying extents, reduced fetal mobility, severe infantile hypotonia, childhood-onset obesity, hypogonadism, and intellectual disability. MAGEL2 mutations have been recently reported in affected individuals with features resembling PWS and called Schaaf-Yang syndrome. Here, we show that paternal MAGEL2 mutations are also responsible for lethal AMC, recapitulating the clinical spectrum of PWS and suggesting that MAGEL2 is a PWS-determining gene. PMID:26365340

Unlocking the Bottleneck in Forward Genetics Using Whole-Genome Sequencing and Identity by Descent to Isolate Causative Mutations

PubMed Central

Siggs, Owen M.; Miosge, Lisa A.; Roots, Carla M.; Enders, Anselm; Bertram, Edward M.; Crockford, Tanya L.; Whittle, Belinda; Potter, Paul K.; Simon, Michelle M.; Mallon, Ann-Marie; Brown, Steve D. M.; Beutler, Bruce; Goodnow, Christopher C.; Lunter, Gerton; Cornall, Richard J.

2013-01-01

Forward genetics screens with N-ethyl-N-nitrosourea (ENU) provide a powerful way to illuminate gene function and generate mouse models of human disease; however, the identification of causative mutations remains a limiting step. Current strategies depend on conventional mapping, so the propagation of affected mice requires non-lethal screens; accurate tracking of phenotypes through pedigrees is complex and uncertain; out-crossing can introduce unexpected modifiers; and Sanger sequencing of candidate genes is inefficient. Here we show how these problems can be efficiently overcome using whole-genome sequencing (WGS) to detect the ENU mutations and then identify regions that are identical by descent (IBD) in multiple affected mice. In this strategy, we use a modification of the Lander-Green algorithm to isolate causative recessive and dominant mutations, even at low coverage, on a pure strain background. Analysis of the IBD regions also allows us to calculate the ENU mutation rate (1.54 mutations per Mb) and to model future strategies for genetic screens in mice. The introduction of this approach will accelerate the discovery of causal variants, permit broader and more informative lethal screens to be used, reduce animal costs, and herald a new era for ENU mutagenesis. PMID:23382690

Synthetically lethal nanoparticles for treatment of endometrial cancer

NASA Astrophysics Data System (ADS)

Ebeid, Kareem; Meng, Xiangbing; Thiel, Kristina W.; Do, Anh-Vu; Geary, Sean M.; Morris, Angie S.; Pham, Erica L.; Wongrakpanich, Amaraporn; Chhonker, Yashpal S.; Murry, Daryl J.; Leslie, Kimberly K.; Salem, Aliasger K.

2018-01-01

Uterine serous carcinoma, one of the most aggressive types of endometrial cancer, is characterized by poor outcomes and mutations in the tumour suppressor p53. Our objective was to engender synthetic lethality to paclitaxel (PTX), the frontline treatment for endometrial cancer, in tumours with mutant p53 and enhance the therapeutic efficacy using polymeric nanoparticles (NPs). First, we identified the optimal NP formulation through comprehensive analyses of release profiles and cellular-uptake and cell viability studies. Not only were PTX-loaded NPs superior to PTX in solution, but the combination of PTX-loaded NPs with the antiangiogenic molecular inhibitor BIBF 1120 (BIBF) promoted synthetic lethality specifically in cells with the loss-of-function (LOF) p53 mutation. In a xenograft model of endometrial cancer, this combinatorial therapy resulted in a marked inhibition of tumour progression and extended survival. Together, our data provide compelling evidence for future studies of BIBF- and PTX-loaded NPs as a therapeutic opportunity for LOF p53 cancers.

A familial case of Keratitis-Ichthyosis-Deafness (KID) syndrome with the GJB2 mutation G45E.

PubMed

Jonard, Laurence; Feldmann, Delphine; Parsy, Christophe; Freitag, Sylvie; Sinico, Martine; Koval, Céleste; Grati, Mhamed; Couderc, Remy; Denoyelle, Françoise; Bodemer, Christine; Marlin, Sandrine; Hadj-Rabia, Smail

2008-01-01

Keratitis-Ichthyosis-Deafness (KID) syndrome (OMIM 148210) is a congenital ectodermal defect. KID consists of an atypical ichthyosiform erythroderma associated with congenital sensorineural deafness. A rare form of the KID syndrome is a fatal course in the first year of life due to severe skin lesion infections and septicaemia. KID appears to be genetically heterogeneous and may be caused by mutations in connexin 26 or connexin 30 genes. GJB2 mutations in the connexin 26 gene are the main cause of the disease. Most of the cases caused by GJB2 mutations are sporadic, but dominant transmission has also been described. To date, the rare lethal form of the disease has been only observed in two Caucasian sporadic patients with the GJB2 mutation, with the p.Gly45Glu (G45E) arising de novo. We have reported an African family with dizygotic twins suffering from a lethal form of KID. The dizygosity of the twins was confirmed by microsatellite markers. The two patients were heterozygous for the G45E mutation of GJB2, whereas the mutation was not detected in the two parents. The unusual transmission of the disease observed in this family could be explained by the occurrence of a somatic or more probably a germinal mosaic in one of the parents.

Ehlers-Danlos syndrome with lethal cardiac valvular dystrophy in males carrying a novel splice mutation in FLNA.

PubMed

Ritelli, Marco; Morlino, Silvia; Giacopuzzi, Edoardo; Carini, Giulia; Cinquina, Valeria; Chiarelli, Nicola; Majore, Silvia; Colombi, Marina; Castori, Marco

2017-01-01

Filamin A is an X-linked, ubiquitous actin-binding protein whose mutations are associated to multiple disorders with limited genotype-phenotype correlations. While gain-of-function mutations cause various bone dysplasias, loss-of-function variants are the most common cause of periventricular nodular heterotopias with variable soft connective tissue involvement, as well as X-linked cardiac valvular dystrophy (XCVD). The term "Ehlers-Danlos syndrome (EDS) with periventricular heterotopias" has been used in females with neurological, cardiovascular, integument and joint manifestations, but this nosology is still a matter of debate. We report the clinical and molecular update of an Italian family with an X-linked recessive soft connective tissue disorder and which was described, in 1975, as the first example of EDS type V of the Berlin nosology. The cutaneous phenotype of the index patient was close to classical EDS and all males died for a lethal cardiac valvular dystrophy. Whole exome sequencing identified the novel c.1829-1G>C splice variation in FLNA in two affected cousins. The nucleotide change was predicted to abolish the canonical splice acceptor site of exon 13 and to activate a cryptic acceptor site 15 bp downstream, leading to in frame deletion of five amino acid residues (p.Phe611_Gly615del). The predicted in frame deletion clusters with all the mutations previously identified in XCVD and falls within the N-terminus rod 1 domain of filamin A. Our findings expand the male-specific phenotype of FLNA mutations that now includes classical-like EDS with lethal cardiac valvular dystrophy, and offer further insights for the genotype-phenotype correlations within this spectrum. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Mutation of the dengue virus type 2 envelope protein heparan sulfate binding sites or the domain III lateral ridge blocks replication in Vero cells prior to membrane fusion

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

Roehrig, John T., E-mail: jtr1@cdc.gov; Butrapet, Siritorn; Liss, Nathan M.

Using an infectious cDNA clone we engineered seven mutations in the putative heparan sulfate- and receptor-binding motifs of the envelope protein of dengue virus serotype 2, strain 16681. Four mutant viruses, KK122/123EE, E202K, G304K, and KKK305/307/310EEE, were recovered following transfection of C6/36 cells. A fifth mutant, KK291/295EE, was recovered from C6/36 cells with a compensatory E295V mutation. All mutants grew in and mediated fusion of virus-infected C6/36 cells, but three of the mutants, KK122/123EE, E202K, G304K, did not grow in Vero cells without further modification. Two Vero cell lethal mutants, KK291/295EV and KKK307/307/310EEE, failed to replicate in DC-SIGN-transformed Raji cellsmore » and did not react with monoclonal antibodies known to block DENV attachment to Vero cells. Additionally, both mutants were unable to initiate negative-strand vRNA synthesis in Vero cells by 72 h post-infection, suggesting that the replication block occurred prior to virus-mediated membrane fusion. - Highlights: • Heparan sulfate- and receptor-binding motifs of DENV2 envelope protein were mutated. • Four mutant viruses were isolated—all could fuse C6/36 cells. • Two of these mutants were lethal in Vero cells without further modification. • Lethal mutations were KK291/295EV and KKK305/307/310EEE. • Cell attachment was implicated as the replication block for both mutants.« less

Topology of evolving, mutagenized viral populations: quasispecies expansion, compression, and operation of negative selection

PubMed Central

2008-01-01

Background The molecular events and evolutionary forces underlying lethal mutagenesis of virus (or virus extinction through an excess of mutations) are not well understood. Here we apply for the first time phylogenetic methods and Partition Analysis of Quasispecies (PAQ) to monitor genetic distances and intra-population structures of mutant spectra of foot-and-mouth disease virus (FMDV) quasispecies subjected to mutagenesis by base and nucleoside analogues. Results Phylogenetic and PAQ analyses have revealed a highly dynamic variation of intrapopulation diversity of FMDV quasispecies. The population diversity first suffers striking expansions in the presence of mutagens and then compressions either when the presence of the mutagenic analogue was discontinued or when a mutation that decreased sensitivity to a mutagen was selected. The pattern of mutations found in the populations was in agreement with the behavior of the corresponding nucleotide analogues with FMDV in vitro. Mutations accumulated at preferred genomic sites, and dn/ds ratios indicate the operation of negative (or purifying) selection in populations subjected to mutagenesis. No evidence of unusually elevated genetic distances has been obtained for FMDV populations approaching extinction. Conclusion Phylogenetic and PAQ analysis provide adequate procedures to describe the evolution of viral sequences subjected to lethal mutagenesis. These methods define the changes of intra-population structure more precisely than mutation frequencies and Shannon entropies. PAQ is very sensitive to variations of intrapopulation genetic distances. Strong negative (or purifying) selection operates in FMDV populations subjected to enhanced mutagenesis. The quantifications provide evidence that extinction does not imply unusual increases of intrapopulation complexity, in support of the lethal defection model of virus extinction. PMID:18637173

A missense mutation in PFAS (phosphoribosylformylglycinamidine synthase) is likely causal for embryonic lethality associated with the MH1 haplotype in Montbéliarde dairy cattle.

PubMed

Michot, Pauline; Fritz, Sébastien; Barbat, Anne; Boussaha, Mekki; Deloche, Marie-Christine; Grohs, Cécile; Hoze, Chris; Le Berre, Laurène; Le Bourhis, Daniel; Desnoes, Olivier; Salvetti, Pascal; Schibler, Laurent; Boichard, Didier; Capitan, Aurélien

2017-10-01

A candidate mutation in the sex hormone binding globulin gene was proposed in 2013 to be responsible for the MH1 recessive embryonic lethal locus segregating in the Montbéliarde breed. In this follow-up study, we excluded this candidate variant because healthy homozygous carriers were observed in large-scale genotyping data generated in the framework of the genomic selection program. We fine mapped the MH1 locus in a 702-kb interval and analyzed genome sequence data from the 1,000 bull genomes project and 54 Montbéliarde bulls (including 14 carriers and 40 noncarriers). We report the identification of a strong candidate mutation in the gene encoding phosphoribosylformylglycinamidine synthase (PFAS), a protein involved in de novo purine synthesis. This mutation, located in a class I glutamine amidotransferase-like domain, results in the substitution of an arginine residue that is entirely conserved among eukaryotes by a cysteine (p.R1205C). No homozygote for the cysteine-encoding allele was observed in a large population of more than 25,000 individuals despite a 6.7% allelic frequency and 122 expected homozygotes under neutrality assumption. Genotyping of 18 embryos collected from heterozygous parents as well as analysis on nonreturn rates suggested that most homozygous carriers died between 7 and 35 d postinsemination. The identification of this strong candidate mutation will enable the accurate testing of the reproducers and the efficient selection against this lethal recessive embryonic defect in the Montbéliarde breed. Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Phenotypic characterization of spontaneously mutated rats showing lethal dwarfism and epilepsy.

PubMed

Suzuki, Hiroetsu; Takenaka, Motoo; Suzuki, Katsushi

2007-08-01

We have characterized the phenotype of spontaneously mutated rats, found during experimental inbreeding in a closed colony of Wistar Imamichi rats. Mutant rats showed severe dwarfism, short lifespan (early postnatal lethality), and high incidence of epileptic seizures. Mutant rats showed growth retardation after 3 d of age, and at 21 d their weight was about 56% that of normal rats. Most mutant rats died without reaching maturity, and 95% of the mutant rats had an ataxic gait. About 34% of the dwarf rats experienced epileptic seizures, most of which started as 'wild running' convulsions, progressing to generalized tonic-clonic convulsions. At age 28 d, the relative weight of the testes was significantly lower, and the relative weight of the brain was significantly higher, in mutant than in normal rats. Histologically, increased apoptotic germ cells, lack of spermatocytes, and immature Leydig cells were found in the mutant testes, and extracellular vacuoles of various sizes were present in the hippocampus and amygdala of the mutant brain. Mutant rats had significantly increased concentrations of plasma urea nitrogen, creatinine, and inorganic phosphate, as well as decreased concentrations of plasma growth hormone. Hereditary analysis showed that the defects were inherited as a single recessive trait. We have named the hypothetically mutated gene as lde (lethal dwarfism with epilepsy).

Katz model prediction of Caenorhabditis elegans mutagenesis on STS-42

NASA Technical Reports Server (NTRS)

Cucinotta, Francis A.; Wilson, John W.; Katz, Robert; Badhwar, Gautam D.

1992-01-01

Response parameters that describe the production of recessive lethal mutations in C. elegans from ionizing radiation are obtained with the Katz track structure model. The authors used models of the space radiation environment and radiation transport to predict and discuss mutation rates for C. elegans on the IML-1 experiment aboard STS-42.

An active site mutation increases the polymerase activity of the guinea pig-lethal Marburg virus.

PubMed

Koehler, Alexander; Kolesnikova, Larissa; Becker, Stephan

2016-10-01

Marburg virus (MARV) causes severe, often fatal, disease in humans and transient illness in rodents. Sequential passaging of MARV in guinea pigs resulted in selection of a lethal virus containing 4 aa changes. A D184N mutation in VP40 (VP40D184N), which leads to a species-specific gain of viral fitness, and three mutations in the active site of viral RNA-dependent RNA polymerase L, which were investigated in the present study for functional significance in human and guinea pig cells. The transcription/replication activity of L mutants was strongly enhanced by a substitution at position 741 (S741C), and inhibited by other substitutions (D758A and A759D) in both species. The polymerase activity of L carrying the S741C substitution was eightfold higher in guinea pig cells than in human cells upon co-expression with VP40D184N, suggesting that the additive effect of the two mutations provides MARV a replicative advantage in the new host.

Metabolic synthetic lethality in cancer therapy.

PubMed

Zecchini, Vincent; Frezza, Christian

2017-08-01

Our understanding of cancer has recently seen a major paradigm shift resulting in it being viewed as a metabolic disorder, and altered cellular metabolism being recognised as a hallmark of cancer. This concept was spurred by the findings that the oncogenic mutations driving tumorigenesis induce a reprogramming of cancer cell metabolism that is required for unrestrained growth and proliferation. The recent discovery that mutations in key mitochondrial enzymes play a causal role in tumorigenesis suggested that dysregulation of metabolism could also be a driver of tumorigenesis. These mutations induce profound adaptive metabolic alterations that are a prerequisite for the survival of the mutated cells. Because these metabolic events are specific to cancer cells, they offer an opportunity to develop new therapies that specifically target tumour cells without affecting healthy tissue. Here, we will describe recent developments in metabolism-based cancer therapy, in particular focusing on the concept of metabolic synthetic lethality. This article is part of a Special Issue entitled Mitochondria in Cancer, edited by Giuseppe Gasparre, Rodrigue Rossignol and Pierre Sonveaux. Copyright © 2016 Elsevier B.V. All rights reserved.

Lethal genes surviving by mosaicism: a possible explanation for sporadic birth defects involving the skin.

PubMed

Happle, R

1987-04-01

A genetic concept is advanced to explain the origin of several sporadic syndromes characterized by a mosaic distribution of skin defects. It is postulated that these disorders are due to the action of a lethal gene surviving by mosaicism. The presence of the mutation in the zygote will lead to death of the embryo at an early stage of development. Cells bearing the mutation can survive only in a mosaic state, in close proximity with normal cells. The mosaic may arise either from a gametic half chromatid mutation or from an early somatic mutation. This concept of origin is proposed to apply to the Schimmelpenning-Feuerstein-Mims syndrome, the McCune-Albright syndrome, the Klippel-Trenaunay syndrome, the Sturge-Weber syndrome, and neurocutaneous melanosis. Moreover, this etiologic hypothesis may apply to two other birth defects that have recently been delineated, the Proteus syndrome (partial gigantism of hands or feet, hemihypertrophy, macrocephaly, linear papillomatous epidermal nevus, subcutaneous hemangiomas and lipomas, accelerated growth, and visceral anomalies), and the Delleman-Oorthuys syndrome (orbital cyst, porencephaly, periorbital appendages, and focal aplasia of the skin.

THE ACTION OF RADIATION AND OTHER MUTAGENIC AGENTS (1) IN INDUCING MUTATION IN DROSOPHILA FEMALES, AND (2) IN CONTROLLING THE ACTION OF SPECIFIC GENES RESPONSIBLE FOR SUPPRESSING UNCONTROLLED GROWTH. Report Covering 9-Year Period, May 1, 1953-April 30, 1962

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

Glass, H.B.

1962-02-01

Studies of the comparative mutagenic effects of ionizing radiations on males and females of Drosophila melanogaster are described. Sex-linked recessive lethal mutations were induced in nitrogen, air, and oxygen at doses of obtained in spermatozoa were uniformly about one-third higher than the frequencies obtained for the same dose and condition of atmosphere in mature oocytes. The relative frequencies of recessive autosomal lethals in mature male and female germ cells were identical with the relative fre quencies of sex-linked recessive lethals. In studies of point mutations and deficiencies involving specific loci, the rates in the male germ cells exceeded those inmore » the female germ cells by a proportion equal to that found to apply to autosomal and sex-linked recessive lethals. Spontaneous mutation rates were determined for a number of specific loci marked by recessive genes used in the tested stocks. Fertility was lost in both males and females when they were x-rayed as 80-hr-old larvae and bred upon emerging as adults. Females recovered their fertility rapidly but the males did so at a much slower rate. The brown; scarlet'' stock was found to carry two mutants each suppressed by a particular suppressor gene. It was concluded that the two suppressors act along different metabolic pathways departing from tryplophan, but both involving an x-ray-sensitive step. A study was made of the effects on the life span of two different mating regimens: immediate and deferred. It was found that the lines previously subjected to immediate mating significantly outlived the lines previously subjected to deferred mating when the mating regimen in the test was immediate mating. Exactly the opposite happened when the mating regimen in the test was deferred mating. (M.C.G.)« less

Advances in Radiation Mutagenesis through Studies on Drosophila

DOE R&D Accomplishments Database

Muller, H. J.

1958-06-01

The approximately linear relation between radiation dose and induced lethals known for Drosophila spermatozoa, is now extended to spermatids. Data are included regarding oogonia. The linearity principle has been confined for minute structural changes in sperm as multi-hit events, on about the 1.5 power of the dose, long known for spermatozoa, is now extended to spermatids and late oocytes, for relatively short exposures. are found to allow union of broken chromosomes. Therefore, the frequencies are lower for more dispersed exposures of varies with lethals induced in late oocytes follow the same frequency pattern and there fore are multi-hit events. Yet han spermatozoan irradiation that two broken ends derived from nonreciprocal. The following is the order of decreasing radiation mutability of different stages found by ourselves and others: spermatids, spermatozoa in females, spermatozoa 0 to 1 day before ejaculation, earlier spermatozoa, late oocytes, gonia of either sex. Lethal frequencies for these stages range over approximately an order of magnitude, gross structural changes far more widely. Of potential usefulness is our extension of genesis by anoxia, known for spermatozoa in adult males, to those in pupal males and in females, to sperion is especially marked but the increase caused by substituting oxygen for air is less marked, perhaps because of enzymatic differences. In contrast, the induction of gross structural changes in oocytes, but not in spermatids, is markedly reduced by oxygen post-treatment; it is increased by dehydration. The efficacy of induction of structural changes by treatment of spermatozoa, whether with radiation or chemical mutagen, is correlated with the conditions of sperm utilization and egg production. Improving our perspective on radiation effects, some 800,000 offspring have been scored for spontaneous visible mutations of 13 specific loci. The average point-mutation rate was 0.5 to 1.0 per locus among 10/sup 5/ germ cells. Most mutation occurred in peri- fertilization stages. All loci studied mutated from one to nine times. Loci mutating oftener spontaneously also gave more radiation mutation, in other studies, Spectra of individual loci prove similar for spontaneous and induced mutation. Studies on back-mutation also showed similarity of spontaneous and radiation mutations. The doubling dose for back-mutations of forked induced in spermatozoa was several hundred roentgens, gonia at diverse loci. Recent analyses of human mutational load lead to mutation-rate estimated like those earlier based on extrapolations from Drosophila, thus supporting the significance for man of the present studies. (auth)

Emergence of DNA Polymerase ε Antimutators That Escape Error-Induced Extinction in Yeast

PubMed Central

Williams, Lindsey N.; Herr, Alan J.; Preston, Bradley D.

2013-01-01

DNA polymerases (Pols) ε and δ perform the bulk of yeast leading- and lagging-strand DNA synthesis. Both Pols possess intrinsic proofreading exonucleases that edit errors during polymerization. Rare errors that elude proofreading are extended into duplex DNA and excised by the mismatch repair (MMR) system. Strains that lack Pol proofreading or MMR exhibit a 10- to 100-fold increase in spontaneous mutation rate (mutator phenotype), and inactivation of both Pol δ proofreading (pol3-01) and MMR is lethal due to replication error-induced extinction (EEX). It is unclear whether a similar synthetic lethal relationship exists between defects in Pol ε proofreading (pol2-4) and MMR. Using a plasmid-shuffling strategy in haploid Saccharomyces cerevisiae, we observed synthetic lethality of pol2-4 with alleles that completely abrogate MMR (msh2Δ, mlh1Δ, msh3Δ msh6Δ, or pms1Δ mlh3Δ) but not with partial MMR loss (msh3Δ, msh6Δ, pms1Δ, or mlh3Δ), indicating that high levels of unrepaired Pol ε errors drive extinction. However, variants that escape this error-induced extinction (eex mutants) frequently emerged. Five percent of pol2-4 msh2Δ eex mutants encoded second-site changes in Pol ε that reduced the pol2-4 mutator phenotype between 3- and 23-fold. The remaining eex alleles were extragenic to pol2-4. The locations of antimutator amino-acid changes in Pol ε and their effects on mutation spectra suggest multiple mechanisms of mutator suppression. Our data indicate that unrepaired leading- and lagging-strand polymerase errors drive extinction within a few cell divisions and suggest that there are polymerase-specific pathways of mutator suppression. The prevalence of suppressors extragenic to the Pol ε gene suggests that factors in addition to proofreading and MMR influence leading-strand DNA replication fidelity. PMID:23307893

Atelosteogenesis type 2.

PubMed Central

Newbury-Ecob, R

1998-01-01

Atelosteogenesis type 2 (AO2) (MIM 256050) is a neonatally lethal chondrodysplasia characterised by severe limb shortening and deficient ossification of parts of the skeleton. Other features include facial dysmorphism, cleft palate, talipes, and abducted thumbs and toes. Phenotypic overlap with non-lethal diastrophic dysplasia (DTD) suggested a common aetiology and it has recently been confirmed that both syndromes result from mutations in the DTDST (diastrophic dysplasia sulphate transporter) gene. Images PMID:9475095

[Identification of new genes that affect [PSI^(+)] prion toxicity in Saccharomyces cerevisiae yeast].

PubMed

Matveenko, A G; Belousov, M V; Bondarev, S A; Moskalenko, S E; Zhouravleva, G A

2016-01-01

Translation termination is an important step in gene expression. Its correct processing is governed by eRF1 (Sup45) and eRF3 (Sup35) proteins. In Saccharomyces cerevisiae, mutations in the corresponding genes, as well as Sup35 aggregation in [PSI^(+)] cells that propagate the prion form of Sup35 lead to inaccurate stop codon recognition and, consequently, nonsense suppression. The presence of stronger prion variants results in the more efficient suppression of nonsense mutations. Previously, we proposed a synthetic lethality test that enables the identification of genes that may influence either translation termination factors or [PSI^(+)] manifestation. This is based on the fact that the combination of sup45 mutations with the strong [PSI^(+)] prion variant in diploids is lethal. In this work, a set of genes that were previously shown to enhance nonsense suppression was analyzed. It was found that ABF1, FKH2, and REB1 overexpression decreased the growth of strains in a prion-dependent manner and, thus, might influence [PSI^(+)] prion toxicity. It was also shown that the synthetic lethality of [PSI^(+)] and sup45 mutations increased with the overexpression of GLN3 and MOT3 that encode Q/N-rich transcription factors. An analysis of the effects of their expression on the transcription of the release factors genes revealed an increase in SUP35 transcription in both cases. Since SUP35 overexpression is known to be toxic in [PSI^(+)] strains, these genes apparently enhance [PSI^(+)] toxicity via the regulation of SUP35 transcription.

The Role of BRCA1/BARD1 Heterodimers in the Mitosis-Interphase Transition

DTIC Science & Technology

2007-05-01

53 4 INTRODUCTION Germ line mutations in the BRCA1 gene predispose to breast and/or ovarian cancer (Miki, et al...Targeted mutations of breast cancer susceptibility gene homologs in mice: lethal phenotypes of Brca1, Brca2, Brca1/Brca2, Brca1/p53, and Brca2/p53...Ludwig, T., Chapman, D.L., Papaioannou, V.E., and Efstratiadis, A. (1997). Targeted mutations of breast cancer susceptibility gene homo- logs in mice

Inhibition of BRCA2 and Thymidylate Synthase Creates Multidrug Sensitive Tumor Cells via the Induction of Combined "Complementary Lethality".

PubMed

Rytelewski, Mateusz; Ferguson, Peter J; Maleki Vareki, Saman; Figueredo, Rene; Vincent, Mark; Koropatnick, James

2013-03-12

A high mutation rate leading to tumor cell heterogeneity is a driver of malignancy in human cancers. Paradoxically, however, genomic instability can also render tumors vulnerable to therapeutic attack. Thus, targeting DNA repair may induce an intolerable level of DNA damage in tumor cells. BRCA2 mediates homologous recombination repair, and BRCA2 polymorphisms increase cancer risk. However, tumors with BRCA2 mutations respond better to chemotherapy and are associated with improved patient prognosis. Thymidylate synthase (TS) is also involved in DNA maintenance and generates cellular thymidylate. We determined that antisense downregulation of BRCA2 synergistically potentiated drugs with mechanisms of action related to BRCA2 function (cisplatin, melphalan), a phenomenon we named "complementary lethality." TS knockdown induced complementary lethality to TS-targeting drugs (5-FUdR and pemetrexed) but not DNA cross-linking agents. Combined targeting of BRCA2 and TS induced complementary lethality to both DNA-damaging and TS-targeting agents, thus creating multidrug sensitive tumors. In addition, we demonstrated for the first time that simultaneous downregulation of both targets induced combined complementary lethality to multiple mechanistically different drugs in the same cell population. In this study, we propose and define the concept of "complementary lethality" and show that actively targeting BRCA2 and TS is of potential therapeutic benefit in multidrug treatment of human tumors. This work has contributed to the development of a BRCA2-targeting antisense oligdeoxynucleotide (ASO) "BR-1" which we will test in vivo in combination with our TS-targeting ASO "SARI 83" and attempt early clinical trials in the future.Molecular Therapy - Nucleic Acids (2013) 2, e78; doi:10.1038/mtna.2013.7 published online 12 March 2013.

piRNA-mediated transposon regulation and the germ-line mutation rate in Drosophila melanogaster males.

PubMed

Simmons, Michael J; Peterson, Mark P; Thorp, Michael W; Buschette, Jared T; DiPrima, Stephanie N; Harter, Christine L; Skolnick, Matthew J

2015-03-01

Transposons, especially retrotransposons, are abundant in the genome of Drosophila melanogaster. These mobile elements are regulated by small RNAs that interact with the Piwi family of proteins-the piwi-interacting or piRNAs. The Piwi proteins are encoded by the genes argonaute3 (ago3), aubergine (aub), and piwi. Heterochromatin Protein 1 (HP1), a chromatin-organizing protein encoded by the Suppressor of variegation 205 [Su(var)205] gene, also plays a role in this regulation. To assess the mutational impact of weakening the system for transposon regulation, we measured the frequency of recessive X-linked lethal mutations occurring in the germ lines of males from stocks that were heterozygous for mutant alleles of the ago3, aub, piwi, or Su(var)205 genes. These mutant alleles are expected to deplete the wild-type proteins encoded by these genes by as much as 50%. The mutant alleles of piwi and Su(var)205 significantly increased the X-linked lethal mutation frequency, whereas the mutant alleles of ago3 did not. An increased mutation frequency was also observed in males from one of two mutant aub stocks, but this increase may not have been due to the aub mutant. The increased mutation frequency caused by depleting Piwi or HP1suggests that chromatin-organizing proteins play important roles in minimizing the germ-line mutation rate, possibly by stabilizing the structure of the heterochromatin in which many transposons are situated. Copyright © 2015 Elsevier B.V. All rights reserved.

Candidate synthetic lethality partners to PARP inhibitors in the treatment of ovarian clear cell cancer

PubMed Central

Kawahara, Naoki; Ogawa, Kenji; Nagayasu, Mika; Kimura, Mai; Sasaki, Yoshikazu; Kobayashi, Hiroshi

2017-01-01

Inhibitors of poly(ADP-ribose) polymerase (PARP) are new types of personalized treatment of relapsed platinum-sensitive ovarian cancer harboring BRCA1/2 mutations. Ovarian clear cell cancer (CCC), a subset of ovarian cancer, often appears as low-stage disease with a higher incidence among Japanese. Advanced CCC is highly aggressive with poor patient outcome. The aim of the present study was to determine the potential synthetic lethality gene pairs for PARP inhibitions in patients with CCC through virtual and biological screenings as well as clinical studies. We conducted a literature review for putative PARP sensitivity genes that are associated with the CCC pathophysiology. Previous studies identified a variety of putative target genes from several pathways associated with DNA damage repair, chromatin remodeling complex, PI3K-AKT-mTOR signaling, Notch signaling, cell cycle checkpoint signaling, BRCA-associated complex and Fanconi's anemia susceptibility genes that could be used as biomarkers or therapeutic targets for PARP inhibition. BRCA1/2, ATM, ATR, BARD1, CCNE1, CHEK1, CKS1B, DNMT1, ERBB2, FGFR2, MRE11A, MYC, NOTCH1 and PTEN were considered as candidate genes for synthetic lethality gene partners for PARP interactions. When considering the biological background underlying PARP inhibition, we hypothesized that PARP inhibitors would be a novel synthetic lethal therapeutic approach for CCC tumors harboring homologous recombination deficiency and activating oncogene mutations. The results showed that the majority of CCC tumors appear to have indicators of DNA repair dysfunction similar to those in BRCA-mutation carriers, suggesting the possible utility of PARP inhibitors in a subset of CCC. PMID:29109859

Tracking the origins and drivers of subclonal metastatic expansion in prostate cancer

DOE PAGES

Hong, Matthew K. H.; Macintyre, Geoff; Wedge, David C.; ...

2015-04-01

Tumour heterogeneity in primary prostate cancer is a well-established phenomenon. However, how the subclonal diversity of tumours changes during metastasis and progression to lethality is poorly understood. Here we reveal the precise direction of metastatic spread across four lethal prostate cancer patients using whole-genome and ultra-deep targeted sequencing of longitudinally collected primary and metastatic tumours. We find one case of metastatic spread to the surgical bed causing local recurrence, and another case of cross-metastatic site seeding combining with dynamic remoulding of subclonal mixtures in response to therapy. By ultra-deep sequencing end-stage blood, we detect both metastatic and primary tumour clones,more » even years after removal of the prostate. As a result, analysis of mutations associated with metastasis reveals an enrichment of TP53 mutations, and additional sequencing of metastases from 19 patients demonstrates that acquisition of TP53 mutations is linked with the expansion of subclones with metastatic potential which we can detect in the blood.« less

Tracking the origins and drivers of subclonal metastatic expansion in prostate cancer.

PubMed

Hong, Matthew K H; Macintyre, Geoff; Wedge, David C; Van Loo, Peter; Patel, Keval; Lunke, Sebastian; Alexandrov, Ludmil B; Sloggett, Clare; Cmero, Marek; Marass, Francesco; Tsui, Dana; Mangiola, Stefano; Lonie, Andrew; Naeem, Haroon; Sapre, Nikhil; Phal, Pramit M; Kurganovs, Natalie; Chin, Xiaowen; Kerger, Michael; Warren, Anne Y; Neal, David; Gnanapragasam, Vincent; Rosenfeld, Nitzan; Pedersen, John S; Ryan, Andrew; Haviv, Izhak; Costello, Anthony J; Corcoran, Niall M; Hovens, Christopher M

2015-04-01

Tumour heterogeneity in primary prostate cancer is a well-established phenomenon. However, how the subclonal diversity of tumours changes during metastasis and progression to lethality is poorly understood. Here we reveal the precise direction of metastatic spread across four lethal prostate cancer patients using whole-genome and ultra-deep targeted sequencing of longitudinally collected primary and metastatic tumours. We find one case of metastatic spread to the surgical bed causing local recurrence, and another case of cross-metastatic site seeding combining with dynamic remoulding of subclonal mixtures in response to therapy. By ultra-deep sequencing end-stage blood, we detect both metastatic and primary tumour clones, even years after removal of the prostate. Analysis of mutations associated with metastasis reveals an enrichment of TP53 mutations, and additional sequencing of metastases from 19 patients demonstrates that acquisition of TP53 mutations is linked with the expansion of subclones with metastatic potential which we can detect in the blood.

Cationic lipid-assisted polymeric nanoparticle mediated GATA2 siRNA delivery for synthetic lethal therapy of KRAS mutant non-small-cell lung carcinoma.

PubMed

Shen, Song; Mao, Chong-Qiong; Yang, Xian-Zhu; Du, Xiao-Jiao; Liu, Yang; Zhu, Yan-Hua; Wang, Jun

2014-08-04

Synthetic lethal interaction provides a conceptual framework for the development of wiser cancer therapeutics. In this study, we exploited a therapeutic strategy based on the interaction between GATA binding protein 2 (GATA2) downregulation and the KRAS mutation status by delivering small interfering RNA targeting GATA2 (siGATA2) with cationic lipid-assisted polymeric nanoparticles for treatment of non-small-cell lung carcinoma (NSCLC) harboring oncogenic KRAS mutations. Nanoparticles carrying siGATA2 (NPsiGATA2) were effectively taken up by NSCLC cells and resulted in targeted gene suppression. NPsiGATA2 selectively inhibited cell proliferation and induced cell apoptosis in KRAS mutant NSCLC cells. However, this intervention was harmless to normal KRAS wild-type NSCLC cells and HL7702 hepatocytes, confirming the advantage of synthetic lethality-based therapy. Moreover, systemic delivery of NPsiGATA2 significantly inhibited tumor growth in the KRAS mutant A549 NSCLC xenograft murine model, suggesting the therapeutic promise of NPsiGATA2 delivery in KRAS mutant NSCLC therapy.

Nutritional supplement chromium picolinate causes sterility and lethal mutations in Drosophila melanogaster

PubMed Central

Hepburn, Dion D. D.; Xiao, Jiarong; Bindom, Sharell; Vincent, John B.; O'Donnell, Janis

2003-01-01

The nutritional dietary supplement chromium picolinate, [Cr(pic)3], has gained much notoriety as a safe supplement that supposedly promotes fat loss and muscle enhancement in humans. Thus, a significant industry has materialized around the incorporation of [Cr(pic)3] in many sports foods and drinks and a variety of weight loss products. However, in vitro studies have suggested that low levels of [Cr(pic)3] in the presence of biological reducing agents can catalytically generate reactive oxygen species, and recent in vivo studies have detected oxidative damage in rats receiving the supplement. The potential deleterious in vivo effects of this activity were examined by using Drosophila melanogaster. [Cr(pic)3], but not CrCl3, at levels of 260 μg Cr/kg food or less were found to lower the success rate of pupation and eclosion and to arrest development of pupae in a concentration dependent fashion. X-linked lethal analysis indicates that the supplement greatly enhances the rate of appearance of lethal mutations and dominant female sterility. PMID:12649323

Familial Ehlers-Danlos syndrome with lethal arterial events caused by a mutation in COL5A1.

PubMed

Monroe, Glen R; Harakalova, Magdalena; van der Crabben, Saskia N; Majoor-Krakauer, Danielle; Bertoli-Avella, Aida M; Moll, Frans L; Oranen, Björn I; Dooijes, Dennis; Vink, Aryan; Knoers, Nine V; Maugeri, Alessandra; Pals, Gerard; Nijman, Isaac J; van Haaften, Gijs; Baas, Annette F

2015-06-01

Different forms of Ehlers-Danlos syndrome (EDS) exist, with specific phenotypes and associated genes. Vascular EDS, caused by heterozygous mutations in the COL3A1 gene, is characterized by fragile vasculature with a high risk of catastrophic vascular events at a young age. Classic EDS, caused by heterozygous mutations in the COL5A1 or COL5A2 genes, is characterized by fragile, hyperextensible skin and joint laxity. To date, vessel rupture in four unrelated classic EDS patients with a confirmed COL5A1 mutation has been reported. We describe familial occurrence of a phenotype resembling vascular EDS in a mother and her two sons, who all died at an early age from arterial ruptures. Diagnostic Sanger sequencing in the proband failed to detect aberrations in COL3A1, COL1A1, COL1A2, TGFBR1, TGFBR2, SMAD3, and ACTA2. Next, the proband's DNA was analyzed using a next-generation sequencing approach targeting 554 genes linked to vascular disease (VASCULOME project). A novel heterozygous mutation in COL5A1 was detected, resulting in an essential glycine substitution at the C-terminal end of the triple helix domain (NM_000093.4:c.4610G>T; p.Gly1537Val). This mutation was also present in DNA isolated from autopsy material of the index's brother. No material was available from the mother, but the mutation was excluded in her parents, siblings and in the father of her sons, suggesting that the COL5A1 mutation occurred in the mother's genome de novo. In conclusion, we report familial occurrence of lethal arterial events caused by a COL5A1 mutation. © 2015 Wiley Periodicals, Inc.

Mutation of Breast Cancer Cell Genomic DNA by APOBEC3B

DTIC Science & Technology

2013-09-01

lethal prostate cancers. Proc. Natl Acad. Sci. USA 108, 17087–17092 (2011). 5. Parsons, D. W. et al. The genetic landscape of the childhood cancer...7. Stransky, N. et al. The mutational landscape of head and neck squamous cell carcinoma. Science 333, 1157–1160 (2011). 8. Nik-Zainal, S. et al...Mutational processes molding the genomes of 21 breast cancers. Cell 149, 979–993 (2012). 9. Stephens, P. J. et al. The landscape of cancer genes and

The Drosophila mitochondrial translation elongation factor G1 contains a nuclear localization signal and inhibits growth and DPP signaling.

PubMed

Trivigno, Catherine; Haerry, Theodor E

2011-02-25

Mutations in the human mitochondrial elongation factor G1 (EF-G1) are recessive lethal and cause death shortly after birth. We have isolated mutations in iconoclast (ico), which encodes the highly conserved Drosophila orthologue of EF-G1. We find that EF-G1 is essential during fly development, but its function is not required in every tissue. In contrast to null mutations, missense mutations exhibit stronger, possibly neomorphic phenotypes that lead to premature death during embryogenesis. Our experiments show that EF-G1 contains a secondary C-terminal nuclear localization signal. Expression of missense mutant forms of EF-G1 can accumulate in the nucleus and cause growth and patterning defects and animal lethality. We find that transgenes that encode mutant human EF-G1 proteins can rescue ico mutants, indicating that the underlying problem of the human disease is not just the loss of enzymatic activity. Our results are consistent with a model where EF-G1 acts as a retrograde signal from mitochondria to the nucleus to slow down cell proliferation if mitochondrial energy output is low.

The Drosophila Mitochondrial Translation Elongation Factor G1 Contains a Nuclear Localization Signal and Inhibits Growth and DPP Signaling

PubMed Central

Trivigno, Catherine; Haerry, Theodor E.

2011-01-01

Mutations in the human mitochondrial elongation factor G1 (EF-G1) are recessive lethal and cause death shortly after birth. We have isolated mutations in iconoclast (ico), which encodes the highly conserved Drosophila orthologue of EF-G1. We find that EF-G1 is essential during fly development, but its function is not required in every tissue. In contrast to null mutations, missense mutations exhibit stronger, possibly neomorphic phenotypes that lead to premature death during embryogenesis. Our experiments show that EF-G1 contains a secondary C-terminal nuclear localization signal. Expression of missense mutant forms of EF-G1 can accumulate in the nucleus and cause growth and patterning defects and animal lethality. We find that transgenes that encode mutant human EF-G1 proteins can rescue ico mutants, indicating that the underlying problem of the human disease is not just the loss of enzymatic activity. Our results are consistent with a model where EF-G1 acts as a retrograde signal from mitochondria to the nucleus to slow down cell proliferation if mitochondrial energy output is low. PMID:21364917

FGFR3-related condition: a skeletal dysplasia with similarities to thanatophoric dysplasia and SADDAN due to Lys650Met.

PubMed

Farmakis, Shannon G; Shinawi, Marwan; Miller-Thomas, Michelle; Radmanesh, Alireza; Herman, Thomas E

2015-03-01

Mutations in the fibroblast growth factor receptor 3 (FGFR3) gene account for six related skeletal dysplasia conditions: achondroplasia, hypochondroplasia, thanatophoric dysplasia types 1 and 2, SADDAN (severe achondroplasia with developmental delay and acanthosis nigricans), and platyspondylic lethal skeletal dysplasia, San Diego type. This group of disorders has very characteristic clinical and radiologic features, which distinguish them from other skeletal dysplasias. They display a spectrum of severity in the skeletal findings, ranging from relatively mild hypochondroplasia to lethal thanatophoric dysplasia. We report a patient who has the missense FGFR3 mutation, Lys650Met, previously reported in association only with SADDAN, who exhibits some findings similar to both thanatophoric dysplasia (types 1 and 2) in addition to those findings characteristic of SADDAN.

Novel Lethal Form of Congenital Hypopituitarism Associated With the First Recessive LHX4 Mutation

PubMed Central

Gregory, L. C.; Humayun, K. N.; Turton, J. P. G.; McCabe, M. J.; Rhodes, S. J.

2015-01-01

Background: LHX4 encodes a member of the LIM-homeodomain family of transcription factors that is required for normal development of the pituitary gland. To date, only incompletely penetrant heterozygous mutations in LHX4 have been described in patients with variable combined pituitary hormone deficiencies. Objective/Hypothesis: To report a unique family with a novel recessive variant in LHX4 associated with a lethal form of congenital hypopituitarism that was identified through screening a total of 97 patients. Method: We screened 97 unrelated patients with combined pituitary hormone deficiency, including 65% with an ectopic posterior pituitary, for variants in the LHX4 gene using Sanger sequencing. Control databases (1000 Genomes, dbSNP, Exome Variant Server, ExAC Browser) were consulted upon identification of variants. Results: We identified the first novel homozygous missense variant (c.377C>T, p.T126M) in two deceased male patients of Pakistani origin with severe panhypopituitarism associated with anterior pituitary aplasia and posterior pituitary ectopia. Both were born small for gestational age with a small phallus, undescended testes, and mid-facial hypoplasia. The parents' first-born child was a female with mid-facial hypoplasia (DNA was unavailable). Despite rapid commencement of hydrocortisone and T4 in the brothers, all three children died within the first week of life. The LHX4(p.T126M) variant is located within the LIM2 domain, in a highly conserved location. The absence of homozygosity for the variant in over 65 000 controls suggests that it is likely to be responsible for the phenotype. Conclusion: We report, for the first time to our knowledge, a novel homozygous mutation in LHX4 associated with a lethal phenotype, implying that recessive mutations in LHX4 may be incompatible with life. PMID:25871839

Vaccination With a Highly Attenuated Recombinant Vesicular Stomatitis Virus Vector Protects Against Challenge With a Lethal Dose of Ebola Virus

PubMed Central

Matassov, Demetrius; Marzi, Andrea; Latham, Terri; Xu, Rong; Ota-Setlik, Ayuko; Feldmann, Friederike; Geisbert, Joan B.; Mire, Chad E.; Hamm, Stefan; Nowak, Becky; Egan, Michael A.; Geisbert, Thomas W.; Eldridge, John H.; Feldmann, Heinz; Clarke, David K.

2015-01-01

Previously, recombinant vesicular stomatitis virus (rVSV) pseudotypes expressing Ebolavirus glycoproteins (GPs) in place of the VSV G protein demonstrated protection of nonhuman primates from lethal homologous Ebolavirus challenge. Those pseudotype vectors contained no additional attenuating mutations in the rVSV genome. Here we describe rVSV vectors containing a full complement of VSV genes and expressing the Ebola virus (EBOV) GP from an additional transcription unit. These rVSV vectors contain the same combination of attenuating mutations used previously in the clinical development pathway of an rVSV/human immunodeficiency virus type 1 vaccine. One of these rVSV vectors (N4CT1-EBOVGP1), which expresses membrane-anchored EBOV GP from the first position in the genome (GP1), elicited a balanced cellular and humoral GP-specific immune response in mice. Guinea pigs immunized with a single dose of this vector were protected from any signs of disease following lethal EBOV challenge, while control animals died in 7–9 days. Subsequently, N4CT1-EBOVGP1 demonstrated complete, single-dose protection of 2 macaques following lethal EBOV challenge. A single sham-vaccinated macaque died from disease due to EBOV infection. These results demonstrate that highly attenuated rVSV vectors expressing EBOV GP may provide safer alternatives to current EBOV vaccines. PMID:26109675

The population genetics of X-autosome synthetic lethals and steriles.

PubMed

Lachance, Joseph; Johnson, Norman A; True, John R

2011-11-01

Epistatic interactions are widespread, and many of these interactions involve combinations of alleles at different loci that are deleterious when present in the same individual. The average genetic environment of sex-linked genes differs from that of autosomal genes, suggesting that the population genetics of interacting X-linked and autosomal alleles may be complex. Using both analytical theory and computer simulations, we analyzed the evolutionary trajectories and mutation-selection balance conditions for X-autosome synthetic lethals and steriles. Allele frequencies follow a set of fundamental trajectories, and incompatible alleles are able to segregate at much higher frequencies than single-locus expectations. Equilibria exist, and they can involve fixation of either autosomal or X-linked alleles. The exact equilibrium depends on whether synthetic alleles are dominant or recessive and whether fitness effects are seen in males, females, or both sexes. When single-locus fitness effects and synthetic incompatibilities are both present, population dynamics depend on the dominance of alleles and historical contingency (i.e., whether X-linked or autosomal mutations occur first). Recessive synthetic lethality can result in high-frequency X-linked alleles, and dominant synthetic lethality can result in high-frequency autosomal alleles. Many X-autosome incompatibilities in natural populations may be cryptic, appearing to be single-locus effects because one locus is fixed. We also discuss the implications of these findings with respect to standing genetic variation and the origins of Haldane's rule.

Mutation dynamics and fitness effects followed in single cells.

PubMed

Robert, Lydia; Ollion, Jean; Robert, Jerome; Song, Xiaohu; Matic, Ivan; Elez, Marina

2018-03-16

Mutations have been investigated for more than a century but remain difficult to observe directly in single cells, which limits the characterization of their dynamics and fitness effects. By combining microfluidics, time-lapse imaging, and a fluorescent tag of the mismatch repair system in Escherichia coli , we visualized the emergence of mutations in single cells, revealing Poissonian dynamics. Concomitantly, we tracked the growth and life span of single cells, accumulating ~20,000 mutations genome-wide over hundreds of generations. This analysis revealed that 1% of mutations were lethal; nonlethal mutations displayed a heavy-tailed distribution of fitness effects and were dominated by quasi-neutral mutations with an average cost of 0.3%. Our approach has enabled the investigation of single-cell individuality in mutation rate, mutation fitness costs, and mutation interactions. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Computational analysis of histidine mutations on the structural stability of human tyrosinases leading to albinism insurgence.

PubMed

Hassan, Mubashir; Abbas, Qamar; Raza, Hussain; Moustafa, Ahmed A; Seo, Sung-Yum

2017-07-25

Misfolding and structural alteration in proteins lead to serious malfunctions and cause various diseases in humans. Mutations at the active binding site in tyrosinase impair structural stability and cause lethal albinism by abolishing copper binding. To evaluate the histidine mutational effect, all mutated structures were built using homology modelling. The protein sequence was retrieved from the UniProt database, and 3D models of original and mutated human tyrosinase sequences were predicted by changing the residual positions within the target sequence separately. Structural and mutational analyses were performed to interpret the significance of mutated residues (N 180 , R 202 , Q 202 , R 211 , Y 363 , R 367 , Y 367 and D 390 ) at the active binding site of tyrosinases. CSpritz analysis depicted that 23.25% residues actively participate in the instability of tyrosinase. The accuracy of predicted models was confirmed through online servers ProSA-web, ERRAT score and VERIFY 3D values. The theoretical pI and GRAVY generated results also showed the accuracy of the predicted models. The CCA negative correlation results depicted that the replacement of mutated residues at His within the active binding site disturbs the structural stability of tyrosinases. The predicted CCA scores of Tyr 367 (-0.079) and Q/R 202 (0.032) revealed that both mutations have more potential to disturb the structural stability. MD simulation analyses of all predicted models justified that Gln 202 , Arg 202 , Tyr 367 and D 390 replacement made the protein structures more susceptible to destabilization. Mutational results showed that the replacement of His with Q/R 202 and Y/R 363 has a lethal effect and may cause melanin associated diseases such as OCA1. Taken together, our computational analysis depicts that the mutated residues such as Q/R 202 and Y/R 363 actively participate in instability and misfolding of tyrosinases, which may govern OCA1 through disturbing the melanin biosynthetic pathway.

[Chlorophyll mutations induced by gamma radiation in Phaseolus vulgaris L].

PubMed

Meoño, M E

1975-07-01

In a study of chlorophyll mutants of Phaseolus vulgaris L. through Co60 gamma radiation, five types of mutants, classified as albino, cream, yellow, yellow-green and light green were obtained; all were lethal; their segregation was always proportionally lower than the Mendelian. Gamma radiation-induced mutations in black beans do not depart significantly from those obtained elsewhere in barley and wheat.

Modeling synthetic lethality

PubMed Central

Le Meur, Nolwenn; Gentleman, Robert

2008-01-01

Background Synthetic lethality defines a genetic interaction where the combination of mutations in two or more genes leads to cell death. The implications of synthetic lethal screens have been discussed in the context of drug development as synthetic lethal pairs could be used to selectively kill cancer cells, but leave normal cells relatively unharmed. A challenge is to assess genome-wide experimental data and integrate the results to better understand the underlying biological processes. We propose statistical and computational tools that can be used to find relationships between synthetic lethality and cellular organizational units. Results In Saccharomyces cerevisiae, we identified multi-protein complexes and pairs of multi-protein complexes that share an unusually high number of synthetic genetic interactions. As previously predicted, we found that synthetic lethality can arise from subunits of an essential multi-protein complex or between pairs of multi-protein complexes. Finally, using multi-protein complexes allowed us to take into account the pleiotropic nature of the gene products. Conclusions Modeling synthetic lethality using current estimates of the yeast interactome is an efficient approach to disentangle some of the complex molecular interactions that drive a cell. Our model in conjunction with applied statistical methods and computational methods provides new tools to better characterize synthetic genetic interactions. PMID:18789146

Genetics Home Reference: oculofaciocardiodental syndrome

MedlinePlus

... the signs and symptoms of OFCD syndrome. In males (who have only one X chromosome ), mutations result ... be lethal very early in development, so no males are born with OFCD syndrome. Related Information What ...

Clinical and molecular characterization of Diastrophic Dysplasia in the Portuguese population.

PubMed

Barbosa, M; Sousa, A B; Medeira, A; Lourenço, T; Saraiva, J; Pinto-Basto, J; Soares, G; Fortuna, A M; Superti-Furga, A; Mittaz, L; Reis-Lima, M; Bonafé, L

2011-12-01

SLC26A2-related dysplasias encompass a spectrum of diseases: from lethal achondrogenesis type 1B (ACG1B; MIM #600972) and atelosteogenesis type 2 (AO2; MIM #256050) to classical diastrophic dysplasia (cDTD; MIM #222600) and recessive multiple epiphyseal dysplasia (rMED; MIM #226900). This study aimed at characterizing clinically, radiologically and molecularly 14 patients affected by non-lethal SLC26A2-related dysplasias and at evaluating genotype-phenotype correlation. Phenotypically, eight patients were classified as cDTD, four patients as rMED and two patients had an intermediate phenotype (mild DTD - mDTD, previously 'DTD variant'). The Arg279Trp mutation was present in all patients, either in homozygosity (resulting in rMED) or in compound heterozygosity with the known severe alleles Arg178Ter or Asn425Asp (resulting in DTD) or with the mutation c.727-1G>C (causing mDTD). The 'Finnish mutation', c.-26+2T>C, and the p.Cys653Ser, both frequent mutations in non-Portuguese populations, were not identified in any of the patients of our cohort and are probably very rare in the Portuguese population. A targeted mutation analysis for p.Arg279Trp and p.Arg178Ter in the Portuguese population allows the identification of approximately 90% of the pathogenic alleles. © 2010 John Wiley & Sons A/S.

Radiosensitivity of jute

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

Joshua, D.C.; Thakare, R.G.; Rao, N.S.

1972-11-01

The differential effects of fast and thermal neutrons and gamma rays on diploid ond autotetraploid of C. olitorius cv JRO 632 were studied. The frequency and spectrum of lethal chlorophyll mutations were studied in the diploid variety. (auth)

Advances in radiation mutagenesis through studies on Drosophila

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

Muller, H. J.

The approximately linear relation between radiation dose and induced lethals, known for Drosophila spermatozoa, is now extended to spermatids. Data are included regarding oogonia. The linearity principle has been confirmed for minute structural changes in spermatozoa. The dependence of gross structural changes, as multi-hit events, on about the 1.5 power of the dose, long known for spermatozoa, is now extended to spermatids and late oocytes, for relatively short exposures. However, these stages unlike spermatozoa are found to allow union of broken chromosomes. Therefore, the frequencies are lower for more dispersed exposures of these stages, and the precise dose relation variesmore » with the timing. Part of the dominant and even recessive lethals induced in late oocytes follow the same frequency pattern and therefore are multi-hit events. Yet there is a much lower chance after oocytic than spermatozoan irradiation that two broken ends derived from different hits will unite, hence most such unions are nonreciprocal. The following is the order of decreasing radiation mutability of different stages found by ourselves and others: spermatids, spermatozoa in females, spermatozoa 0 to 1 day before ejaculation, earlier spermatozoa, late oocytes, gonia of either sex. Lethal frequencies for these stages range over approximately an order of magnitude, gross structural changes far more widely. Of potential usefulness is our extension of the principle of marked reduction of radiation mutagenesis by anoxia, known for spermatozoa in adult males, to those in pupal males and in females to spermatids and to oocytes. In spermatids this reduction is especially marked but the increase caused by substituting oxygen for air is less marked, perhaps because of enzymatic differences. In contrast, the induction of gross structural changes in oocytes, but not in spermatids, is markedly reduced by oxygen post-treatment; it is increased by dehydration. The efficacy of induction of structural changes by treatment of spermatozoa, whether with radiation or chemical mutagens, is correlated with the conditions of sperm utilization and egg production. Improving our perspective on radiation effects, some 800,000 offspring have been scored for spontaneous visible mutations of 13 specific loci. The average point-mutation rate was 0.5 to 1.0 per locus among 10/sup 5/ germ cells. Most mutations occurred in peri-fertilization stages. All loci studied mutated from one to nine times. Loci mutating oftener spontaneously also gave more radiation mutation, in other studies. Spectra of individual loci prove similar for spontaneous and induced mutation. Studies on back-mutations also showed similarity of spontaneous and radiation mutations. The doubling dose for back-mutations of forked induced in spermatozoa was several hundred roentgens, similar to that for direct point-mutations induced in gonia at diverse loci. Recent analyses of human mutational load lead to mutation-rate estimates like those earlier based on extrapolations from Drosophila, thus supporting the significance for man of the present studies. (auth)« less

A case of thanatophoric dysplasia type 2: a novel mutation.

PubMed

Gülaşı, Selvi; Atıcı, Aytuğ; Çelik, Yalçın

2015-03-01

Thanatophoric dysplasia (TD) is a lethal form of skeletal dysplasia with short-limb dwarfism. Two types distinguished with their radiological characteristics have been defined clinically. The femur is curved in type 1, while it is straight in type 2. TD is known to be due to a mutation in the fibroblast growth factor receptor 3 (FGFR3) gene. We report a male patient who showed clinical findings congruent with TD type 2 and a new mutation in the FGFR3 gene, a finding which has not been reported previously.

Utilization of a quantitative mammalian cell mutation system, CHO/HGPRT, in experimental mutagenesis and genetic toxicology

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

Hsie, A. W.; Couch, D. B.; O'Neill, J. P.

1977-01-01

Development of the CHO/HGPRT system is described and a host-mediated CHO/HGPRT assay is discussed. The following topics are discussed: evidence for the genetic origin of mutation induction in the CHO/HGPRT system; dose-response relationship for EMS-mediated mutation induction and cell lethality; apparent dosimetry of EMS-induced mutagenesis; structure-activity relationship of alkylating agents and ICR compounds; mutagenicity and cytotoxicity of congeners of two classes of nitrosi compounds; and preliminary validation of the CHO/HGPRT assay in predicting chemical carcinogenicity. (HLW)

The effect of radiation on the long term productivity of a plant based CELSS

NASA Technical Reports Server (NTRS)

Thompson, B. G.; Lake, B. H.

1987-01-01

Mutations occur at a higher rate in space than under terrestrial conditions, primarily due to an increase in radiation levels. These mutations may effect the productivity of plants found in a controlled ecological life support system (CELSS). Computer simulations of plants with different ploidies, modes of reproduction, lethality thresholds, viability thresholds and susceptibilities to radiation induced mutations were performed under space normal and solar flare conditions. These simulations identified plant characteristics that would enable plants to retain high productivities over time in a CELSS.

CHILD syndrome in a boy

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

Happle, R.; Effendy, I., Megahed, M.; Orlow, S.J.

CHILD syndrome (congential hemidysplasia with ichthyosiform nevus and limb defects) occurs, as a rule, exclusively in girls because of the underlying X-linked gene exerts a lethal effect on male embryos. In this report the characteristic manifestations of CHILD syndrome are described in a 2-year-old boy with a normal chromosome constitution 46,XY. This exceptional case is best explained by the assumption of an early somatic mutation and thus compatible with the concept of X-linked dominant male-lethal inheritance of this trait. 18 refs., 6 figs.

A case of boomerang dysplasia with a novel causative mutation in filamin B: identification of typical imaging findings on ultrasonography and 3D-CT imaging.

PubMed

Tsutsumi, Seiji; Maekawa, Ayako; Obata, Miyuki; Morgan, Timothy; Robertson, Stephen P; Kurachi, Hirohisa

2012-01-01

Boomerang dysplasia is a rare lethal osteochondrodysplasia characterized by disorganized mineralization of the skeleton, leading to complete nonossification of some limb bones and vertebral elements, and a boomerang-like aspect to some of the long tubular bones. Like many short-limbed skeletal dysplasias with accompanying thoracic hypoplasia, the potential lethality of the phenotype can be difficult to ascertain prenatally. We report a case of boomerang dysplasia prenatally diagnosed by use of ultrasonography and 3D-CT imaging, and identified a novel mutation in the gene encoding the cytoskeletal protein filamin B (FLNB) postmortem. Findings that aided the radiological diagnosis of this condition in utero included absent ossification of two out of three long bones in each limb and elements of the vertebrae and a boomerang-like shape to the ulnae. The identified mutation is the third described for this disorder and is predicted to lead to amino acid substitution in the actin-binding domain of the filamin B molecule. Copyright © 2012 S. Karger AG, Basel.

A functional cancer genomics screen identifies a druggable synthetic lethal interaction between MSH3 and PRKDC.

PubMed

Dietlein, Felix; Thelen, Lisa; Jokic, Mladen; Jachimowicz, Ron D; Ivan, Laura; Knittel, Gero; Leeser, Uschi; van Oers, Johanna; Edelmann, Winfried; Heukamp, Lukas C; Reinhardt, H Christian

2014-05-01

Here, we use a large-scale cell line-based approach to identify cancer cell-specific mutations that are associated with DNA-dependent protein kinase catalytic subunit (DNA-PKcs) dependence. For this purpose, we profiled the mutational landscape across 1,319 cancer-associated genes of 67 distinct cell lines and identified numerous genes involved in homologous recombination-mediated DNA repair, including BRCA1, BRCA2, ATM, PAXIP, and RAD50, as being associated with non-oncogene addiction to DNA-PKcs. Mutations in the mismatch repair gene MSH3, which have been reported to occur recurrently in numerous human cancer entities, emerged as the most significant predictors of DNA-PKcs addiction. Concordantly, DNA-PKcs inhibition robustly induced apoptosis in MSH3-mutant cell lines in vitro and displayed remarkable single-agent efficacy against MSH3-mutant tumors in vivo. Thus, we here identify a therapeutically actionable synthetic lethal interaction between MSH3 and the non-homologous end joining kinase DNA-PKcs. Our observations recommend DNA-PKcs inhibition as a therapeutic concept for the treatment of human cancers displaying homologous recombination defects.

cea-kil operon of the ColE1 plasmid.

PubMed Central

Sabik, J F; Suit, J L; Luria, S E

1983-01-01

We isolated a series of Tn5 transposon insertion mutants and chemically induced mutants with mutations in the region of the ColE1 plasmid that includes the cea (colicin) and imm (immunity) genes. Bacterial cells harboring each of the mutant plasmids were tested for their response to the colicin-inducing agent mitomycin C. All insertion mutations within the cea gene failed to bring about cell killing after mitomycin C treatment. A cea- amber mutation exerted a polar effect on killing by mitomycin C. Two insertions beyond the cea gene but within or near the imm gene also prevented the lethal response to mitomycin C. These findings suggest the presence in the ColE1 plasmid of an operon containing the cea and kil genes whose product is needed for mitomycin C-induced lethality. Bacteria carrying ColE1 plasmids with Tn5 inserted within the cea gene produced serologically cross-reacting fragments of the colicin E1 molecule, the lengths of which were proportional to the distance between the insertion and the promoter end of the cea gene. Images PMID:6298187

Long survival in patients with leigh syndrome and the m.10191T>C mutation in MT-ND3 : a case report and review of the literature.

PubMed

Levy, Rebecca J; Ríos, Purificación Gutierrez; Akman, Hasan O; Sciacco, Monica; Vivo, Darryl C De; DiMauro, Salvatore

2014-10-01

We report an unusual case of Leigh syndrome due to the m.10191T>C mutation in the complex I gene MT-ND3. This mutation has been associated with a spectrum of clinical phenotypes ranging from infant lethality to adult onset. Despite infantile onset and severe symptoms, our patient has survived to early adulthood because of a strict dietary regimen and parental care. This patient is an extreme example of the frequently prolonged course of Leigh syndrome due to this particular mutation. © The Author(s) 2013.

Two O-linked N-acetylglucosamine transferase genes of Arabidopsis thaliana L. Heynh. have overlapping functions necessary for gamete and seed development.

PubMed Central

Hartweck, Lynn M; Scott, Cheryl L; Olszewski, Neil E

2002-01-01

The Arabidopsis SECRET AGENT (SEC) and SPINDLY (SPY) proteins are similar to animal O-linked N-acetylglucosamine transferases (OGTs). OGTs catalyze the transfer of N-acetylglucosamine (GlcNAc) from UDP-GlcNAc to Ser/Thr residues of proteins. In animals, O-GlcNAcylation has been shown to affect protein activity, stability, and/or localization. SEC protein expressed in Escherichia coli had autocatalytic OGT activity. To determine the function of SEC in plants, two tDNA insertional mutants were identified and analyzed. Although sec mutant plants did not exhibit obvious phenotypes, sec and spy mutations had a synthetic lethal interaction. This lethality was incompletely penetrant in gametes and completely penetrant postfertilization. The rate of both female and male sec spy gamete transmission was higher in plants heterozygous for both mutations than in plants heterozygous for sec and homozygous for spy. Double-mutant embryos aborted at various stages of development and no double-mutant seedlings were obtained. These results indicate that OGT activity is required during gametogenesis and embryogenesis with lethality occurring when parentally derived SEC, SPY, and/or O-GlcNAcylated proteins become limiting. PMID:12136030

Gene expression and mutation-guided synthetic lethality eradicates proliferating and quiescent leukemia cells

PubMed Central

Nieborowska-Skorska, Margaret; Sullivan, Katherine; Dasgupta, Yashodhara; Podszywalow-Bartnicka, Paulina; Maifrede, Silvia; Di Marcantonio, Daniela; Bolton-Gillespie, Elisabeth; Cramer-Morales, Kimberly; Lee, Jaewong; Li, Min; Slupianek, Artur; Gritsyuk, Daniel; Cerny-Reiterer, Sabine; Seferynska, Ilona; Bullinger, Lars; Gorbunova, Vera; Piwocka, Katarzyna; Valent, Peter; Civin, Curt I.; Muschen, Markus; Dick, John E.; Wang, Jean C.Y.; Bhatia, Smita; Bhatia, Ravi; Eppert, Kolja; Minden, Mark D.; Sykes, Stephen M.

2017-01-01

Quiescent and proliferating leukemia cells accumulate highly lethal DNA double-strand breaks that are repaired by 2 major mechanisms: BRCA-dependent homologous recombination and DNA-dependent protein kinase–mediated (DNA-PK–mediated) nonhomologous end-joining, whereas DNA repair pathways mediated by poly(ADP)ribose polymerase 1 (PARP1) serve as backups. Here we have designed a personalized medicine approach called gene expression and mutation analysis (GEMA) to identify BRCA- and DNA-PK–deficient leukemias either directly, using reverse transcription-quantitative PCR, microarrays, and flow cytometry, or indirectly, by the presence of oncogenes such as BCR-ABL1. DNA-PK–deficient quiescent leukemia cells and BRCA/DNA-PK–deficient proliferating leukemia cells were sensitive to PARP1 inhibitors that were administered alone or in combination with current antileukemic drugs. In conclusion, GEMA-guided targeting of PARP1 resulted in dual cellular synthetic lethality in quiescent and proliferating immature leukemia cells, and is thus a potential approach to eradicate leukemia stem and progenitor cells that are responsible for initiation and manifestation of the disease. Further, an analysis of The Cancer Genome Atlas database indicated that this personalized medicine approach could also be applied to treat numerous solid tumors from individual patients. PMID:28481221

Mutations in GLDN, Encoding Gliomedin, a Critical Component of the Nodes of Ranvier, Are Responsible for Lethal Arthrogryposis.

PubMed

Maluenda, Jérôme; Manso, Constance; Quevarec, Loic; Vivanti, Alexandre; Marguet, Florent; Gonzales, Marie; Guimiot, Fabien; Petit, Florence; Toutain, Annick; Whalen, Sandra; Grigorescu, Romulus; Coeslier, Anne Dieux; Gut, Marta; Gut, Ivo; Laquerrière, Annie; Devaux, Jérôme; Melki, Judith

2016-10-06

Arthrogryposis multiplex congenita (AMC) is a developmental condition characterized by multiple joint contractures resulting from reduced or absent fetal movements. Through linkage analysis, homozygosity mapping, and exome sequencing in four unrelated families affected by lethal AMC, we identified biallelic mutations in GLDN in the affected individuals. GLDN encodes gliomedin, a secreted cell adhesion molecule involved in the formation of the nodes of Ranvier. Transmission electron microscopy of the sciatic nerve from one of the affected individuals showed a marked lengthening defect of the nodes. The GLDN mutations found in the affected individuals abolish the cell surface localization of gliomedin and its interaction with its axonal partner, neurofascin-186 (NF186), in a cell-based assay. The axoglial contact between gliomedin and NF186 is essential for the initial clustering of Na + channels at developing nodes. These results indicate a major role of gliomedin in node formation and the development of the peripheral nervous system in humans. These data indicate that mutations of GLDN or CNTNAP1 (MIM: 616286), encoding essential components of the nodes of Ranvier and paranodes, respectively, lead to inherited nodopathies, a distinct disease entity among peripheral neuropathies. Copyright © 2016 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

A Strategy To Isolate Modifiers of Caenorhabditis elegans Lethal Mutations: Investigating the Endoderm Specifying Ability of the Intestinal Differentiation GATA Factor ELT-2.

PubMed

Wiesenfahrt, Tobias; Duanmu, Jingjie; Snider, Frances; Moerman, Don; Au, Vinci; Li-Leger, Erica; Flibotte, Stephane; Parker, Dylan M; Marshall, Craig J; Nishimura, Erin Osborne; Mains, Paul E; McGhee, James D

2018-05-04

The ELT-2 GATA factor normally functions in differentiation of the C. elegans endoderm, downstream of endoderm specification. We have previously shown that, if ELT-2 is expressed sufficiently early, it is also able to specify the endoderm and to replace all other members of the core GATA-factor transcriptional cascade (END-1, END-3, ELT-7). However, such rescue requires multiple copies (and presumably overexpression) of the end-1p :: elt-2 cDNA transgene; a single copy of the transgene does not rescue. We have made this observation the basis of a genetic screen to search for genetic modifiers that allow a single copy of the end-1p :: elt-2 cDNA transgene to rescue the lethality of the end-1 end-3 double mutant. We performed this screen on a strain that has a single copy insertion of the transgene in an end-1 end-3 background. These animals are kept alive by virtue of an extrachromosomal array containing multiple copies of the rescuing transgene; the extrachromosomal array also contains a toxin under heat shock control to counterselect for mutagenized survivors that have been able to lose the rescuing array. A screen of ∼14,000 mutagenized haploid genomes produced 17 independent surviving strains. Whole genome sequencing was performed to identify genes that incurred independent mutations in more than one surviving strain. The C. elegans gene tasp-1 was mutated in four independent strains. tasp-1 encodes the C. elegans homolog of Taspase, a threonine-aspartic acid protease that has been found, in both mammals and insects, to cleave several proteins involved in transcription, in particular MLL1/trithorax and TFIIA. A second gene, pqn-82 , was mutated in two independent strains and encodes a glutamine-asparagine rich protein. tasp-1 and pqn-82 were verified as loss-of-function modifiers of the end-1p :: elt-2 transgene by RNAi and by CRISPR/Cas9-induced mutations. In both cases, gene loss leads to modest increases in the level of ELT-2 protein in the early endoderm although ELT-2 levels do not strictly correlate with rescue. We suggest that tasp-1 and pqn-82 represent a class of genes acting in the early embryo to modulate levels of critical transcription factors or to modulate the responsiveness of critical target genes. The screen's design, rescuing lethality with an extrachromosomal transgene followed by counterselection, has a background survival rate of <10 -4 without mutagenesis and should be readily adapted to the general problem of identifying suppressors of C. elegans lethal mutations. Copyright © 2018 Wiesenfahrt et al.

Mutations blocking side chain assembly, polymerization, or transport of a Wzy-dependent Streptococcus pneumoniae capsule are lethal in the absence of suppressor mutations and can affect polymer transfer to the cell wall.

PubMed

Xayarath, Bobbi; Yother, Janet

2007-05-01

Extracellular polysaccharides of many bacteria are synthesized by the Wzy polymerase-dependent mechanism, where long-chain polymers are assembled from undecaprenyl-phosphate-linked repeat units on the outer face of the cytoplasmic membrane. In gram-positive bacteria, Wzy-dependent capsules remain largely cell associated via membrane and peptidoglycan linkages. Like many Wzy-dependent capsules, the Streptococcus pneumoniae serotype 2 capsule is branched. In this study, we found that deletions of cps2K, cps2J, or cps2H, which encode a UDP-glucose dehydrogenase necessary for side chain synthesis, the putative Wzx transporter (flippase), and the putative Wzy polymerase, respectively, were obtained only in the presence of suppressor mutations. Most of the suppressor mutations were in cps2E, which encodes the initiating glycosyltransferase for capsule synthesis. The cps2K mutants containing the suppressor mutations produced low levels of high-molecular-weight polymer that was detected only in membrane fractions. cps2K-repaired mutants exhibited only modest increases in capsule production due to the effect of the secondary mutation, but capsule was detectable in both membrane and cell wall fractions. Lethality of the cps2K, cps2J, and cps2H mutations was likely due to sequestration of undecaprenyl-phosphate in the capsule pathway and either preclusion of its turnover for utilization in essential pathways or destabilization of the membrane due to an accumulation of lipid-linked intermediates. The results demonstrate that proper polymer assembly requires not only a functional transporter and polymerase but also complete repeat units. A central role for the initiating glycosyltransferase in controlling capsule synthesis is also suggested.

Hypophosphatemia, hyperphosphaturia, and bisphosphonate treatment are associated with survival beyond infancy in generalized arterial calcification of infancy.

PubMed

Rutsch, Frank; Böyer, Petra; Nitschke, Yvonne; Ruf, Nico; Lorenz-Depierieux, Bettina; Wittkampf, Tanja; Weissen-Plenz, Gabriele; Fischer, Rudolf-Josef; Mughal, Zulf; Gregory, John W; Davies, Justin H; Loirat, Chantal; Strom, Tim M; Schnabel, Dirk; Nürnberg, Peter; Terkeltaub, Robert

2008-12-01

Generalized arterial calcification of infancy has been reported to be frequently lethal, and the efficiency of any therapy, including bisphosphonates, is unknown. A phosphate-poor diet markedly increases survival of NPP1 null mice, a model of generalized arterial calcification of infancy. We performed a multicenter genetic study and retrospective observational analysis of 55 subjects affected by generalized arterial calcification of infancy to identify prognostic factors. Nineteen (34%) patients survived the critical period of infancy. In all 8 surviving patients tested, hypophosphatemia due to reduced renal tubular phosphate reabsorption developed during childhood. Eleven of 17 (65%) patients treated with bisphosphonates survived. Of 26 patients who survived their first day of life and were not treated with bisphosphonates only 8 (31%) patients survived beyond infancy. Forty different homozygous or compound heterozygous mutations, including 16 novel mutations in ENPP1, were found in 41 (75%) of the 55 patients. Twenty-nine (71%) of these 41 patients died in infancy (median, 30 days). Seven of the 14 (50%) patients without ENPP1 mutations died in infancy (median, 9 days). When present on both alleles, the mutation p.P305T was associated with death in infancy in all 5 cases; otherwise, no clear genotype-phenotype correlation was seen. ENPP1 coding region mutations are associated with generalized arterial calcification of infancy in approximately 75% of subjects. Except for the p.P305T mutation, which was universally lethal when present on both alleles, the identified ENPP1 mutations per se have no discernable effect on survival. However, survival seems to be associated with hypophosphatemia linked with hyperphosphaturia and also with bisphosphonate treatment.

Hypophosphatemia, Hyperphosphaturia, and Bisphosphonate Treatment Are Associated With Survival Beyond Infancy in Generalized Arterial Calcification of Infancy

PubMed Central

Rutsch, Frank; Böyer, Petra; Nitschke, Yvonne; Ruf, Nico; Lorenz-Depierieux, Bettina; Wittkampf, Tanja; Weissen-Plenz, Gabriele; Fischer, Rudolf-Josef; Mughal, Zulf; Gregory, John W.; Davies, Justin H.; Loirat, Chantal; Strom, Tim M.; Schnabel, Dirk; Nürnberg, Peter; Terkeltaub, Robert

2009-01-01

Background Generalized arterial calcification of infancy has been reported to be frequently lethal, and the efficiency of any therapy, including bisphosphonates, is unknown. A phosphate-poor diet markedly increases survival of NPP1 null mice, a model of generalized arterial calcification of infancy. Methods and Results We performed a multicenter genetic study and retrospective observational analysis of 55 subjects affected by generalized arterial calcification of infancy to identify prognostic factors. Nineteen (34%) patients survived the critical period of infancy. In all 8 surviving patients tested, hypophosphatemia due to reduced renal tubular phosphate reabsorption developed during childhood. Eleven of 17 (65%) patients treated with bisphosphonates survived. Of 26 patients who survived their first day of life and were not treated with bisphosphonates only 8 (31%) patients survived beyond infancy. Forty different homozygous or compound heterozygous mutations, including 16 novel mutations in ENPP1, were found in 41 (75%) of the 55 patients. Twenty-nine (71%) of these 41 patients died in infancy (median, 30 days). Seven of the 14 (50%) patients without ENPP1 mutations died in infancy (median, 9 days). When present on both alleles, the mutation p.P305T was associated with death in infancy in all 5 cases; otherwise, no clear genotype-phenotype correlation was seen. Conclusion ENPP1 coding region mutations are associated with generalized arterial calcification of infancy in ≈75% of subjects. Except for the p.P305T mutation, which was universally lethal when present on both alleles, the identified ENPP1 mutations per se have no discernable effect on survival. However, survival seems to be associated with hypophosphatemia linked with hyperphosphaturia and also with bisphosphonate treatment. PMID:20016754

Ras1 interacts with multiple new signaling and cytoskeletal loci in Drosophila eggshell patterning and morphogenesis.

PubMed Central

Schnorr, J D; Holdcraft, R; Chevalier, B; Berg, C A

2001-01-01

Little is known about the genes that interact with Ras signaling pathways to regulate morphogenesis. The synthesis of dorsal eggshell structures in Drosophila melanogaster requires multiple rounds of Ras signaling followed by dramatic epithelial sheet movements. We took advantage of this process to identify genes that link patterning and morphogenesis; we screened lethal mutations on the second chromosome for those that could enhance a weak Ras1 eggshell phenotype. Of 1618 lethal P-element mutations tested, 13 showed significant enhancement, resulting in forked and fused dorsal appendages. Our genetic and molecular analyses together with information from the Berkeley Drosophila Genome Project reveal that 11 of these lines carry mutations in previously characterized genes. Three mutations disrupt the known Ras1 cell signaling components Star, Egfr, and Blistered, while one mutation disrupts Sec61beta, implicated in ligand secretion. Seven lines represent cell signaling and cytoskeletal components that are new to the Ras1 pathway; these are Chickadee (Profilin), Tec29, Dreadlocks, POSH, Peanut, Smt3, and MESK2, a suppressor of dominant-negative Ksr. A twelfth insertion disrupts two genes, Nrk, a "neurospecific" receptor tyrosine kinase, and Tpp, which encodes a neuropeptidase. These results suggest that Ras1 signaling during oogenesis involves novel components that may be intimately associated with additional signaling processes and with the reorganization of the cytoskeleton. To determine whether these Ras1 Enhancers function upstream or downstream of the Egf receptor, four mutations were tested for their ability to suppress an activated Egfr construct (lambdatop) expressed in oogenesis exclusively in the follicle cells. Mutations in Star and l(2)43Bb had no significant effect upon the lambdatop eggshell defect whereas smt3 and dock alleles significantly suppressed the lambdatop phenotype. PMID:11606538

Ras1 interacts with multiple new signaling and cytoskeletal loci in Drosophila eggshell patterning and morphogenesis.

PubMed

Schnorr, J D; Holdcraft, R; Chevalier, B; Berg, C A

2001-10-01

Little is known about the genes that interact with Ras signaling pathways to regulate morphogenesis. The synthesis of dorsal eggshell structures in Drosophila melanogaster requires multiple rounds of Ras signaling followed by dramatic epithelial sheet movements. We took advantage of this process to identify genes that link patterning and morphogenesis; we screened lethal mutations on the second chromosome for those that could enhance a weak Ras1 eggshell phenotype. Of 1618 lethal P-element mutations tested, 13 showed significant enhancement, resulting in forked and fused dorsal appendages. Our genetic and molecular analyses together with information from the Berkeley Drosophila Genome Project reveal that 11 of these lines carry mutations in previously characterized genes. Three mutations disrupt the known Ras1 cell signaling components Star, Egfr, and Blistered, while one mutation disrupts Sec61beta, implicated in ligand secretion. Seven lines represent cell signaling and cytoskeletal components that are new to the Ras1 pathway; these are Chickadee (Profilin), Tec29, Dreadlocks, POSH, Peanut, Smt3, and MESK2, a suppressor of dominant-negative Ksr. A twelfth insertion disrupts two genes, Nrk, a "neurospecific" receptor tyrosine kinase, and Tpp, which encodes a neuropeptidase. These results suggest that Ras1 signaling during oogenesis involves novel components that may be intimately associated with additional signaling processes and with the reorganization of the cytoskeleton. To determine whether these Ras1 Enhancers function upstream or downstream of the Egf receptor, four mutations were tested for their ability to suppress an activated Egfr construct (lambdatop) expressed in oogenesis exclusively in the follicle cells. Mutations in Star and l(2)43Bb had no significant effect upon the lambdatop eggshell defect whereas smt3 and dock alleles significantly suppressed the lambdatop phenotype.

Mitochondrial uncoupler exerts a synthetic lethal effect against β-catenin mutant tumor cells.

PubMed

Shikata, Yuki; Kiga, Masaki; Futamura, Yushi; Aono, Harumi; Inoue, Hiroyuki; Kawada, Manabu; Osada, Hiroyuki; Imoto, Masaya

2017-04-01

The wingless/int-1 (Wnt) signal transduction pathway plays a central role in cell proliferation, survival, differentiation and apoptosis. When β-catenin: a component of the Wnt pathway, is mutated into an active form, cell growth signaling is hyperactive and drives oncogenesis. As β-catenin is mutated in a wide variety of tumors, including up to 10% of all sporadic colon carcinomas and 20% of hepatocellular carcinomas, it has been considered a promising target for therapeutic interventions. Therefore, we screened an in-house natural product library for compounds that exhibited synthetic lethality towards β-catenin mutations and isolated nonactin, an antibiotic mitochondrial uncoupler, as a hit compound. Nonactin, as well as other mitochondrial uncouplers, induced apoptosis selectively in β-catenin mutated tumor cells. Significant tumor regression was observed in the β-catenin mutant HCT 116 xenograft model, but not in the β-catenin wild type A375 xenograft model, in response to daily administration of nonactin in vivo. Furthermore, we found that expression of an active mutant form of β-catenin induced a decrease in the glycolysis rate. Taken together, our results demonstrate that tumor cells with mutated β-catenin depend on mitochondrial oxidative phosphorylation for survival. Therefore, they undergo apoptosis in response to mitochondrial dysfunction following the addition of mitochondrial uncouplers, such as nonactin. These results suggest that targeting mitochondria is a potential chemotherapeutic strategy for tumor cells that harbor β-catenin mutations. © 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

Cloning of the neurodegeneration gene drop-dead and characterization of additional phenotypes of its mutation.

PubMed

Blumenthal, Edward M

2008-01-01

Mutations in the Drosophila gene drop-dead (drd) result in early adult lethality and neurodegeneration, but the molecular identity of the drd gene and its mechanism of action are not known. This paper describes the characterization of a new X-linked recessive adult-lethal mutation, originally called lot's wife (lwf(1)) but subsequently identified as an allele of drd (drd(lwf)); drd(lwf) mutants die within two weeks of eclosion. Through mapping and complementation, the drd gene has been identified as CG33968, which encodes a putative integral membrane protein of unknown function. The drd(lwf) allele is associated with a nonsense mutation that eliminates nearly 80% of the CG33968 gene product; mutations in the same gene were also found in two previously described drd alleles. Characterization of drd (lwf) flies revealed additional phenotypes of drd, most notably, defects in food processing by the digestive system and in oogenesis. Mutant flies store significantly more food in their crops and defecate less than wild-type flies, suggesting that normal transfer of ingested food from the crop into the midgut is dependent upon the DRD gene product. The defect in oogenesis results in the sterility of homozygous mutant females and is associated with a reduction in the number of vitellogenic egg chambers. The disruption in vitellogenesis is far more severe than that seen in starved flies and so is unlikely to be a secondary consequence of the digestive phenotype. This study demonstrates that mutation of the drd gene CG33968 results in a complex phenotype affecting multiple physiological systems within the fly.

Kunjin Virus Replicon-Based Vaccines Expressing Ebola Virus Glycoprotein GP Protect the Guinea Pig Against Lethal Ebola Virus Infection

PubMed Central

Reynard, O.; Mokhonov, V.; Mokhonova, E.; Leung, J.; Page, A.; Mateo, M.; Pyankova, O.; Georges-Courbot, M. C.; Raoul, H.; Khromykh, A. A.

2011-01-01

Pre- or postexposure treatments against the filoviral hemorrhagic fevers are currently not available for human use. We evaluated, in a guinea pig model, the immunogenic potential of Kunjin virus (KUN)–derived replicons as a vaccine candidate against Ebola virus (EBOV). Virus like particles (VLPs) containing KUN replicons expressing EBOV wild-type glycoprotein GP, membrane anchor-truncated GP (GP/Ctr), and mutated GP (D637L) with enhanced shedding capacity were generated and assayed for their protective efficacy. Immunization with KUN VLPs expressing full-length wild-type and D637L-mutated GPs but not membrane anchor–truncated GP induced dose-dependent protection against a challenge of a lethal dose of recombinant guinea pig-adapted EBOV. The surviving animals showed complete clearance of the virus. Our results demonstrate the potential for KUN replicon vectors as vaccine candidates against EBOV infection. PMID:21987742

Production of maternal-zygotic mutant zebrafish by germ-line replacement.

PubMed

Ciruna, Brian; Weidinger, Gilbert; Knaut, Holger; Thisse, Bernard; Thisse, Christine; Raz, Erez; Schier, Alexander F

2002-11-12

We report a generally applicable strategy for transferring zygotic lethal mutations through the zebrafish germ line. By using a morpholino oligonucleotide that blocks primordial germ cell (PGC) development, we generate embryos devoid of endogenous PGCs to serve as hosts for the transplantation of germ cells derived from homozygous mutant donors. Successful transfers are identified by the localization of specifically labeled donor PGCs to the region of the developing gonad in chimeric embryos. This strategy, which results in the complete replacement of the host germ line with donor PGCs, was validated by the generation of maternal and maternal-zygotic mutants for the miles apart locus. This germ-line replacement technique provides a powerful tool for studying the maternal effects of zygotic lethal mutations. Furthermore, the ability to generate large clutches of purely mutant embryos will greatly facilitate embryological, genetic, genomic, and biochemical studies.

Production of maternal-zygotic mutant zebrafish by germ-line replacement

PubMed Central

Ciruna, Brian; Weidinger, Gilbert; Knaut, Holger; Thisse, Bernard; Thisse, Christine; Raz, Erez; Schier, Alexander F.

2002-01-01

We report a generally applicable strategy for transferring zygotic lethal mutations through the zebrafish germ line. By using a morpholino oligonucleotide that blocks primordial germ cell (PGC) development, we generate embryos devoid of endogenous PGCs to serve as hosts for the transplantation of germ cells derived from homozygous mutant donors. Successful transfers are identified by the localization of specifically labeled donor PGCs to the region of the developing gonad in chimeric embryos. This strategy, which results in the complete replacement of the host germ line with donor PGCs, was validated by the generation of maternal and maternal-zygotic mutants for the miles apart locus. This germ-line replacement technique provides a powerful tool for studying the maternal effects of zygotic lethal mutations. Furthermore, the ability to generate large clutches of purely mutant embryos will greatly facilitate embryological, genetic, genomic, and biochemical studies. PMID:12397179

An essential role of intestinal cell kinase in lung development is linked to the perinatal lethality of human ECO syndrome

PubMed Central

Tong, Yixin; Park, So Hyun; Wu, Di; Xu, Wenhao; Guillot, Stacey J.; Jin, Li; Li, Xudong; Wang, Yalin; Lin, Chyuan-Sheng; Fu, Zheng

2017-01-01

Human endocrine-cerebro-osteodysplasia (ECO) syndrome, caused by the loss-of-function mutation R272Q in the ICK (intestinal cell kinase) gene, is a neonatal-lethal developmental disorder. To elucidate the molecular basis of ECO syndrome, we constructed an Ick R272Q knock-in mouse model that recapitulates ECO pathological phenotypes. Newborns bearing Ick R272Q homozygous mutations die at birth due to respiratory distress. Ick mutant lungs exhibit not only impaired branching morphogenesis associated with reduced mesenchymal proliferation, but also significant airspace deficiency in primitive alveoli concomitant with abnormal interstitial mesenchymal differentiation. ICK dysfunction induces elongated primary cilia and perturbs ciliary Hedgehog signaling and autophagy during lung sacculation. Our study identifies an essential role for ICK in lung development and advances the mechanistic understanding of ECO syndrome. PMID:28380258

An essential role of intestinal cell kinase in lung development is linked to the perinatal lethality of human ECO syndrome.

PubMed

Tong, Yixin; Park, So Hyun; Wu, Di; Xu, Wenhao; Guillot, Stacey J; Jin, Li; Li, Xudong; Wang, Yalin; Lin, Chyuan-Sheng; Fu, Zheng

2017-05-01

Human endocrine-cerebro-osteodysplasia (ECO) syndrome, caused by the loss-of-function mutation R272Q in the intestinal cell kinase (ICK) gene, is a neonatal-lethal developmental disorder. To elucidate the molecular basis of ECO syndrome, we constructed an Ick R272Q knock-in mouse model that recapitulates ECO pathological phenotypes. Newborns bearing Ick R272Q homozygous mutations die at birth due to respiratory distress. Ick mutant lungs exhibit not only impaired branching morphogenesis associated with reduced mesenchymal proliferation but also significant airspace deficiency in primitive alveoli concomitant with abnormal interstitial mesenchymal differentiation. ICK dysfunction induces elongated primary cilia and perturbs ciliary Hedgehog signaling and autophagy during lung sacculation. Our study identifies an essential role for ICK in lung development and advances the mechanistic understanding of ECO syndrome. © 2017 Federation of European Biochemical Societies.

dNTP pool levels modulate mutator phenotypes of error-prone DNA polymerase ε variants.

PubMed

Williams, Lindsey N; Marjavaara, Lisette; Knowels, Gary M; Schultz, Eric M; Fox, Edward J; Chabes, Andrei; Herr, Alan J

2015-05-12

Mutator phenotypes create genetic diversity that fuels tumor evolution. DNA polymerase (Pol) ε mediates leading strand DNA replication. Proofreading defects in this enzyme drive a number of human malignancies. Here, using budding yeast, we show that mutator variants of Pol ε depend on damage uninducible (Dun)1, an S-phase checkpoint kinase that maintains dNTP levels during a normal cell cycle and up-regulates dNTP synthesis upon checkpoint activation. Deletion of DUN1 (dun1Δ) suppresses the mutator phenotype of pol2-4 (encoding Pol ε proofreading deficiency) and is synthetically lethal with pol2-M644G (encoding altered Pol ε base selectivity). Although pol2-4 cells cycle normally, pol2-M644G cells progress slowly through S-phase. The pol2-M644G cells tolerate deletions of mediator of the replication checkpoint (MRC) 1 (mrc1Δ) and radiation sensitive (Rad) 9 (rad9Δ), which encode mediators of checkpoint responses to replication stress and DNA damage, respectively. The pol2-M644G mutator phenotype is partially suppressed by mrc1Δ but not rad9Δ; neither deletion suppresses the pol2-4 mutator phenotype. Thus, checkpoint activation augments the Dun1 effect on replication fidelity but is not required for it. Deletions of genes encoding key Dun1 targets that negatively regulate dNTP synthesis, suppress the dun1Δ pol2-M644G synthetic lethality and restore the mutator phenotype of pol2-4 in dun1Δ cells. DUN1 pol2-M644G cells have constitutively high dNTP levels, consistent with checkpoint activation. In contrast, pol2-4 and POL2 cells have similar dNTP levels, which decline in the absence of Dun1 and rise in the absence of the negative regulators of dNTP synthesis. Thus, dNTP pool levels correlate with Pol ε mutator severity, suggesting that treatments targeting dNTP pools could modulate mutator phenotypes for therapy.

Minimal Contribution of APOBEC3-Induced G-to-A Hypermutation to HIV-1 Recombination and Genetic Variation

PubMed Central

Nikolaitchik, Olga A.; Burdick, Ryan C.; Gorelick, Robert J.; Keele, Brandon F.; Hu, Wei-Shau; Pathak, Vinay K.

2016-01-01

Although the predominant effect of host restriction APOBEC3 proteins on HIV-1 infection is to block viral replication, they might inadvertently increase retroviral genetic variation by inducing G-to-A hypermutation. Numerous studies have disagreed on the contribution of hypermutation to viral genetic diversity and evolution. Confounding factors contributing to the debate include the extent of lethal (stop codon) and sublethal hypermutation induced by different APOBEC3 proteins, the inability to distinguish between G-to-A mutations induced by APOBEC3 proteins and error-prone viral replication, the potential impact of hypermutation on the frequency of retroviral recombination, and the extent to which viral recombination occurs in vivo, which can reassort mutations in hypermutated genomes. Here, we determined the effects of hypermutation on the HIV-1 recombination rate and its contribution to genetic variation through recombination to generate progeny genomes containing portions of hypermutated genomes without lethal mutations. We found that hypermutation did not significantly affect the rate of recombination, and recombination between hypermutated and wild-type genomes only increased the viral mutation rate by 3.9 × 10−5 mutations/bp/replication cycle in heterozygous virions, which is similar to the HIV-1 mutation rate. Since copackaging of hypermutated and wild-type genomes occurs very rarely in vivo, recombination between hypermutated and wild-type genomes does not significantly contribute to the genetic variation of replicating HIV-1. We also analyzed previously reported hypermutated sequences from infected patients and determined that the frequency of sublethal mutagenesis for A3G and A3F is negligible (4 × 10−21 and1 × 10−11, respectively) and its contribution to viral mutations is far below mutations generated during error-prone reverse transcription. Taken together, we conclude that the contribution of APOBEC3-induced hypermutation to HIV-1 genetic variation is substantially lower than that from mutations during error-prone replication. PMID:27186986

Minimal Contribution of APOBEC3-Induced G-to-A Hypermutation to HIV-1 Recombination and Genetic Variation.

PubMed

Delviks-Frankenberry, Krista A; Nikolaitchik, Olga A; Burdick, Ryan C; Gorelick, Robert J; Keele, Brandon F; Hu, Wei-Shau; Pathak, Vinay K

2016-05-01

Although the predominant effect of host restriction APOBEC3 proteins on HIV-1 infection is to block viral replication, they might inadvertently increase retroviral genetic variation by inducing G-to-A hypermutation. Numerous studies have disagreed on the contribution of hypermutation to viral genetic diversity and evolution. Confounding factors contributing to the debate include the extent of lethal (stop codon) and sublethal hypermutation induced by different APOBEC3 proteins, the inability to distinguish between G-to-A mutations induced by APOBEC3 proteins and error-prone viral replication, the potential impact of hypermutation on the frequency of retroviral recombination, and the extent to which viral recombination occurs in vivo, which can reassort mutations in hypermutated genomes. Here, we determined the effects of hypermutation on the HIV-1 recombination rate and its contribution to genetic variation through recombination to generate progeny genomes containing portions of hypermutated genomes without lethal mutations. We found that hypermutation did not significantly affect the rate of recombination, and recombination between hypermutated and wild-type genomes only increased the viral mutation rate by 3.9 × 10-5 mutations/bp/replication cycle in heterozygous virions, which is similar to the HIV-1 mutation rate. Since copackaging of hypermutated and wild-type genomes occurs very rarely in vivo, recombination between hypermutated and wild-type genomes does not significantly contribute to the genetic variation of replicating HIV-1. We also analyzed previously reported hypermutated sequences from infected patients and determined that the frequency of sublethal mutagenesis for A3G and A3F is negligible (4 × 10-21 and1 × 10-11, respectively) and its contribution to viral mutations is far below mutations generated during error-prone reverse transcription. Taken together, we conclude that the contribution of APOBEC3-induced hypermutation to HIV-1 genetic variation is substantially lower than that from mutations during error-prone replication.

Modelling vemurafenib resistance in melanoma reveals a strategy to forestall drug resistance

PubMed Central

Thakur, Meghna Das; Salangsang, Fernando; Landman, Allison S.; Sellers, William R.; Pryer, Nancy K.; Levesque, Mitchell P.; Dummer, Reinhard; McMahon, Martin; Stuart, Darrin D.

2014-01-01

Mutational activation of BRAF is the most prevalent genetic alteration in human melanoma, with ≥ 50% of tumours expressing the BRAF(V600E) oncoprotein1,2. Moreover, the marked tumour regression and improved survival of late-stage BRAF-mutated melanoma patients in response to treatment with vemurafenib demonstrates the essential role of oncogenic BRAF in melanoma maintenance3,4. However, as most patients relapse with lethal drug-resistant disease, understanding and preventing mechanism(s) of resistance is critical to providing improved therapy5. Here we investigate the cause and consequences of vemurafenib resistance using two independently derived primary human melanoma xeno-graft models in which drugresistanceisselected by continuous vemurafenib administration. In one of these models, resistant tumours show continued dependency on BRAF(V600E) → MEK → ERK signalling owing to elevated BRAF(V600E) expression. Most importantly, we demonstrate that vemurafenib-resistant melanomas become drug dependent for their continued proliferation, such that cessation of drug administration leads to regression of established drug-resistant tumours. We further demonstrate that a discontinuous dosing strategy, which exploits the fitness disadvantage displayed by drug-resistant cells in the absence of the drug, forestalls the onset of lethal drug-resistant disease. These data highlight the concept that drug-resistant cells may also display drug dependency, such that altered dosing may prevent the emergence of lethal drug resistance. Such observations may contribute to sustaining the durability of the vemurafenib response with the ultimate goal of curative therapy for the subset of melanoma patients with BRAF mutations. PMID:23302800

APOBEC3G-Induced Hypermutation of Human Immunodeficiency Virus Type-1 Is Typically a Discrete “All or Nothing” Phenomenon

PubMed Central

Armitage, Andrew E.; Deforche, Koen; Chang, Chih-hao; Wee, Edmund; Kramer, Beatrice; Welch, John J.; Gerstoft, Jan; Fugger, Lars; McMichael, Andrew; Rambaut, Andrew; Iversen, Astrid K. N.

2012-01-01

The rapid evolution of Human Immunodeficiency Virus (HIV-1) allows studies of ongoing host–pathogen interactions. One key selective host factor is APOBEC3G (hA3G) that can cause extensive and inactivating Guanosine-to-Adenosine (G-to-A) mutation on HIV plus-strand DNA (termed hypermutation). HIV can inhibit this innate anti-viral defense through binding of the viral protein Vif to hA3G, but binding efficiency varies and hypermutation frequencies fluctuate in patients. A pivotal question is whether hA3G-induced G-to-A mutation is always lethal to the virus or if it may occur at sub-lethal frequencies that could increase viral diversification. We show in vitro that limiting-levels of hA3G-activity (i.e. when only a single hA3G-unit is likely to act on HIV) produce hypermutation frequencies similar to those in patients and demonstrate in silico that potentially non-lethal G-to-A mutation rates are ∼10-fold lower than the lowest observed hypermutation levels in vitro and in vivo. Our results suggest that even a single incorporated hA3G-unit is likely to cause extensive and inactivating levels of HIV hypermutation and that hypermutation therefore is typically a discrete “all or nothing” phenomenon. Thus, therapeutic measures that inhibit the interaction between Vif and hA3G will likely not increase virus diversification but expand the fraction of hypermutated proviruses within the infected host. PMID:22457633

GPR98 mutations cause Usher syndrome type 2 in males.

PubMed

Ebermann, I; Wiesen, M H J; Zrenner, E; Lopez, I; Pigeon, R; Kohl, S; Löwenheim, H; Koenekoop, R K; Bolz, H J

2009-04-01

Mutations in the large GPR98 gene underlie Usher syndrome type 2C (USH2C), and all patients described to date have been female. It was speculated that GPR98 mutations cause a more severe, and eventually lethal, phenotype in males. We describe for the first time two male patients with USH2 with novel GPR98 mutations. Clinical characterization of a male patient and his affected sister revealed a typical USH2 phenotype in both. GPR98 may have been excluded from systematic investigation in previous studies, and the proportion of patients with USH2C probably underestimated. GPR98 should be considered in patients with USH2 of both sexes.

[Mutants of the yeast Saccharomyces cerevisiae characterized by enhanced induced mutagenesis. III. Effect of the him mutation on the effectiveness and specificity of UF-induced mutagenesis].

PubMed

Ivanov, E L; Koval'tsova, S V; Korolev, V G

1987-09-01

We have studied the influence of him1-1, him2-1, him3-1 and himX mutations on induction frequency and specificity of UV-induced adenine-dependent mutations in the yeast Saccharomyces cerevisiae. Him mutations do not render haploid cells more sensitive to the lethal action of UV-light; however, in him strains adenine-dependent mutations (ade1, ade2) were induced more frequently (1.5--2-fold), as compared to the HIM strain. An analysis of the molecular nature of ade2 mutants revealed that him1-1, him2-1 and himX mutations increase specifically the yield of transitions (AT----GC and GC----AT), whereas in the him3-1 strain the yield of transversions was enhanced as well. We suggest him mutations analysed to affect specific repair pathway for mismatch correction.

Arginine-glycine-aspartic acid motif is critical for human parechovirus 1 entry.

PubMed

Boonyakiat, Y; Hughes, P J; Ghazi, F; Stanway, G

2001-10-01

The human parechovirus 1 RGD motif in VP1 was studied by mutagenesis. An RGD-to-RGE change gave only revertant viruses with a restored RGD, while deletion of GD was lethal and nonrevertable. Mutations at the +1 and +2 positions had some effect on growth properties and a +1 M-to-P change was lethal. These studies indicate that the RGD motif plays a critical role in infectivity, presumably by interacting with integrins, and that downstream amino acids can have an influence on function.

A Mutation in the Rett Syndrome Gene, MECP2, Causes X-Linked Mental Retardation and Progressive Spasticity in Males

PubMed Central

Meloni, Ilaria; Bruttini, Mirella; Longo, Ilaria; Mari, Francesca; Rizzolio, Flavio; D’Adamo, Patrizia; Denvriendt, Koenraad; Fryns, Jean-Pierre; Toniolo, Daniela; Renieri, Alessandra

2000-01-01

Heterozygous mutations in the X-linked MECP2 gene cause Rett syndrome, a severe neurodevelopmental disorder of young females. Only one male presenting an MECP2 mutation has been reported; he survived only to age 1 year, suggesting that mutations in MECP2 are male lethal. Here we report a three-generation family in which two affected males showed severe mental retardation and progressive spasticity, previously mapped in Xq27.2-qter. Two obligate carrier females showed either normal or borderline intelligence, simulating an X-linked recessive trait. The two males and the two obligate carrier females presented a mutation in the MECP2 gene, demonstrating that, in males, MECP2 can be responsible for severe mental retardation associated with neurological disorders. PMID:10986043

Gene Mutations and Genomic Rearrangements in the Mouse as a Result of Transposon Mobilization from Chromosomal Concatemers

PubMed Central

Geurts, Aron M; Collier, Lara S; Geurts, Jennifer L; Oseth, Leann L; Bell, Matthew L; Mu, David; Lucito, Robert; Godbout, Susan A; Green, Laura E; Lowe, Scott W; Hirsch, Betsy A; Leinwand, Leslie A; Largaespada, David A

2006-01-01

Previous studies of the Sleeping Beauty (SB) transposon system, as an insertional mutagen in the germline of mice, have used reverse genetic approaches. These studies have led to its proposed use for regional saturation mutagenesis by taking a forward-genetic approach. Thus, we used the SB system to mutate a region of mouse Chromosome 11 in a forward-genetic screen for recessive lethal and viable phenotypes. This work represents the first reported use of an insertional mutagen in a phenotype-driven approach. The phenotype-driven approach was successful in both recovering visible and behavioral mutants, including dominant limb and recessive behavioral phenotypes, and allowing for the rapid identification of candidate gene disruptions. In addition, a high frequency of recessive lethal mutations arose as a result of genomic rearrangements near the site of transposition, resulting from transposon mobilization. The results suggest that the SB system could be used in a forward-genetic approach to recover interesting phenotypes, but that local chromosomal rearrangements should be anticipated in conjunction with single-copy, local transposon insertions in chromosomes. Additionally, these mice may serve as a model for chromosome rearrangements caused by transposable elements during the evolution of vertebrate genomes. PMID:17009875

A genetic screen for zygotic embryonic lethal mutations affecting cuticular morphology in the wasp Nasonia vitripennis.

PubMed Central

Pultz, M A; Zimmerman, K K; Alto, N M; Kaeberlein, M; Lange, S K; Pitt, J N; Reeves, N L; Zehrung, D L

2000-01-01

We have screened for zygotic embryonic lethal mutations affecting cuticular morphology in Nasonia vitripennis (Hymenoptera; Chalcidoidea). Our broad goal was to investigate the use of Nasonia for genetically surveying conservation and change in regulatory gene systems, as a means to understand the diversity of developmental strategies that have arisen during the course of evolution. Specifically, we aim to compare anteroposterior patterning gene functions in two long germ band insects, Nasonia and Drosophila. In Nasonia, unfertilized eggs develop as haploid males while fertilized eggs develop as diploid females, so the entire genome can be screened for recessive zygotic mutations by examining the progeny of F1 females. We describe 74 of >100 lines with embryonic cuticular mutant phenotypes, including representatives of coordinate, gap, pair-rule, segment polarity, homeotic, and Polycomb group functions, as well as mutants with novel phenotypes not directly comparable to those of known Drosophila genes. We conclude that Nasonia is a tractable experimental organism for comparative developmental genetic study. The mutants isolated here have begun to outline the extent of conservation and change in the genetic programs controlling embryonic patterning in Nasonia and Drosophila. PMID:10866651

Icebox, a recessive X-linked mutation in Drosophila causing low sexual receptivity.

PubMed

Kerr, C; Ringo, J; Dowse, H; Johnson, E

1997-11-01

The X-linked recessive mutation icebox (ibx; 1-23, 7F1) of Drosophila melanogaster lowers the sexual receptivity of females. The probability of mating with mature wild-type males is reduced in ibx homozygotes, and the frequency of rejection behavior (rate per minute) towards courting males is increased. ibx fails to complement In(1)RA35, which is a lethal allele of Neuroglian (Nrg, which encodes a transmembrane protein found in embryonic tissues including the nervous system) due to a breakpoint in that gene; however, both l(1)B4 and l(1)VA142, other lethal mutations of Nrg, do complement ibx. 12-h ibx embryos exhibit a normal pattern of staining for the Neuroglian-specific antibody, Mab BP104. Males and females mutant for ibx have normal egg-to-adult survival and appear normal in several "general" behavioral traits including olfaction, phototaxis, locomotor activity, and heartbeat. ibx males court normally, and are successful in mating. These characteristics suggest that ibx does not cause sensory or motor defects. Ovarian growth and sperm storage are wild-type in ibx/ibx females. Treatment with the JH analog methoprene increases the receptivity of ibx/ibx females.

A genetic screen for temperature-sensitive cell-division mutants of Caenorhabditis elegans.

PubMed Central

O'Connell, K F; Leys, C M; White, J G

1998-01-01

A novel screen to isolate conditional cell-division mutants in Caenorhabditis elegans has been developed. The screen is based on the phenotypes associated with existing cell-division mutations: some disrupt postembryonic divisions and affect formation of the gonad and ventral nerve cord-resulting in sterile, uncoordinated animals-while others affect embryonic divisions and result in lethality. We obtained 19 conditional mutants that displayed these phenotypes when shifted to the restrictive temperature at the appropriate developmental stage. Eighteen of these mutations have been mapped; 17 proved to be single alleles of newly identified genes, while 1 proved to be an allele of a previously identified gene. Genetic tests on the embryonic lethal phenotypes indicated that for 13 genes, embryogenesis required maternal expression, while for 6, zygotic expression could suffice. In all cases, maternal expression of wild-type activity was found to be largely sufficient for embryogenesis. Cytological analysis revealed that 10 mutants possessed embryonic cell-division defects, including failure to properly segregate DNA, failure to assemble a mitotic spindle, late cytokinesis defects, prolonged cell cycles, and improperly oriented mitotic spindles. We conclude that this approach can be used to identify mutations that affect various aspects of the cell-division cycle. PMID:9649522

Unbiased Combinatorial Genomic Approaches to Identify Alternative Therapeutic Targets within the TSC Signaling Network

DTIC Science & Technology

2015-09-01

assessed the specificity of mutation in Drosophila S2R+ cells. We generated a quantitative mutation reporter vector in which an sgRNA target sequence ...phosphatases (563 genes) in the Drosophila genome (Figure 4). 65 samples that displayed synthetic lethality (15 genes) or synthetic increases in viability...targeting all kinases and phosphatases (563 genes) in the Drosophila genome . . Identified three hits (mRNA-Cap, Pitslre and CycT) that scored as

Hydrophobic imbalance in the cytoplasmic domain of phospholamban is a determinant for lethal dilated cardiomyopathy.

PubMed

Ceholski, Delaine K; Trieber, Catharine A; Young, Howard S

2012-05-11

The sarco(endo)plasmic reticulum calcium ATPase (SERCA) and its regulatory partner phospholamban (PLN) are essential for myocardial contractility. Arg(9) → Cys (R9C) and Arg(14) deletion (R14del) mutations in PLN are associated with lethal dilated cardiomyopathy in humans. To better understand these mutations, we made a series of amino acid substitutions in the cytoplasmic domain of PLN and tested their ability to inhibit SERCA. R9C is a complete loss-of-function mutant of PLN, whereas R14del is a mild loss-of-function mutant. When combined with wild-type PLN to simulate heterozygous conditions, the mutants had a dominant negative effect on SERCA function. A series of targeted mutations in this region of the PLN cytoplasmic domain ((8)TRSAIRR(14)) demonstrated the importance of hydrophobic balance in proper PLN regulation of SERCA. We found that Arg(9) → Leu and Thr(8) → Cys substitutions mimicked the behavior of the R9C mutant, and an Arg(14) → Ala substitution mimicked the behavior of the R14del mutant. The results reveal that the change in hydrophobicity resulting from the R9C and R14del mutations is sufficient to explain the loss of function and persistent interaction with SERCA. Hydrophobic imbalance in the cytoplasmic domain of PLN appears to be a predictor for the development and progression of dilated cardiomyopathy.

Delineating the requirements for spontaneous DNA damage resistance pathways in genome maintenance and viability in Saccharomyces cerevisiae.

PubMed

Morey, Natalie J; Doetsch, Paul W; Jinks-Robertson, Sue

2003-06-01

Cellular metabolic processes constantly generate reactive species that damage DNA. To counteract this relentless assault, cells have developed multiple pathways to resist damage. The base excision repair (BER) and nucleotide excision repair (NER) pathways remove damage whereas the recombination (REC) and postreplication repair (PRR) pathways bypass the damage, allowing deferred removal. Genetic studies in yeast indicate that these pathways can process a common spontaneous lesion(s), with mutational inactivation of any pathway increasing the burden on the remaining pathways. In this study, we examine the consequences of simultaneously compromising three or more of these pathways. Although the presence of a functional BER pathway alone is able to support haploid growth, retention of the NER, REC, or PRR pathway alone is not, indicating that BER is the key damage resistance pathway in yeast and may be responsible for the removal of the majority of either spontaneous DNA damage or specifically those lesions that are potentially lethal. In the diploid state, functional BER, NER, or REC alone can support growth, while PRR alone is insufficient for growth. In diploids, the presence of PRR alone may confer a lethal mutation load or, alternatively, PRR alone may be insufficient to deal with potentially lethal, replication-blocking lesions.

ESCRT-Dependent Cell Death in a Caenorhabditis elegans Model of the Lysosomal Storage Disorder Mucolipidosis Type IV

PubMed Central

Huynh, Julie M.; Dang, Hope; Munoz-Tucker, Isabel A.; O’Ketch, Marvin; Liu, Ian T.; Perno, Savannah; Bhuyan, Natasha; Crain, Allison; Borbon, Ivan; Fares, Hanna

2016-01-01

Mutations in MCOLN1, which encodes the cation channel protein TRPML1, result in the neurodegenerative lysosomal storage disorder Mucolipidosis type IV. Mucolipidosis type IV patients show lysosomal dysfunction in many tissues and neuronal cell death. The ortholog of TRPML1 in Caenorhabditis elegans is CUP-5; loss of CUP-5 results in lysosomal dysfunction in many tissues and death of developing intestinal cells that results in embryonic lethality. We previously showed that a null mutation in the ATP-Binding Cassette transporter MRP-4 rescues the lysosomal defect and embryonic lethality of cup-5(null) worms. Here we show that reducing levels of the Endosomal Sorting Complex Required for Transport (ESCRT)-associated proteins DID-2, USP-50, and ALX-1/EGO-2, which mediate the final de-ubiquitination step of integral membrane proteins being sequestered into late endosomes, also almost fully suppresses cup-5(null) mutant lysosomal defects and embryonic lethality. Indeed, we show that MRP-4 protein is hypo-ubiquitinated in the absence of CUP-5 and that reducing levels of ESCRT-associated proteins suppresses this hypo-ubiquitination. Thus, increased ESCRT-associated de-ubiquitinating activity mediates the lysosomal defects and corresponding cell death phenotypes in the absence of CUP-5. PMID:26596346

Detection of haplotypes associated with prenatal death in dairy cattle and identification of deleterious mutations in GART, SHBG and SLC37A2.

PubMed

Fritz, Sébastien; Capitan, Aurelien; Djari, Anis; Rodriguez, Sabrina C; Barbat, Anne; Baur, Aurélia; Grohs, Cécile; Weiss, Bernard; Boussaha, Mekki; Esquerré, Diane; Klopp, Christophe; Rocha, Dominique; Boichard, Didier

2013-01-01

The regular decrease of female fertility over time is a major concern in modern dairy cattle industry. Only half of this decrease is explained by indirect response to selection on milk production, suggesting the existence of other factors such as embryonic lethal genetic defects. Genomic regions harboring recessive deleterious mutations were detected in three dairy cattle breeds by identifying frequent haplotypes (>1%) showing a deficit in homozygotes among Illumina Bovine 50k Beadchip haplotyping data from the French genomic selection database (47,878 Holstein, 16,833 Montbéliarde, and 11,466 Normande animals). Thirty-four candidate haplotypes (p<10(-4)) including previously reported regions associated with Brachyspina, CVM, HH1, and HH3 in Holstein breed were identified. Haplotype length varied from 1 to 4.8 Mb and frequencies from 1.7 up to 9%. A significant negative effect on calving rate, consistent in heifers and in lactating cows, was observed for 9 of these haplotypes in matings between carrier bulls and daughters of carrier sires, confirming their association with embryonic lethal mutations. Eight regions were further investigated using whole genome sequencing data from heterozygous bull carriers and control animals (45 animals in total). Six strong candidate causative mutations including polymorphisms previously reported in FANCI (Brachyspina), SLC35A3 (CVM), APAF1 (HH1) and three novel mutations with very damaging effect on the protein structure, according to SIFT and Polyphen-2, were detected in GART, SHBG and SLC37A2 genes. In conclusion, this study reveals a yet hidden consequence of the important inbreeding rate observed in intensively selected and specialized cattle breeds. Counter-selection of these mutations and management of matings will have positive consequences on female fertility in dairy cattle.

An initiator codon mutation in SDE2 causes recessive embryonic lethality in Holstein cattle.

PubMed

Fritz, Sébastien; Hoze, Chris; Rebours, Emmanuelle; Barbat, Anne; Bizard, Méline; Chamberlain, Amanda; Escouflaire, Clémentine; Vander Jagt, Christy; Boussaha, Mekki; Grohs, Cécile; Allais-Bonnet, Aurélie; Philippe, Maëlle; Vallée, Amélie; Amigues, Yves; Hayes, Benjamin J; Boichard, Didier; Capitan, Aurélien

2018-04-18

Researching depletions in homozygous genotypes for specific haplotypes among the large cohorts of animals genotyped for genomic selection is a very efficient strategy to map recessive lethal mutations. In this study, by analyzing real or imputed Illumina BovineSNP50 (Illumina Inc., San Diego, CA) genotypes from more than 250,000 Holstein animals, we identified a new locus called HH6 showing significant negative effects on conception rate and nonreturn rate at 56 d in at-risk versus control mating. We fine-mapped this locus in a 1.1-Mb interval and analyzed genome sequence data from 12 carrier and 284 noncarrier Holstein bulls. We report the identification of a strong candidate mutation in the gene encoding SDE2 telomere maintenance homolog (SDE2), a protein essential for genomic stability in eukaryotes. This A-to-G transition changes the initiator ATG (methionine) codon to ACG because the gene is transcribed on the reverse strand. Using RNA sequencing and quantitative reverse-transcription PCR, we demonstrated that this mutation does not significantly affect SDE2 splicing and expression level in heterozygous carriers compared with control animals. Initiation of translation at the closest in-frame methionine codon would truncate the SDE2 precursor by 83 amino acids, including the cleavage site necessary for its activation. Finally, no homozygote for the G allele was observed in a large population of nearly 29,000 individuals genotyped for the mutation. The low frequency (1.3%) of the derived allele in the French population and the availability of a diagnostic test on the Illumina EuroG10K SNP chip routinely used for genomic evaluation will enable rapid and efficient selection against this deleterious mutation. Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

[Genetic control of mitotic crossing-over in yeasts. III. Induction by 8-methoxypsoralen and long-wave UV irradiation (lambda=365 nm)].

PubMed

Fedorova, I V; Marfin, S V

1982-02-01

The lethal effect of 8-methoxypsoralen (8-MOP) plus 365 nm light has been studied in haploid radiosensitive strains of Saccharomyces cerevisiae. The diploid of wild type and the diploid homozygous for the rad2 mutation (this mutation blocks the excision of UV-induced pyrimidine dimers) were more resistant to the lethal effect of 8-MOP plus 365 nm light than the haploid of wild type and rad2 haploid, respectively. The diploid homozygous for rad54 mutation (the mutation blocks the repair of double-strand breaks in DNA) was more sensitive than haploid rad54. The method of repeated irradiation allowed to study the capacity of radiosensitive diploids to remove monoadducts induced by 8-MOP in DNA. This process was very effective in diploids of wild type and in the rad54 rad54 diploid, while the rad2 rad2 diploid was characterized by nearly complete absence of monoadduct excision. The study of mitotic crossing over and mitotic segregation in yeast diploids, containing a pair of complementing alleles of the ade2 gene (red/pink) has shown a very high recombinogenic effect of 8-MOP plus 365 nm light. The rad2 mutation slightly increased the frequency of mitotic segregation and mitotic crossing over. The rad54 mutation decreased the frequency of mitotic segregation and entirely suppressed mitotic crossing over. The method of repeated irradiation showed that the cross-links, but not monoadducts, are the main cause of high recombinogenic effect of 8-MOP plus 365 nm light. The possible participation of different repair systems in recombinational processes induced by 8-MOP in yeast cells is discussed.

Biological damage induced by ionizing cosmic rays in dry Arabidopsis seeds.

PubMed

Kranz, A R; Bork, U; Bucker, H; Reitz, G

1990-01-01

In September 1987 dry seeds containing embryos of the crucifer plant Arabidopsis thaliana (L.) Heynh, were flown in orbit for 13 days on the Kosmos 1887 satellite. The seeds were fixed on CNd detectors and stored in units of Biorack type I/O. One unit was exposed inside, another one outside the satellite. The temperature profile of the flown seeds inside the satellite was simulated on earth in an identical backup control sample (BC). An additional control (SC) was studied with the original seeds sample. By use of the CNd-detector, HZE-tracks were measured with a PC-assisted microscope. The biological damages were investigated by growing the seeds under controlled climatic conditions. The following biological endpoints of the cosmic radiation damage were studied: germination, radicle length, sublethality, morphological aberrations, flower development, tumorization, embryo lethality inside the siliques. The summarized damage (D) and the mutation frequencies of embyronic lethal genes were calculated. The following results were obtained: the damages increase significantly in orbit at all biological endpoints; germination and fiowerings especially, as well as embryo lethality of fruits and lethal mutation frequency, were maximum mostly for HZE-hit seeds. Additionally, an increase of damage was observed for the seeds of the outside-exposed Biorack in comparison to the inside ones, which was probably caused by less radiation shielding and free space vacuum. The significance of the results obtained is discussed with respect to stress and risk and, thus, the quality of the RBE-factors and heavy ionizing radiation all needed for the very definition of radiation protection standards in space.

Characterization of potential driver mutations involved in human breast cancer by computational approaches

PubMed Central

Rajendran, Barani Kumar; Deng, Chu-Xia

2017-01-01

Breast cancer is the second most frequently occurring form of cancer and is also the second most lethal cancer in women worldwide. A genetic mutation is one of the key factors that alter multiple cellular regulatory pathways and drive breast cancer initiation and progression yet nature of these cancer drivers remains elusive. In this article, we have reviewed various computational perspectives and algorithms for exploring breast cancer driver mutation genes. Using both frequency based and mutational exclusivity based approaches, we identified 195 driver genes and shortlisted 63 of them as candidate drivers for breast cancer using various computational approaches. Finally, we conducted network and pathway analysis to explore their functions in breast tumorigenesis including tumor initiation, progression, and metastasis. PMID:28477017

AMPA receptor desensitization mutation results in severe developmental phenotypes and early postnatal lethality

PubMed Central

Christie, Louisa A.; Russell, Theron A.; Xu, Jian; Wood, Lydia; Shepherd, Gordon M. G.; Contractor, Anis

2010-01-01

AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole-propionate) recep-tors desensitize rapidly and completely in the continued presence of their endogenous ligand glutamate; however, it is not clear what role AMPA receptor desensitization plays in the brain. We generated a knock-in mouse in which a single amino acid residue, which controls desensitization, was mutated in the GluA2 (GluR2) receptor subunit (GluA2L483Y). This mutation was homozygous lethal. However, mice carrying a single mutated allele, GluA2L483Y/wt, survived past birth, but displayed severe and progressive neurological deficits including seizures and, ultimately, increased mortality. The expression of the AMPA receptor subunits GluA1 and GluA2 was decreased, whereas NMDA receptor protein expression was increased in GluA2L483Y/wt mice. Despite this, basal synaptic transmission and plasticity in the hippocampus were largely unaffected, suggesting that neurons preferentially target receptors to synapses to normalize synaptic weight. We found no gross neuroanatomical alterations in GluA2L483Y/wt mice. Moreover, there was no accumulation of AMPA receptor subunits in intracellular compartments, suggesting that folding and assembly of AMPA receptors are not affected by this mutation. Interestingly, EPSC paired pulse ratios in the CA1 were enhanced without a change in synaptic release probability, demonstrating that postsynaptic receptor properties can contribute to facilitation. The dramatic phenotype observed in this study by the introduction of a single amino acid change demonstrates an essential role in vivo for AMPA receptor desensitization. PMID:20439731

Genetics Home Reference: platyspondylic lethal skeletal dysplasia, Torrance type

MedlinePlus

... type of collagen in the body. Instead of forming collagen molecules, the abnormal COL2A1 protein builds up ... Y, Nagai T, Yamaguchi T, Kosaki R, Ohashi H, Makita Y, Ikegawa S. Identification of COL2A1 mutations in ...

The Heterozygous Disproportionate Micromelia (Dmm) Mouse: Morphological Changes in Fetal Cartilage Precede Postnatal Dwarfism and Compared With Lethal Homozygotes Can Explain the Mild Phenotype

PubMed Central

Seegmiller, Robert E.; Bomsta, Brandon D.; Bridgewater, Laura C.; Niederhauser, Cindy M.; Montaño, Carolina; Sudweeks, Sterling; Eyre, David R.; Fernandes, Russell J.

2008-01-01

The disproportionate micromelia (Dmm) mouse has a mutation in the C-propeptide coding region of the Col2a1 gene that causes lethal dwarfism when homozygous (Dmm/Dmm) but causes only mild dwarfism observable ∼1-week postpartum when heterozygous (Dmm/+). The purpose of this study was 2-fold: first, to analyze and quantify morphological changes that precede the expression of mild dwarfism in Dmm/+ animals, and second, to compare morphological alterations between Dmm/+ and Dmm/Dmm fetal cartilage that may correlate with the marked skeletal differences between mild and lethal dwarfism. Light and electron transmission microscopy were used to visualize structure of chondrocytes and extracellular matrix (ECM) of fetal rib cartilage. Both Dmm/+ and Dmm/Dmm fetal rib cartilage had significantly larger chondrocytes, greater cell density, and less ECM per unit area than +/+ littermates. Quantitative RT-PCR showed a decrease in aggrecan mRNA in Dmm/+ vs +/+ cartilage. Furthermore, the cytoplasm of chondrocytes in Dmm/+ and Dmm/Dmm cartilage was occupied by significantly more distended rough endoplasmic reticulum (RER) compared with wild-type chondrocytes. Fibril diameters and packing densities of +/+ and Dmm/+ cartilage were similar, but Dmm/Dmm cartilage showed thinner, sparsely distributed fibrils. These findings support the prevailing hypothesis that a C-propeptide mutation could interrupt the normal assembly and secretion of Type II procollagen trimers, resulting in a buildup of proα1(II) chains in the RER and a reduced rate of matrix synthesis. Thus, intracellular entrapment of proα1(II) seems to be primarily responsible for the dominant-negative effect of the Dmm mutation in the expression of dwarfism. (J Histochem Cytochem 56:1003–1011, 2008) PMID:18678883

The heterozygous disproportionate micromelia (dmm) mouse: morphological changes in fetal cartilage precede postnatal dwarfism and compared with lethal homozygotes can explain the mild phenotype.

PubMed

Seegmiller, Robert E; Bomsta, Brandon D; Bridgewater, Laura C; Niederhauser, Cindy M; Montaño, Carolina; Sudweeks, Sterling; Eyre, David R; Fernandes, Russell J

2008-11-01

The disproportionate micromelia (Dmm) mouse has a mutation in the C-propeptide coding region of the Col2a1 gene that causes lethal dwarfism when homozygous (Dmm/Dmm) but causes only mild dwarfism observable approximately 1-week postpartum when heterozygous (Dmm/+). The purpose of this study was 2-fold: first, to analyze and quantify morphological changes that precede the expression of mild dwarfism in Dmm/+ animals, and second, to compare morphological alterations between Dmm/+ and Dmm/Dmm fetal cartilage that may correlate with the marked skeletal differences between mild and lethal dwarfism. Light and electron transmission microscopy were used to visualize structure of chondrocytes and extracellular matrix (ECM) of fetal rib cartilage. Both Dmm/+ and Dmm/Dmm fetal rib cartilage had significantly larger chondrocytes, greater cell density, and less ECM per unit area than +/+ littermates. Quantitative RT-PCR showed a decrease in aggrecan mRNA in Dmm/+ vs +/+ cartilage. Furthermore, the cytoplasm of chondrocytes in Dmm/+ and Dmm/Dmm cartilage was occupied by significantly more distended rough endoplasmic reticulum (RER) compared with wild-type chondrocytes. Fibril diameters and packing densities of +/+ and Dmm/+ cartilage were similar, but Dmm/Dmm cartilage showed thinner, sparsely distributed fibrils. These findings support the prevailing hypothesis that a C-propeptide mutation could interrupt the normal assembly and secretion of Type II procollagen trimers, resulting in a buildup of proalpha1(II) chains in the RER and a reduced rate of matrix synthesis. Thus, intracellular entrapment of proalpha1(II) seems to be primarily responsible for the dominant-negative effect of the Dmm mutation in the expression of dwarfism.

A pharmacological screen for compounds that rescue the developmental lethality of a Drosophila ATM mutant.

PubMed

Rimkus, Stacey A; Wassarman, David A

2018-01-01

Ataxia-telangiectasia (A-T) is a neurodegenerative disease caused by mutation of the A-T mutated (ATM) gene. ATM encodes a protein kinase that is activated by DNA damage and phosphorylates many proteins, including those involved in DNA repair, cell cycle control, and apoptosis. Characteristic biological and molecular functions of ATM observed in mammals are conserved in Drosophila melanogaster. As an example, conditional loss-of-function ATM alleles in flies cause progressive neurodegeneration through activation of the innate immune response. However, unlike in mammals, null alleles of ATM in flies cause lethality during development. With the goals of understanding biological and molecular roles of ATM in a whole animal and identifying candidate therapeutics for A-T, we performed a screen of 2400 compounds, including FDA-approved drugs, natural products, and bioactive compounds, for modifiers of the developmental lethality caused by a temperature-sensitive ATM allele (ATM8) that has reduced kinase activity at non-permissive temperatures. Ten compounds reproducibly suppressed the developmental lethality of ATM8 flies, including Ronnel, which is an organophosphate. Ronnel and other suppressor compounds are known to cause mitochondrial dysfunction or to inhibit the enzyme acetylcholinesterase, which controls the levels of the neurotransmitter acetylcholine, suggesting that detrimental consequences of reduced ATM kinase activity can be rescued by inhibiting the function of mitochondria or increasing acetylcholine levels. We carried out further studies of Ronnel because, unlike the other compounds that suppressed the developmental lethality of homozygous ATM8 flies, Ronnel was toxic to the development of heterozygous ATM8 flies. Ronnel did not affect the innate immune response of ATM8 flies, and it further increased the already high levels of DNA damage in brains of ATM8 flies, but its effects were not harmful to the lifespan of rescued ATM8 flies. These results provide new leads for understanding the biological and molecular roles of ATM and for the treatment of A-T.

Functional Analysis of Variants of Unknown Significance in BRCA1 and BRCA2 Using Complementation of a Synthetic Lethal Interaction with PARP Inhibition

DTIC Science & Technology

2014-12-01

general population3-5. A pathogenic mutation in BRCA1 or BRCA2 is an important genetic biomarker for a high ovarian cancer risk in breast cancer patients...doxycycline induces cytological signs of synthetic lethality with Parp inhibitor by RAD51 and RH2AX focus formation. REPORTABLE OUTCOMES None RH2AX... genetics . Mar 2001;68(3):700-710. 3. Chen S, Parmigiani G. Meta-analysis of BRCA1 and BRCA2 penetrance. Journal of clinical oncology : official journal of

AP2 hemicomplexes contribute independently to synaptic vesicle endocytosis

PubMed Central

Gu, Mingyu; Liu, Qiang; Watanabe, Shigeki; Sun, Lin; Hollopeter, Gunther; Grant, Barth D; Jorgensen, Erik M

2013-01-01

The clathrin adaptor complex AP2 is thought to be an obligate heterotetramer. We identify null mutations in the α subunit of AP2 in the nematode Caenorhabditis elegans. α-adaptin mutants are viable and the remaining μ2/β hemicomplex retains some function. Conversely, in μ2 mutants, the alpha/sigma2 hemicomplex is localized and is partially functional. α-μ2 double mutants disrupt both halves of the complex and are lethal. The lethality can be rescued by expression of AP2 components in the skin, which allowed us to evaluate the requirement for AP2 subunits at synapses. Mutations in either α or μ2 subunits alone reduce the number of synaptic vesicles by about 30%; however, simultaneous loss of both α and μ2 subunits leads to a 70% reduction in synaptic vesicles and the presence of large vacuoles. These data suggest that AP2 may function as two partially independent hemicomplexes. DOI: http://dx.doi.org/10.7554/eLife.00190.001 PMID:23482940

Effect of different laser irradiation on the dysentery bacilli

NASA Astrophysics Data System (ADS)

Ou, Lin; Chen, Rong; Chen, Yanjiao; Li, Depin; Wen, Caixia

1998-08-01

The S. flexnesi, which have high drug-resistance especially in Cm, Sm, Tc, SD, were irradiated by Ar+ laser at 488 nm and semiconductor laser at 808 nm. The experiment results have shown that both Ar+ laser and semiconductor laser with power density of 1.7 w/cm2 and irradiation dose of 2000 J/cm2 can conduce to the bacterial lethality and increase the mutation rates of the bacterial drug-sensitivity, and 'Colony Count' method have the superiority over the 'Inhibacteria Ring' method. At the mean time it further indicate that the high power semiconductor laser would play an important role in the sciences of laser biological medicine. But the effect of the near infrared semiconductor laser is far lower than that of Ar+ laser of shorter wavelength at the same irradiation dose. It is clear that the output and irradiation dose of near infrared semiconductor laser shall be increased in order to get the same rates of the bacterial lethality and the drug-sensitivity mutation as Ar+ laser's.

WT1: a weak spot in KRAS-induced transformation

PubMed Central

Licciulli, Silvia; Kissil, Joseph L.

2010-01-01

Activating mutations in the Ras alleles are found frequently in tumors, making the proteins they encode highly attractive candidate therapeutic targets. However, Ras proteins have proven difficult to target directly. Recent approaches have therefore focused on identifying indirect targets to inhibit Ras-induced oncogenesis. For example, RNAi-based negative selection screens to identify genes that when silenced in concert with activating Ras mutations are incompatible with cellular proliferation, a concept known as synthetic lethality. In this issue of the JCI, Vicent et al. report on the identification of Wilms tumor 1 (Wt1) as a Kras synthetic-lethal gene in a mouse model of lung adenocarcinoma. Silencing of Wt1 in cells expressing an endogenous allele of activated Kras triggers senescence in vitro and has an impact on tumor progression in vivo. These findings are of significant interest given previous studies suggesting that the ability of oncogenic Kras to induce senescence versus proliferation depends on its levels of expression. PMID:20972324

[Hygienic evaluation of the total mutagenic activity of snow samples from Magnitogorsk].

PubMed

Legostaeva, T B; Ingel', F I; Antipanova, N A; Iurchenko, V V; Iuretseva, N A; Kotliar, N N

2010-01-01

The paper gives the results of 4-year monitoring of the total mutagenic activity of snow samples from different Magnitogork areas in a test for induction of dominant lethal mutations (DLM) in the gametes of Drosophila melanogaster. An association was first found between the rate of DLM and the content of some chemical compounds in the ambient air and snow samples; moreover all the substances present in the samples, which had found genotoxic effects, showed a positive correlation with the rate of DLM. Furthermore, direct correlations were first established between the rate of DLM and the air pollution index and morbidity rates in 5-7-year-old children residing in the areas under study. The findings allow the test for induction of dominant lethal mutations (DLM) in the gametes of Drosophila melanogaster to be recommended due to its unique informative and prognostic value for monitoring ambient air pollution and for extensive use in the risk assessment system.

CRISPR/Cas9 mediated genome editing in ES cells and its application for chimeric analysis in mice.

PubMed

Oji, Asami; Noda, Taichi; Fujihara, Yoshitaka; Miyata, Haruhiko; Kim, Yeon Joo; Muto, Masanaga; Nozawa, Kaori; Matsumura, Takafumi; Isotani, Ayako; Ikawa, Masahito

2016-08-17

Targeted gene disrupted mice can be efficiently generated by expressing a single guide RNA (sgRNA)/CAS9 complex in the zygote. However, the limited success of complicated genome editing, such as large deletions, point mutations, and knockins, remains to be improved. Further, the mosaicism in founder generations complicates the genotypic and phenotypic analyses in these animals. Here we show that large deletions with two sgRNAs as well as dsDNA-mediated point mutations are efficient in mouse embryonic stem cells (ESCs). The dsDNA-mediated gene knockins are also feasible in ESCs. Finally, we generated chimeric mice with biallelic mutant ESCs for a lethal gene, Dnajb13, and analyzed their phenotypes. Not only was the lethal phenotype of hydrocephalus suppressed, but we also found that Dnajb13 is required for sperm cilia formation. The combination of biallelic genome editing in ESCs and subsequent chimeric analysis provides a useful tool for rapid gene function analysis in the whole organism.

Validation of Deleterious Mutations in Vorderwald Cattle

PubMed Central

Reinartz, Sina; Distl, Ottmar

2016-01-01

In Montbéliarde cattle two candidate mutations on bovine chromosomes 19 and 29 responsible for embryonic lethality have been detected. Montbéliarde bulls have been introduced into Vorderwald cattle to improve milk and fattening performance. Due to the small population size of Vorderwald cattle and the wide use of a few Montbéliarde bulls through artificial insemination, inbreeding on Montbéliarde bulls in later generations was increasing. Therefore, we genotyped an aborted fetus which was inbred on Montbéliarde as well as Vorderwald x Montbéliarde crossbred bulls for both deleterious mutations. The abortion was observed in an experimental herd of Vorderwald cattle. The objectives of the present study were to prove if one or both lethal mutations may be assumed to have caused this abortion and to show whether these deleterious mutations have been introduced into the Vorderwald cattle population through Montbéliarde bulls. The aborted fetus was homozygous for the SLC37A2:g.28879810C>T mutation (ss2019324563) on BTA29 and both parents as well as the paternal and maternal grandsire were heterozygous for this mutation. In addition, the parents and the paternal grandsire were carriers of the MH2-haplotype linked with the T-allele of the SLC37A2:g.28879810C>T mutation. For the SHBG:g.27956790C>T mutation (rs38377500) on BTA19 (MH1), the aborted fetus and its sire were heterozygous. Among all further 341 Vorderwald cattle genotyped we found 27 SLC37A2:g.28879810C>T heterozygous animals resulting in an allele frequency of 0.0396. Among the 120 male Vorderwald cattle, there were 12 heterozygous with an allele frequency of 0.05. The SLC37A2:g.28879810C>T mutation could not be found in further nine cattle breeds nor in Vorderwald cattle with contributions from Ayrshire bulls. In 69 Vorderwald cattle without genes from Montbéliarde bulls the mutated allele of SLC37A2:g.28879810C>T could not be detected. The SHBG:g.27956790C>T mutation appeared unlikely to be responsible for the present case of abortion and, in addition, we observed this mutation in a homozygous state in a living animal. In conclusion, we could demonstrate the first case of an aborted fetus carrying the deleterious SLC37A2:g.28879810C>T mutation homozygous and show that this deleterious mutation had been introduced through Montbéliarde bulls into Vorderwald cattle. PMID:27472836

Adaptive tuning of mutation rates allows fast response to lethal stress in Escherichia coli

PubMed Central

Swings, Toon; Van den Bergh, Bram; Wuyts, Sander; Oeyen, Eline; Voordeckers, Karin; Verstrepen, Kevin J; Fauvart, Maarten; Verstraeten, Natalie; Michiels, Jan

2017-01-01

While specific mutations allow organisms to adapt to stressful environments, most changes in an organism's DNA negatively impact fitness. The mutation rate is therefore strictly regulated and often considered a slowly-evolving parameter. In contrast, we demonstrate an unexpected flexibility in cellular mutation rates as a response to changes in selective pressure. We show that hypermutation independently evolves when different Escherichia coli cultures adapt to high ethanol stress. Furthermore, hypermutator states are transitory and repeatedly alternate with decreases in mutation rate. Specifically, population mutation rates rise when cells experience higher stress and decline again once cells are adapted. Interestingly, we identified cellular mortality as the major force driving the quick evolution of mutation rates. Together, these findings show how organisms balance robustness and evolvability and help explain the prevalence of hypermutation in various settings, ranging from emergence of antibiotic resistance in microbes to cancer relapses upon chemotherapy. DOI: http://dx.doi.org/10.7554/eLife.22939.001 PMID:28460660

Tissue-specific mosaicism for a lethal osteogenesis imperfecta COL1A1 mutation causes mild OI/EDS overlap syndrome.

PubMed

Symoens, Sofie; Steyaert, Wouter; Demuynck, Lynn; De Paepe, Anne; Diderich, Karin E M; Malfait, Fransiska; Coucke, Paul J

2017-04-01

Type I collagen is the predominant protein of connective tissues such as skin and bone. Mutations in the type I collagen genes (COL1A1 and COL1A2) mainly cause osteogenesis imperfecta (OI). We describe a patient with clinical signs of Ehlers-Danlos syndrome (EDS), including fragile skin, easy bruising, recurrent luxations, and fractures resembling mild OI. Biochemical collagen analysis of the patients' dermal fibroblasts showed faint overmodification of the type I collagen bands, a finding specific for structural defects in type I collagen. Bidirectional Sanger sequencing detected an in-frame deletion in exon 44 of COL1A1 (c.3150_3158del), resulting in the deletion of three amino acids (p.Ala1053_Gly1055del) in the collagen triple helix. This COL1A1 mutation was hitherto identified in four probands with lethal OI, and never in EDS patients. As the peaks on the electropherogram corresponding to the mutant allele were decreased in intensity, we performed next generation sequencing of COL1A1 to study mosaicism in skin and blood. While approximately 9% of the reads originating from fibroblast gDNA harbored the COL1A1 deletion, the deletion was not detected in gDNA from blood. Most likely, the mild clinical symptoms observed in our patient can be explained by the mosaic state of the mutation. © 2017 Wiley Periodicals, Inc.

Mutations in FLNB cause boomerang dysplasia

PubMed Central

Bicknell, L; Morgan, T; Bonafe, L; Wessels, M; Bialer, M; Willems, P; Cohn, D; Krakow, D; Robertson, S

2005-01-01

Boomerang dysplasia (BD) is a perinatal lethal osteochondrodysplasia, characterised by absence or underossification of the limb bones and vertebrae. The BD phenotype is similar to a group of disorders including atelosteogenesis I, atelosteogenesis III, and dominantly inherited Larsen syndrome that we have recently shown to be associated with mutations in FLNB, the gene encoding the actin binding cytoskeletal protein, filamin B. We report the identification of mutations in FLNB in two unrelated individuals with boomerang dysplasia. The resultant substitutions, L171R and S235P, lie within the calponin homology 2 region of the actin binding domain of filamin B and occur at sites that are evolutionarily well conserved. These findings expand the phenotypic spectrum resulting from mutations in FLNB and underline the central role this protein plays during skeletogenesis in humans. PMID:15994868

Mutations in FLNB cause boomerang dysplasia.

PubMed

Bicknell, L S; Morgan, T; Bonafé, L; Wessels, M W; Bialer, M G; Willems, P J; Cohn, D H; Krakow, D; Robertson, S P

2005-07-01

Boomerang dysplasia (BD) is a perinatal lethal osteochondrodysplasia, characterised by absence or underossification of the limb bones and vertebrae. The BD phenotype is similar to a group of disorders including atelosteogenesis I, atelosteogenesis III, and dominantly inherited Larsen syndrome that we have recently shown to be associated with mutations in FLNB, the gene encoding the actin binding cytoskeletal protein, filamin B. We report the identification of mutations in FLNB in two unrelated individuals with boomerang dysplasia. The resultant substitutions, L171R and S235P, lie within the calponin homology 2 region of the actin binding domain of filamin B and occur at sites that are evolutionarily well conserved. These findings expand the phenotypic spectrum resulting from mutations in FLNB and underline the central role this protein plays during skeletogenesis in humans.

Isolation and Genetic Characterization of a Mutation Affecting Ribosomal Resistance to Cycloheximide in Tetrahymena

PubMed Central

Ares, Manuel; Bruns, Peter J.

1978-01-01

A dominant mutation at a new locus affecting resistance to cycloheximide has been isolated by exploiting a synergistic relationship with a previously known mutation for cycloheximide resistance in Tetrahymena. The new mutation (ChxB) was induced in a line homozygous for ChxA and was recovered from that background by a new technique termed interrupted genomic exclusion. Segregation data from the interrupted genomic exclusion suggest that ChxA and ChxB are separate, linked loci showing 30% recombination. Minimal lethal doses of cycloheximide for the four possible combinations of the wild-type and mutant alleles of these two genes are: wild type 6 µg/ml, ChxA 125 µg/ml, ChxB 10 µg/ml, ChxA-ChxB 175 µg/ml. PMID:730051

Coffee mitigates cyclophosphamide-induced genotoxic damage in Drosophila melanogaster germ cells.

PubMed

Nagpal, Isha; Abraham, Suresh K

2018-02-26

In the present study, coffee (CF) was evaluated for its protective effects against genotoxic damage and oxidative stress induced by the chemotherapeutic drug, cyclophosphamide (CPH). The sex-linked recessive lethal (SLRL) test was employed to study the induction of mutations in the larvae as well as in all the successive germ cell stages of treated males. Control and treated third instar larvae were used to monitor the biomarkers of oxidative stress response such as glutathione content (GSH), glutathione-S-transferase (GST), catalase (CAT), superoxide dismutase (SOD) and lipid peroxidation (MDA content). Our results demonstrated that co-administration of CF (2%) with CPH (3 mM) has significantly reduced CPH-induced lethal mutations in the germ cells of larvae and adult flies. The reductions observed in mutation frequencies were: 75% in larvae and 62.4% in the adult. Significant enhancement in antioxidant enzymatic levels: CAT (46.6%) > SOD (43.0%) > GST (42.4%) > GSH (31.6%) and reduction in MDA levels (32.05%) in the pretreated third instar larvae demonstrated the antioxidant activity of CF against CPH-induced oxidative stress. The findings from the present study suggest that the Drosophila model is an ideal one for evaluating the antigenotoxic and antioxidant activity of complex mixtures like CF.

De Novo Emergence of Genetically Resistant Mutants of Mycobacterium tuberculosis from the Persistence Phase Cells Formed against Antituberculosis Drugs In Vitro

PubMed Central

Sebastian, Jees; Swaminath, Sharmada; Nair, Rashmi Ravindran; Jakkala, Kishor; Pradhan, Atul

2016-01-01

ABSTRACT Bacterial persisters are a subpopulation of cells that can tolerate lethal concentrations of antibiotics. However, the possibility of the emergence of genetically resistant mutants from antibiotic persister cell populations, upon continued exposure to lethal concentrations of antibiotics, remained unexplored. In the present study, we found that Mycobacterium tuberculosis cells exposed continuously to lethal concentrations of rifampin (RIF) or moxifloxacin (MXF) for prolonged durations showed killing, RIF/MXF persistence, and regrowth phases. RIF-resistant or MXF-resistant mutants carrying clinically relevant mutations in the rpoB or gyrA gene, respectively, were found to emerge at high frequency from the RIF persistence phase population. A Luria-Delbruck fluctuation experiment using RIF-exposed M. tuberculosis cells showed that the rpoB mutants were not preexistent in the population but were formed de novo from the RIF persistence phase population. The RIF persistence phase M. tuberculosis cells carried elevated levels of hydroxyl radical that inflicted extensive genome-wide mutations, generating RIF-resistant mutants. Consistent with the elevated levels of hydroxyl radical-mediated genome-wide random mutagenesis, MXF-resistant M. tuberculosis gyrA de novo mutants could be selected from the RIF persistence phase cells. Thus, unlike previous studies, which showed emergence of genetically resistant mutants upon exposure of bacteria for short durations to sublethal concentrations of antibiotics, our study demonstrates that continuous prolonged exposure of M. tuberculosis cells to lethal concentrations of an antibiotic generates antibiotic persistence phase cells that form a reservoir for the generation of genetically resistant mutants to the same antibiotic or another antibiotic. These findings may have clinical significance in the emergence of drug-resistant tubercle bacilli. PMID:27895008

Centromere replication timing determines different forms of genomic instability in Saccharomyces cerevisiae checkpoint mutants during replication stress.

PubMed

Feng, Wenyi; Bachant, Jeff; Collingwood, David; Raghuraman, M K; Brewer, Bonita J

2009-12-01

Yeast replication checkpoint mutants lose viability following transient exposure to hydroxyurea, a replication-impeding drug. In an effort to understand the basis for this lethality, we discovered that different events are responsible for inviability in checkpoint-deficient cells harboring mutations in the mec1 and rad53 genes. By monitoring genomewide replication dynamics of cells exposed to hydroxyurea, we show that cells with a checkpoint deficient allele of RAD53, rad53K227A, fail to duplicate centromeres. Following removal of the drug, however, rad53K227A cells recover substantial DNA replication, including replication through centromeres. Despite this recovery, the rad53K227A mutant fails to achieve biorientation of sister centromeres during recovery from hydroxyurea, leading to secondary activation of the spindle assembly checkpoint (SAC), aneuploidy, and lethal chromosome segregation errors. We demonstrate that cell lethality from this segregation defect could be partially remedied by reinforcing bipolar attachment. In contrast, cells with the mec1-1 sml1-1 mutations suffer from severely impaired replication resumption upon removal of hydroxyurea. mec1-1 sml1-1 cells can, however, duplicate at least some of their centromeres and achieve bipolar attachment, leading to abortive segregation and fragmentation of incompletely replicated chromosomes. Our results highlight the importance of replicating yeast centromeres early and reveal different mechanisms of cell death due to differences in replication fork progression.

Monocarboxylate transporter 1 deficiency and ketone utilization.

PubMed

van Hasselt, Peter M; Ferdinandusse, Sacha; Monroe, Glen R; Ruiter, Jos P N; Turkenburg, Marjolein; Geerlings, Maartje J; Duran, Karen; Harakalova, Magdalena; van der Zwaag, Bert; Monavari, Ardeshir A; Okur, Ilyas; Sharrard, Mark J; Cleary, Maureen; O'Connell, Nuala; Walker, Valerie; Rubio-Gozalbo, M Estela; de Vries, Maaike C; Visser, Gepke; Houwen, Roderick H J; van der Smagt, Jasper J; Verhoeven-Duif, Nanda M; Wanders, Ronald J A; van Haaften, Gijs

2014-11-13

Ketoacidosis is a potentially lethal condition caused by the imbalance between hepatic production and extrahepatic utilization of ketone bodies. We performed exome sequencing in a patient with recurrent, severe ketoacidosis and identified a homozygous frameshift mutation in the gene encoding monocarboxylate transporter 1 (SLC16A1, also called MCT1). Genetic analysis in 96 patients suspected of having ketolytic defects yielded seven additional inactivating mutations in MCT1, both homozygous and heterozygous. Mutational status was found to be correlated with ketoacidosis severity, MCT1 protein levels, and transport capacity. Thus, MCT1 deficiency is a novel cause of profound ketoacidosis; the present work suggests that MCT1-mediated ketone-body transport is needed to maintain acid-base balance.

The Essential Gene EMB1611 Maintains Shoot Apical Meristem Function During Arabidopsis Development

USDA-ARS?s Scientific Manuscript database

The Arabidopsis thaliana genome contains hundreds of genes essential for seed development. Because null mutations in these genes cause embryo lethality, their specific molecular and developmental functions are largely unknown. Here, we identify a role for EMB1611/MEE22, an essential gene in Arabidop...

Mechanism of Ovarian Epithelial Tumor Predispostion in Individuals Carrying Germline BRCA1 Mutations

DTIC Science & Technology

2005-01-01

corresponds to the luteal phase. Vaginal smears obtained at the diestrus phase show primarily inflammatory cells. Immature (green) epithelial cells start...Koller, B.H. (1996). BRCA1 deficiency results in early embryonic lethality characterized by neuroepithelial abnormalities. Nat. Genet. 12, 191-194. 22

Accumulation of Spontaneous Mutations in the Ciliate Tetrahymena thermophila

PubMed Central

Long, Hong-An; Paixão, Tiago; Azevedo, Ricardo B. R.; Zufall, Rebecca A.

2013-01-01

Knowledge of the rate and fitness effects of mutations is essential for understanding the process of evolution. Mutations are inherently difficult to study because they are rare and are frequently eliminated by natural selection. In the ciliate Tetrahymena thermophila, mutations can accumulate in the germline genome without being exposed to selection. We have conducted a mutation accumulation (MA) experiment in this species. Assuming that all mutations are deleterious and have the same effect, we estimate that the deleterious mutation rate per haploid germline genome per generation is U = 0.0047 (95% credible interval: 0.0015, 0.0125), and that germline mutations decrease fitness by s = 11% when expressed in a homozygous state (95% CI: 4.4%, 27%). We also estimate that deleterious mutations are partially recessive on average (h = 0.26; 95% CI: –0.022, 0.62) and that the rate of lethal mutations is <10% of the deleterious mutation rate. Comparisons between the observed evolutionary responses in the germline and somatic genomes and the results from individual-based simulations of MA suggest that the two genomes have similar mutational parameters. These are the first estimates of the deleterious mutation rate and fitness effects from the eukaryotic supergroup Chromalveolata and are within the range of those of other eukaryotes. PMID:23934880

Interactive lethal and mutagenic effects of ultraviolet light and bleomycin in yeast: synergism or antagonism?

PubMed

Lillo, O L; Severgnini, A A; Nunes, E M

1997-11-01

The mutagenic interactions of ultraviolet light and bleomycin in haploid populations of Saccharomyces cerevisiae were analyzed. Survival and mutation frequency as a function of different bleomycin concentrations after one conditioning dose of UV radiation were determined. Furthermore, corresponding interaction functions and sensitization factors were calculated. A synergistic interaction between UV light and bleomycin was shown for both lethal and mutagenic events when the cells were in nutrient broth during the treatments. Conversely, the interaction between UV light and bleomycin was antagonistic when the cells were in deionized water during the treatment. The magnitude of lethal and mutagenic interactions depends on dose, and thus presumably on the number of lesions. The observed interactions between UV light and bleomycin suggest that the mechanism that is most likely involved is the induction of repair systems with different error probabilities during the delay of cell division.

NDST1 missense mutations in autosomal recessive intellectual disability.

PubMed

Reuter, Miriam S; Musante, Luciana; Hu, Hao; Diederich, Stefan; Sticht, Heinrich; Ekici, Arif B; Uebe, Steffen; Wienker, Thomas F; Bartsch, Oliver; Zechner, Ulrich; Oppitz, Cornelia; Keleman, Krystyna; Jamra, Rami Abou; Najmabadi, Hossein; Schweiger, Susann; Reis, André; Kahrizi, Kimia

2014-11-01

NDST1 was recently proposed as a candidate gene for autosomal recessive intellectual disability in two families. It encodes a bifunctional GlcNAc N-deacetylase/N-sulfotransferase with important functions in heparan sulfate biosynthesis. In mice, Ndst1 is crucial for embryonic development and homozygous null mutations are perinatally lethal. We now report on two additional unrelated families with homozygous missense NDST1 mutations. All mutations described to date predict the substitution of conserved amino acids in the sulfotransferase domain, and mutation modeling predicts drastic alterations in the local protein conformation. Comparing the four families, we noticed significant overlap in the clinical features, including both demonstrated and apparent intellectual disability, muscular hypotonia, epilepsy, and postnatal growth deficiency. Furthermore, in Drosophila, knockdown of sulfateless, the NDST ortholog, impairs long-term memory, highlighting its function in cognition. Our data confirm NDST1 mutations as a cause of autosomal recessive intellectual disability with a distinctive phenotype, and support an important function of NDST1 in human development. © 2014 Wiley Periodicals, Inc.

Detection of Haplotypes Associated with Prenatal Death in Dairy Cattle and Identification of Deleterious Mutations in GART, SHBG and SLC37A2

PubMed Central

Fritz, Sébastien; Capitan, Aurelien; Djari, Anis; Rodriguez, Sabrina C.; Barbat, Anne; Baur, Aurélia; Grohs, Cécile; Weiss, Bernard; Boussaha, Mekki; Esquerré, Diane; Klopp, Christophe; Rocha, Dominique; Boichard, Didier

2013-01-01

The regular decrease of female fertility over time is a major concern in modern dairy cattle industry. Only half of this decrease is explained by indirect response to selection on milk production, suggesting the existence of other factors such as embryonic lethal genetic defects. Genomic regions harboring recessive deleterious mutations were detected in three dairy cattle breeds by identifying frequent haplotypes (>1%) showing a deficit in homozygotes among Illumina Bovine 50k Beadchip haplotyping data from the French genomic selection database (47,878 Holstein, 16,833 Montbéliarde, and 11,466 Normande animals). Thirty-four candidate haplotypes (p<10−4) including previously reported regions associated with Brachyspina, CVM, HH1, and HH3 in Holstein breed were identified. Haplotype length varied from 1 to 4.8 Mb and frequencies from 1.7 up to 9%. A significant negative effect on calving rate, consistent in heifers and in lactating cows, was observed for 9 of these haplotypes in matings between carrier bulls and daughters of carrier sires, confirming their association with embryonic lethal mutations. Eight regions were further investigated using whole genome sequencing data from heterozygous bull carriers and control animals (45 animals in total). Six strong candidate causative mutations including polymorphisms previously reported in FANCI (Brachyspina), SLC35A3 (CVM), APAF1 (HH1) and three novel mutations with very damaging effect on the protein structure, according to SIFT and Polyphen-2, were detected in GART, SHBG and SLC37A2 genes. In conclusion, this study reveals a yet hidden consequence of the important inbreeding rate observed in intensively selected and specialized cattle breeds. Counter-selection of these mutations and management of matings will have positive consequences on female fertility in dairy cattle. PMID:23762392

Control of cellular morphogenesis by the Ip12/Bem2 GTPase-activating protein: possible role of protein phosphorylation

PubMed Central

1994-01-01

The IPL2 gene is known to be required for normal polarized cell growth in the budding yeast Saccharomyces cerevisiae. We now show that IPL2 is identical to the previously identified BEM2 gene. bem2 mutants are defective in bud site selection at 26 degrees C and localized cell surface growth and organization of the actin cytoskeleton at 37 degrees C. BEM2 encodes a protein with a COOH-terminal domain homologous to sequences found in several GTPase-activating proteins, including human Bcr. The GTPase-activating protein-domain from the Bem2 protein (Bem2p) or human Bcr can functionally substitute for Bem2p. The Rho1 and Rho2 GTPases are the likely in vivo targets of Bem2p because bem2 mutant phenotypes can be partially suppressed by increasing the gene dosage of RHO1 or RHO2. CDC55 encodes the putative regulatory B subunit of protein phosphatase 2A, and mutations in BEM2 have previously been identified as suppressors of the cdc55-1 mutation. We show here that mutations in the previously identified GRR1 gene can suppress bem2 mutations. grr1 and cdc55 mutants are both elongated in shape and cold- sensitive for growth, and cells lacking both GRR1 and CDC55 exhibit a synthetic lethal phenotype. bem2 mutant phenotypes also can be suppressed by the SSD1-vl (also known as SRK1) mutation, which was shown previously to suppress mutations in the protein phosphatase- encoding SIT4 gene. Cells lacking both BEM2 and SIT4 exhibit a synthetic lethal phenotype even in the presence of the SSD1-v1 suppressor. These genetic interactions together suggest that protein phosphorylation and dephosphorylation play an important role in the BEM2-mediated process of polarized cell growth. PMID:7962097

Control of cellular morphogenesis by the Ip12/Bem2 GTPase-activating protein: possible role of protein phosphorylation.

PubMed

Kim, Y J; Francisco, L; Chen, G C; Marcotte, E; Chan, C S

1994-12-01

The IPL2 gene is known to be required for normal polarized cell growth in the budding yeast Saccharomyces cerevisiae. We now show that IPL2 is identical to the previously identified BEM2 gene. bem2 mutants are defective in bud site selection at 26 degrees C and localized cell surface growth and organization of the actin cytoskeleton at 37 degrees C. BEM2 encodes a protein with a COOH-terminal domain homologous to sequences found in several GTPase-activating proteins, including human Bcr. The GTPase-activating protein-domain from the Bem2 protein (Bem2p) or human Bcr can functionally substitute for Bem2p. The Rho1 and Rho2 GTPases are the likely in vivo targets of Bem2p because bem2 mutant phenotypes can be partially suppressed by increasing the gene dosage of RHO1 or RHO2. CDC55 encodes the putative regulatory B subunit of protein phosphatase 2A, and mutations in BEM2 have previously been identified as suppressors of the cdc55-1 mutation. We show here that mutations in the previously identified GRR1 gene can suppress bem2 mutations. grr1 and cdc55 mutants are both elongated in shape and cold-sensitive for growth, and cells lacking both GRR1 and CDC55 exhibit a synthetic lethal phenotype. bem2 mutant phenotypes also can be suppressed by the SSD1-vl (also known as SRK1) mutation, which was shown previously to suppress mutations in the protein phosphatase-encoding SIT4 gene. Cells lacking both BEM2 and SIT4 exhibit a synthetic lethal phenotype even in the presence of the SSD1-v1 suppressor. These genetic interactions together suggest that protein phosphorylation and dephosphorylation play an important role in the BEM2-mediated process of polarized cell growth.

Spontaneous mutations in Streptococcus pyogenes isolates from streptococcal toxic shock syndrome patients play roles in virulence

PubMed Central

Ikebe, Tadayoshi; Matsumura, Takayuki; Nihonmatsu, Hisako; Ohya, Hitomi; Okuno, Rumi; Mitsui, Chieko; Kawahara, Ryuji; Kameyama, Mitsuhiro; Sasaki, Mari; Shimada, Naomi; Ato, Manabu; Ohnishi, Makoto

2016-01-01

Streptococcus pyogenes (group A Streptococcus; GAS) is a widespread human pathogen and causes streptococcal toxic shock syndrome (STSS). STSS isolates have been previously shown to have high frequency mutations in the csrS/csrR (covS/covR) and/or rgg (ropB) genes, which are negative regulators of virulence. However, these mutations were found at somewhat low frequencies in emm1-genotyped isolates, the most prevalent STSS genotype. In this study, we sought to detect causal mutations of enhanced virulence in emm1 isolates lacking mutation(s) in the csrS/csrR and rgg genes. Three mutations associated with elevated virulence were found in the sic (a virulence gene) promoter, the csrR promoter, and the rocA gene (a csrR positive regulator). In vivo contribution of the sic promoter and rocA mutations to pathogenicity and lethality was confirmed in a GAS mouse model. Frequency of the sic promoter mutation was significantly higher in STSS emm1 isolates than in non-invasive STSS isolates; the rocA gene mutation frequency was not significantly different among STSS and non-STSS isolates. STSS emm1 isolates possessed a high frequency mutation in the sic promoter. Thus, this mutation may play a role in the dynamics of virulence and STSS pathogenesis. PMID:27349341

Spontaneous mutations in Streptococcus pyogenes isolates from streptococcal toxic shock syndrome patients play roles in virulence.

PubMed

Ikebe, Tadayoshi; Matsumura, Takayuki; Nihonmatsu, Hisako; Ohya, Hitomi; Okuno, Rumi; Mitsui, Chieko; Kawahara, Ryuji; Kameyama, Mitsuhiro; Sasaki, Mari; Shimada, Naomi; Ato, Manabu; Ohnishi, Makoto

2016-06-28

Streptococcus pyogenes (group A Streptococcus; GAS) is a widespread human pathogen and causes streptococcal toxic shock syndrome (STSS). STSS isolates have been previously shown to have high frequency mutations in the csrS/csrR (covS/covR) and/or rgg (ropB) genes, which are negative regulators of virulence. However, these mutations were found at somewhat low frequencies in emm1-genotyped isolates, the most prevalent STSS genotype. In this study, we sought to detect causal mutations of enhanced virulence in emm1 isolates lacking mutation(s) in the csrS/csrR and rgg genes. Three mutations associated with elevated virulence were found in the sic (a virulence gene) promoter, the csrR promoter, and the rocA gene (a csrR positive regulator). In vivo contribution of the sic promoter and rocA mutations to pathogenicity and lethality was confirmed in a GAS mouse model. Frequency of the sic promoter mutation was significantly higher in STSS emm1 isolates than in non-invasive STSS isolates; the rocA gene mutation frequency was not significantly different among STSS and non-STSS isolates. STSS emm1 isolates possessed a high frequency mutation in the sic promoter. Thus, this mutation may play a role in the dynamics of virulence and STSS pathogenesis.

Mutations in the Drosophila neuroglian cell adhesion molecule affect motor neuron pathfinding and peripheral nervous system patterning.

PubMed

Hall, S G; Bieber, A J

1997-03-01

We have identified and characterized three embryonic lethal mutations that alter or abolish expression of Drosophila Neuroglian and have used these mutations to analyze Neuroglian function during development. Neuroglian is a member of the immunoglobulin superfamily. It is expressed by a variety of cell types during embryonic development, including expression on motoneurons and the muscle cells that they innervate. Examination of the nervous systems of neuroglian mutant embryos reveals that motoneurons have altered pathfinding trajectories. Additionally, the sensory cell bodies of the peripheral nervous system display altered morphology and patterning. Using a temperature-sensitive mutation, the phenocritical period for Neuroglian function was determined to occur during late embryogenesis, an interval which coincides with the period during which neuromuscular connections and the peripheral nervous system pattern are established.

Analysis of mutagenic DNA repair in a thermoconditional mutant of Saccharomyces cerevisiae. IV. Influence of DNA replication and excision repair on REV2 dependent UV-mutagenesis and repair.

PubMed

Siede, W; Eckardt, F

1986-01-01

A double mutant being thermoconditionally defective in mutation induction as well as in repair of pre-lethal UV-induced DNA damage (rev2ts) and deficient in excision repair (rad3-2) was studied in temperature-shift experiments. The influence of inhibitors of DNA replication (hydroxyurea, aphidicolin) was determined. Additionally, an analysis of the dose-response pattern of mutation induction ("mutation kinetics") at several ochre alleles was carried out. It was concluded that the UV-inducible REV2 dependent mutagenic repair process is not induced in excision-deficient cells. In excision-deficient cells, REV2 dependent mutation fixation is slow and mostly post-replicative though not dependent on DNA replication. The REV2 mediated mutagenic process could be separated from the repair function.

Visualizing the origins of selfish de novo mutations in individual seminiferous tubules of human testes

PubMed Central

Maher, Geoffrey J.; McGowan, Simon J.; Giannoulatou, Eleni; Verrill, Clare; Goriely, Anne; Wilkie, Andrew O. M.

2016-01-01

De novo point mutations arise predominantly in the male germline and increase in frequency with age, but it has not previously been possible to locate specific, identifiable mutations directly within the seminiferous tubules of human testes. Using microdissection of tubules exhibiting altered expression of the spermatogonial markers MAGEA4, FGFR3, and phospho-AKT, whole genome amplification, and DNA sequencing, we establish an in situ strategy for discovery and analysis of pathogenic de novo mutations. In 14 testes from men aged 39–90 y, we identified 11 distinct gain-of-function mutations in five genes (fibroblast growth factor receptors FGFR2 and FGFR3, tyrosine phosphatase PTPN11, and RAS oncogene homologs HRAS and KRAS) from 16 of 22 tubules analyzed; all mutations have known associations with severe diseases, ranging from congenital or perinatal lethal disorders to somatically acquired cancers. These results support proposed selfish selection of spermatogonial mutations affecting growth factor receptor-RAS signaling, highlight its prevalence in older men, and enable direct visualization of the microscopic anatomy of elongated mutant clones. PMID:26858415

Visualizing the origins of selfish de novo mutations in individual seminiferous tubules of human testes.

PubMed

Maher, Geoffrey J; McGowan, Simon J; Giannoulatou, Eleni; Verrill, Clare; Goriely, Anne; Wilkie, Andrew O M

2016-03-01

De novo point mutations arise predominantly in the male germline and increase in frequency with age, but it has not previously been possible to locate specific, identifiable mutations directly within the seminiferous tubules of human testes. Using microdissection of tubules exhibiting altered expression of the spermatogonial markers MAGEA4, FGFR3, and phospho-AKT, whole genome amplification, and DNA sequencing, we establish an in situ strategy for discovery and analysis of pathogenic de novo mutations. In 14 testes from men aged 39-90 y, we identified 11 distinct gain-of-function mutations in five genes (fibroblast growth factor receptors FGFR2 and FGFR3, tyrosine phosphatase PTPN11, and RAS oncogene homologs HRAS and KRAS) from 16 of 22 tubules analyzed; all mutations have known associations with severe diseases, ranging from congenital or perinatal lethal disorders to somatically acquired cancers. These results support proposed selfish selection of spermatogonial mutations affecting growth factor receptor-RAS signaling, highlight its prevalence in older men, and enable direct visualization of the microscopic anatomy of elongated mutant clones.

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.

Raine Syndrome (OMIM #259775), Caused By FAM20C Mutation, Is Congenital Sclerosing Osteomalacia With Cerebral Calcification (OMIM 259660).

PubMed

Whyte, Michael P; McAlister, William H; Fallon, Michael D; Pierpont, Mary Ella; Bijanki, Vinieth N; Duan, Shenghui; Otaify, Ghada A; Sly, William S; Mumm, Steven

2017-04-01

In 1985, we briefly reported infant sisters with a unique, lethal, autosomal recessive disorder designated congenital sclerosing osteomalacia with cerebral calcification. In 1986, this condition was entered into Mendelian Inheritance In Man (MIM) as osteomalacia, sclerosing, with cerebral calcification (MIM 259660). However, no attestations followed. Instead, in 1989 Raine and colleagues published an affected neonate considering unprecedented the striking clinical and radiographic features. In 1992, "Raine syndrome" entered MIM formally as osteosclerotic bone dysplasia, lethal (MIM #259775). In 2007, the etiology emerged as loss-of-function mutation of FAM20C that encodes family with sequence similarity 20, member C. FAM20C is highly expressed in embryonic calcified tissues and encodes a kinase (dentin matrix protein 4) for most of the secreted phosphoproteome including FGF23, osteopontin, and other regulators of skeletal mineralization. Herein, we detail the clinical, radiological, biochemical, histopathological, and FAM20C findings of our patients. Following premortem tetracycline labeling, the proposita's non-decalcified skeletal histopathology after autopsy indicated no rickets but documented severe osteomalacia. Archival DNA revealed the sisters were compound heterozygotes for a unique missense mutation and a novel deletion in FAM20C. Individuals heterozygous for the missense mutation seemed to prematurely fuse their metopic suture and develop a metopic ridge sometimes including trigonocephaly. Our findings clarify FAM20C's role in hard tissue formation and mineralization, and show that Raine syndrome is congenital sclerosing osteomalacia with cerebral calcification. © 2016 American Society for Bone and Mineral Research. © 2016 American Society for Bone and Mineral Research.

Development of a chromosome-plasmid balanced lethal system for Lactobacillus acidophilus with thyA gene as selective marker.

PubMed

Fu, X; Xu, J G

2000-01-01

A chromosome-plasmid balanced lethal gene delivery system for Lactobacillus acidophilus based on the thyA gene was developed. The selected L. acidophilus DOM La strain carries a mutated thyA gene and has an obligate requirement for thymidine. This strain can be used as a host for the constructed shuttle vector pFXL03, lacking antibiotic-resistant markers but having the wild-type thyA gene from L. casei which complements the thyA chromosomal mutation. The vector also contains the replicon region from plasmid pUC19 and that of the Lactococcus plasmid pWV01, which allows the transfer between Escherichia coli, L. casei and L. acidophilus. Eight unique restriction sites (i.e., PstI, HindIII, SphI, SalI, AccI, XbaI, KpnI and SacI) are available for cloning. After 40-time transfers in modified MRS medium, no plasmid loss was observed. The vector pFXL03 is potentially useful as a food-grade vaccine delivery system for L. acidophilus.

Crystal structure of APOBEC3A bound to single-stranded DNA reveals structural basis for cytidine deamination and specificity.

PubMed

Kouno, Takahide; Silvas, Tania V; Hilbert, Brendan J; Shandilya, Shivender M D; Bohn, Markus F; Kelch, Brian A; Royer, William E; Somasundaran, Mohan; Kurt Yilmaz, Nese; Matsuo, Hiroshi; Schiffer, Celia A

2017-04-28

Nucleic acid editing enzymes are essential components of the immune system that lethally mutate viral pathogens and somatically mutate immunoglobulins, and contribute to the diversification and lethality of cancers. Among these enzymes are the seven human APOBEC3 deoxycytidine deaminases, each with unique target sequence specificity and subcellular localization. While the enzymology and biological consequences have been extensively studied, the mechanism by which APOBEC3s recognize and edit DNA remains elusive. Here we present the crystal structure of a complex of a cytidine deaminase with ssDNA bound in the active site at 2.2 Å. This structure not only visualizes the active site poised for catalysis of APOBEC3A, but pinpoints the residues that confer specificity towards CC/TC motifs. The APOBEC3A-ssDNA complex defines the 5'-3' directionality and subtle conformational changes that clench the ssDNA within the binding groove, revealing the architecture and mechanism of ssDNA recognition that is likely conserved among all polynucleotide deaminases, thereby opening the door for the design of mechanistic-based therapeutics.

Overcoming myelosuppression due to synthetic lethal toxicity for FLT3-targeted acute myeloid leukemia therapy

PubMed Central

Warkentin, Alexander A; Lopez, Michael S; Lasater, Elisabeth A; Lin, Kimberly; He, Bai-Liang; Leung, Anskar YH; Smith, Catherine C; Shah, Neil P; Shokat, Kevan M

2014-01-01

Activating mutations in FLT3 confer poor prognosis for individuals with acute myeloid leukemia (AML). Clinically active investigational FLT3 inhibitors can achieve complete remissions but their utility has been hampered by acquired resistance and myelosuppression attributed to a ‘synthetic lethal toxicity’ arising from simultaneous inhibition of FLT3 and KIT. We report a novel chemical strategy for selective FLT3 inhibition while avoiding KIT inhibition with the staurosporine analog, Star 27. Star 27 maintains potency against FLT3 in proliferation assays of FLT3-transformed cells compared with KIT-transformed cells, shows no toxicity towards normal human hematopoiesis at concentrations that inhibit primary FLT3-mutant AML blast growth, and is active against mutations that confer resistance to clinical inhibitors. As a more complete understanding of kinase networks emerges, it may be possible to define anti-targets such as KIT in the case of AML to allow improved kinase inhibitor design of clinical agents with enhanced efficacy and reduced toxicity. DOI: http://dx.doi.org/10.7554/eLife.03445.001 PMID:25531068

Seedling lethality in Nicotiana plumbaginifolia conferred by Ds transposable element insertion into a plant-specific gene.

PubMed

Majira, Amel; Domin, Monique; Grandjean, Olivier; Gofron, Krystyna; Houba-Hérin, Nicole

2002-10-01

A seedling lethal mutant of Nicotiana plumbaginifolia (sdl-1) was isolated by transposon tagging using a maize Dissociation (Ds) element. The insertion mutation was produced by direct co-transformation of protoplasts with two plasmids: one containing Ds and a second with an Ac transposase gene. sdl-1 seedlings exhibit several phenotypes: swollen organs, short hypocotyls in light and dark conditions, and enlarged and multinucleated cells, that altogether suggest cell growth defects. Mutant cells are able to proliferate under in vitro culture conditions. Genomic DNA sequences bordering the transposon were used to recover cDNA from the normal allele. Complementation of the mutant phenotype with the cDNA confirmed that the transposon had caused the mutation. The Ds element was inserted into the first exon of the open reading frame and the homozygous mutant lacked detectable transcript. Phenocopies of the mutant were obtained by an antisense approach. SDL-1 encodes a novel protein found in several plant genomes but apparently missingfrom animal and fungal genomes; the protein is highly conserved and has a potential plastid targeting motif.

NORF5/HUG1 is a component of the MEC1-mediated checkpoint response to DNA damage and replication arrest in Saccharomyces cerevisiae.

PubMed

Basrai, M A; Velculescu, V E; Kinzler, K W; Hieter, P

1999-10-01

Analysis of global gene expression in Saccharomyces cerevisiae by the serial analysis of gene expression technique has permitted the identification of at least 302 previously unidentified transcripts from nonannotated open reading frames (NORFs). Transcription of one of these, NORF5/HUG1 (hydroxyurea and UV and gamma radiation induced), is induced by DNA damage, and this induction requires MEC1, a homolog of the ataxia telangiectasia mutated (ATM) gene. DNA damage-specific induction of HUG1, which is independent of the cell cycle stage, is due to the alleviation of repression by the Crt1p-Ssn6p-Tup1p complex. Overexpression of HUG1 is lethal in combination with a mec1 mutation in the presence of DNA damage or replication arrest, whereas a deletion of HUG1 rescues the lethality due to a mec1 null allele. HUG1 is the first example of a NORF with important biological functional properties and defines a novel component of the MEC1 checkpoint pathway.

Mutations Allow JC Polyomaviruses to Elude Antibody Recognition | Center for Cancer Research

Cancer.gov

JC polyomavirus (JCV) infects the urinary tract of most adults. In healthy individuals, JCV infection does not cause noticeable symptoms. However, in those with compromised immune systems, JCV can cause a lethal brain disease called progressive multifocal leukoencephalopathy (PML). Data from a recently approved assay to detect serum antibodies specific for the JCV protein VP1 revealed that patients with antibodies are at increased risk of developing PML. At the same time, sequencing studies of JCV in cerebrospinal fluid (CSF) identified a number of mutations in VP1. Christopher Buck, Ph.D., and Diana Pastrana, Ph.D., of CCR’s Laboratory of Cellular Oncology, and their colleagues hypothesized that the VP1 mutations could allow the virus to evade antibody-mediated elimination.

Error catastrophe and phase transition in the empirical fitness landscape of HIV

NASA Astrophysics Data System (ADS)

Hart, Gregory R.; Ferguson, Andrew L.

2015-03-01

We have translated clinical sequence databases of the p6 HIV protein into an empirical fitness landscape quantifying viral replicative capacity as a function of the amino acid sequence. We show that the viral population resides close to a phase transition in sequence space corresponding to an "error catastrophe" beyond which there is lethal accumulation of mutations. Our model predicts that the phase transition may be induced by drug therapies that elevate the mutation rate, or by forcing mutations at particular amino acids. Applying immune pressure to any combination of killer T-cell targets cannot induce the transition, providing a rationale for why the viral protein can exist close to the error catastrophe without sustaining fatal fitness penalties due to adaptive immunity.

Concomitant Lethal Mutagenesis of Human Immunodeficiency Virus Type 1

PubMed Central

Dapp, Michael J.; Holtz, Colleen M.; Mansky, Louis M.

2012-01-01

RNA virus population dynamics is complex, and sophisticated approaches are needed in many cases for therapeutic intervention. One such approach, termed lethal mutagenesis, is directed at targeting the virus population structure for extinction or error catastrophe. Previous studies have demonstrated the concept of this approach with human immunodeficiency virus type 1 (HIV-1) by use of chemical mutagens (i.e., 5-azacytidine) as well as by host factors with mutagenic properties (i.e., APOBEC3G). In this study, these two unrelated mutagenic agents were used concomitantly to investigate the interplay of these distinct mutagenic mechanisms. Specifically, an HIV-1 was produced from APOBEC3G (A3G)-expressing cells and used to infect permissive target cells treated with 5-azacytidine (5-AZC). Reduced viral infectivity and increased viral mutagenesis was observed with both the viral mutagen (i.e., G-to-C mutations) and the host restriction factor (i.e., G-to-A mutations); however, when combined, had complex interactions. Intriguingly, nucleotide sequence analysis revealed that concomitant HIV-1 exposure to both 5-AZC and A3G resulted in an increase of G-to-A viral mutagenesis at the expense of G-to-C mutagenesis. A3G catalytic activity was required for the diminution in G-to-C mutagenesis. Taken together, our findings provide the first demonstration for potentiation of the mutagenic effect of a cytosine analog by A3G expression, resulting in concomitant HIV-1 lethal mutagenesis. PMID:22426127

Deficiency of CRTAP in non-lethal recessive osteogenesis imperfecta reduces collagen deposition into matrix.

PubMed

Valli, M; Barnes, A M; Gallanti, A; Cabral, W A; Viglio, S; Weis, M A; Makareeva, E; Eyre, D; Leikin, S; Antoniazzi, F; Marini, J C; Mottes, M

2012-11-01

Deficiency of any component of the ER-resident collagen prolyl 3-hydroxylation complex causes recessive osteogenesis imperfecta (OI). The complex modifies the α1(I)Pro986 residue and contains cartilage-associated protein (CRTAP), prolyl 3-hydroxylase 1 (P3H1) and cyclophilin B (CyPB). Fibroblasts normally secrete about 10% of CRTAP. Most CRTAP mutations cause a null allele and lethal type VII OI. We identified a 7-year-old Egyptian boy with non-lethal type VII OI and investigated the effects of his null CRTAP mutation on collagen biochemistry, the prolyl 3-hydroxylation complex, and collagen in extracellular matrix. The proband is homozygous for an insertion/deletion in CRTAP (c.118_133del16insTACCC). His dermal fibroblasts synthesize fully overmodified type I collagen, and 3-hydroxylate only 5% of α1(I)Pro986. CRTAP transcripts are 10% of control. CRTAP protein is absent from proband cells, with residual P3H1 and normal CyPB levels. Dermal collagen fibril diameters are significantly increased. By immunofluorescence of long-term cultures, we identified a severe deficiency (10-15% of control) of collagen deposited in extracellular matrix, with disorganization of the minimal fibrillar network. Quantitative pulse-chase experiments corroborate deficiency of matrix deposition, rather than increased matrix turnover. We conclude that defects of extracellular matrix, as well as intracellular defects in collagen modification, contribute to the pathology of type VII OI. © 2011 John Wiley & Sons A/S.

Targeting the DNA damage response in oncology: past, present and future perspectives.

PubMed

Basu, Bristi; Yap, Timothy A; Molife, L Rhoda; de Bono, Johann S

2012-05-01

The success of poly(ADP-ribose) polymerase inhibition in BRCA1 or BRCA2 deficient tumors as an anticancer strategy provided proof-of-concept for a synthetic lethality approach in oncology. There is therefore now active interest in expanding this approach to include other agents targeting the DNA damage response (DDR). We review lessons learnt from the development of inhibitors against DNA damage response mechanisms and envision the future of DNA repair inhibition in oncology. Preclinical synthetic lethality screens may potentially identify the best combinations of DNA-damaging drugs with inhibitors of DNA repair and the DDR or two agents acting within the DDR. Efforts are currently being made to establish robust and cost-effective assays that may be implemented within appropriate time-scales in parallel with future clinical studies. Detection of relevant mutations in a high-throughput manner, such as with next-generation sequencing for genes implicated in homologous recombination, including BRCA1, BRCA2, and ataxia telangiectasia mutated is anticipated. Novel approaches targeting the DDR are currently being evaluated and inhibitors of ATM, RAD51 and DNA-dependent protein kinase are now in early drug discovery and development. There remains great enthusiasm in oncology practice for pursuing the strategy of synthetic lethality. The future development of antitumor agents targeting the DDR should include detailed correlative biomarker work within early phase clinical studies wherever possible, with clear attempts to identify doses at which robust target modulation is observed.

ACTION OF MUTAGENIC AGENTS ON AUXOTROPHIC STRAINS OF STREPTOMYCES

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

Jarai, M.

1962-01-01

The mutagenic effect on Streptomyces auxotrophs of uv and x irradiation and of some chemical agerts was studied. From the observed reverse mutations it was concluded that uv and probably x irradiation have an optimal mutagenic dose. With nine auxotrophic strains it was shown that under the same conditions different gene loci reacted differently to the same mutagenic agent. With uy radiation, mutations occurred most frequently at doses falling within the range of 3500 to 4000 erg/mm/sup 2/. With such doses, the average mutation frequency for singly deficient mutants was 0.8 x 10/sup -6/, for doubly deficient mutants 8.4 xmore » 10/sup -8/. An analysis of the number of mutations as compared to the number of survivors in two biochemical mutants (N-4 and N-11) showed that with N- 4 the highest number of mutations was obtained at doses of 3500 to 4500 erg/mm/ sup 2/, namely, 12 to 15 per 10 surviving conidia, and with strain N-11, the highest frequency was obtained in the same dose range, namely, three to four mutations per 10/sup 6/ surviving conidia. The optimal dose of irradiation corresponds to 90 to 97% lethality. It was shown that, unlike the results with uv irradiation, with x rays no such definite relation existed between optimal dose and frequency of mutations. The highest mutation frequency occurred at doses of 20,000 to 25,000 r, which corresponded to 85 to 91% lethality. Of the chemical substances examined, a definite mutagenic action was exerted by acridine orange, streptomycin, hydroxylamine, phenyl, isocyannte, and 8-quinolinol. The maximum mutagenic frequency for survivors was 41.4 x 10/sup -6/ after uv irradiation (biochemical mutant arg 3-; frequency of sportaneous back mutation, 0.041 x 10/sup -6/). With x irradiation the maximum mutagenic frequency was 3.42 x 10/sup -6/ (biochemical mutant meth 1-; frequency of spontaneous back mutation, 0.28 X 10/sup -6/). With chemical agents the maximum mutation frequencies for the initial conidia number were as follows: acridine orange, 3.65 x 10/sup -6/ (biochemical mutant arg 3-); streptomycin, 2.05 x 10/sup -6/ (biochemical mutant arg 3-); hydroxylamine, 5.81 x 10/sup -6/ (biochemical mutant meth 1/sup -/); phenyl isocyanate, 6.11 x 10/sup -6/ (biochemical mutant meth 1/sup -/); 8- quinolinol, 1.02 x 10/sup -6/ (biochemical mutant meth 1/sup -/). (BBB)« less

Fos metamorphoses: Lessons from mutants in model organisms (Drosophila).

PubMed

Alfonso-Gonzalez, Carlos; Riesgo-Escovar, Juan Rafael

2018-05-10

The Fos oncogene gene family is evolutionarily conserved throughout Eukarya. Fos proteins characteristically have a leucine zipper and a basic region with a helix-turn-helix motif that binds DNA. In vertebrates, there are several Fos homologs. They can homo- or hetero-dimerize via the leucine zipper domain. Fos homologs coupled with other transcription factors, like Jun oncoproteins, constitute the Activator Protein 1 (AP-1) complex. From its original inception as an oncogene, the subsequent finding that they act as transcription factors binding DNA sequences known as TRE, to the realization that they are activated in many different scenarios, and to loss-of-function analysis, the Fos proteins have traversed a multifarious path in development and physiology. They are instrumental in 'immediate early genes' responses, and activated by a seemingly myriad assemblage of different stimuli. Yet, the majority of these studies were basically gain-of-function studies, since it was thought that Fos genes would be cell lethal. Loss-of-function mutations in vertebrates were recovered later, and were not cell lethal. In fact, c-fos null mutations are viable with developmental defects (osteopetrosis and myeloid lineage abnormalities). It was then hypothesized that vertebrate genomes exhibit partial redundancy, explaining the 'mild' phenotypes, and complicating assessment of complete loss-of-function phenotypes. Due to its promiscuous activation, fos genes (especially c-fos) are now commonly used as markers for cellular responses to stimuli. fos homologs high sequence conservation (including Drosophila) is advantageous as it allows critical assessment of fos genes functions in this genetic model. Drosophila melanogaster contains only one fos homolog, the gene kayak. kayak mutations are lethal, and allow study of all the processes where fos is required. The kayak locus encodes several different isoforms, and is a pleiotropic gene variously required for development involving cell shape changes. In general, fos genes seem to primarily activate programs involved in cellular architectural rearrangements and cell shape changes. Copyright © 2018. Published by Elsevier B.V.

Mutations at the flavin binding site of ETF:QO yield a MADD-like severe phenotype in Drosophila.

PubMed

Alves, Ema; Henriques, Bárbara J; Rodrigues, João V; Prudêncio, Pedro; Rocha, Hugo; Vilarinho, Laura; Martinho, Rui G; Gomes, Cláudio M

2012-08-01

Following a screening on EMS-induced Drosophila mutants defective for formation and morphogenesis of epithelial cells, we have identified three lethal mutants defective for the production of embryonic cuticle. The mutants are allelic to the CG12140 gene, the fly homologue of electron transfer flavoprotein:ubiquinone oxidoreductase (ETF:QO). In humans, inherited defects in this inner membrane protein account for multiple acyl-CoA dehydrogenase deficiency (MADD), a metabolic disease of β-oxidation, with a broad range of clinical phenotypes, varying from embryonic lethal to mild forms. The three mutant alleles carried distinct missense mutations in ETF:QO (G65E, A68V and S104F) and maternal mutant embryos for ETF:QO showed lethal morphogenetic defects and a significant induction of apoptosis following germ-band elongation. This phenotype is accompanied by an embryonic accumulation of short- and medium-chain acylcarnitines (C4, C8 and C12) as well as long-chain acylcarnitines (C14 and C16:1), whose elevation is also found in severe MADD forms in humans under intense metabolic decompensation. In agreement the ETF:QO activity in the mutant embryos is markedly decreased in relation to wild type activity. Amino acid sequence analysis and structural mapping into a molecular model of ETF:QO show that all mutations map at FAD interacting residues, two of which at the nucleotide-binding Rossmann fold. This structural domain is composed by a β-strand connected by a short loop to an α-helix, and its perturbation results in impaired cofactor association via structural destabilisation and consequently enzymatic inactivation. This work thus pinpoints the molecular origins of a severe MADD-like phenotype in the fruit fly and establishes the proof of concept concerning the suitability of this organism as a potential model organism for MADD. © 2012 Elsevier B.V. All rights reserved.

Genetic tests in mice of caffeine alone and in combination with mutagens

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

Thayer, P.S.; Kensler, C.J.

1973-06-01

The possible mutagenicity of caffeine was studied in mice by the dominant-lethal method, in three experiments. Male mice were given caffeine in drinking water for 8 weeks at 3.6, 13.4, 49, and 122 mg/kg/day (comparable to human consumption of 2.8 to 95 cups of coffee per day). Subsequent mating of each of six males from each group to five females per week for 8 weeks showed no significant increase in dominant-lethal mutations (embryonic deaths) whether expressed as early deaths per pregnant female or as mutation index. Although males consuming the two higher levels of caffeine produced fewer pregnancies, litter sizesmore » of females giving birth were not reduced. Single ip injections of caffeine (15 mg/kg) were given to groups of male mice prior to, subsequent to, and immediately at the time of receiving x-rays (100 R). Each of five males from each group was mated to five females per week for 7 weeks. Embryonic deaths did not show any enhancing effect of caffeine on the mutagenicity produced by the irradiation. Three groups of male mice ingested caffeine in water for 16 weeks at levels of 0, 4, and 13 mg/kg/day. Subgroups of five from each group were given either: no further treatment, a single dose of triethylene melamine at 0.2 mg/kg, or 100 R of x ray, and mated for 7 weeks as above. Fertility and litter size were not affected by the caffeine pretreatment, nor did it modify the induction of dominant-lethal mutations by triethylene melamine or x rays. Litter sizes showed no significant preimplantation losses in any experiment. Thus, under the conditions described herein and at the doses employed (higher than human exposure), there was no evidence for the mutagenicity of caffeine or the inhibition of DNA repair mechanisms in these mammalian systems. (auth)« less

The Holstein Friesian Lethal Haplotype 5 (HH5) Results from a Complete Deletion of TBF1M and Cholesterol Deficiency (CDH) from an ERV-(LTR) Insertion into the Coding Region of APOB

PubMed Central

Schütz, Ekkehard; Wehrhahn, Christin; Wanjek, Marius; Bortfeld, Ralf; Wemheuer, Wilhelm E.; Beck, Julia; Brenig, Bertram

2016-01-01

Background With the availability of massive SNP data for several economically important cattle breeds, haplotype tests have been performed to identify unknown recessive disorders. A number of so-called lethal haplotypes, have been uncovered in Holstein Friesian cattle and, for at least seven of these, the causative mutations have been identified in candidate genes. However, several lethal haplotypes still remain elusive. Here we report the molecular genetic causes of lethal haplotype 5 (HH5) and cholesterol deficiency (CDH). A targeted enrichment for the known genomic regions, followed by massive parallel sequencing was used to interrogate for causative mutations in a case/control approach. Methods Targeted enrichment for the known genomic regions, followed by massive parallel sequencing was used in a case/control approach. PCRs for the causing mutations were developed and compared to routine imputing in 2,100 (HH5) and 3,100 (CDH) cattle. Results HH5 is caused by a deletion of 138kbp, spanning position 93,233kb to 93,371kb on chromosome 9 (BTA9), harboring only dimethyl-adenosine transferase 1 (TFB1M). The deletion breakpoints are flanked by bovine long interspersed nuclear elements Bov-B (upstream) and L1ME3 (downstream), suggesting a homologous recombination/deletion event. TFB1M di-methylates adenine residues in the hairpin loop at the 3’-end of mitochondrial 12S rRNA, being essential for synthesis and function of the small ribosomal subunit of mitochondria. Homozygous TFB1M-/- mice reportedly exhibit embryonal lethality with developmental defects. A 2.8% allelic frequency was determined for the German HF population. CDH results from a 1.3kbp insertion of an endogenous retrovirus (ERV2-1-LTR_BT) into exon 5 of the APOB gene at BTA11:77,959kb. The insertion is flanked by 6bp target site duplications as described for insertions mediated by retroviral integrases. A premature stop codon in the open reading frame of APOB is generated, resulting in a truncation of the protein to a length of only <140 amino acids. Such early truncations have been shown to cause an inability of chylomicron excretion from intestinal cells, resulting in malabsorption of cholesterol. The allelic frequency of this mutation in the German HF population was 6.7%, which is substantially higher than reported so far. Compared to PCR assays inferring the genetic variants directly, the routine imputing used so far showed a diagnostic sensitivity of as low as 91% (HH5) and 88% (CDH), with a high specificity for both (≥99.7%). Conclusion With the availability of direct genetic tests it will now be possible to more effectively reduce the carrier frequency and ultimately eliminate the disorders from the HF populations. Beside this, the fact that repetitive genomic elements (RE) are involved in both diseases, underline the evolutionary importance of RE, which can be detrimental as here, but also advantageous over generations. PMID:27128314

SMARCA4-inactivating mutations increase sensitivity to Aurora kinase A inhibitor VX-680 in non-small cell lung cancers. | Office of Cancer Genomics

Cancer.gov

Mutations in the SMARCA4/BRG1 gene resulting in complete loss of its protein (BRG1) occur frequently in non-small cell lung cancer (NSCLC) cells. Currently, no single therapeutic agent has been identified as synthetically lethal with SMARCA4/BRG1 loss. We identify AURKA activity as essential in NSCLC cells lacking SMARCA4/BRG1. In these cells, RNAi-mediated depletion or chemical inhibition of AURKA induces apoptosis and cell death in vitro and in xenograft mouse models.

Phenotypic spectrum of STRA6 mutations: from Matthew-Wood syndrome to non-lethal anophthalmia.

PubMed

Chassaing, Nicolas; Golzio, Christelle; Odent, Sylvie; Lequeux, Léopoldine; Vigouroux, Adeline; Martinovic-Bouriel, Jelena; Tiziano, Francesco Danilo; Masini, Lucia; Piro, Francesca; Maragliano, Giovanna; Delezoide, Anne-Lise; Attié-Bitach, Tania; Manouvrier-Hanu, Sylvie; Etchevers, Heather C; Calvas, Patrick

2009-05-01

Matthew-Wood, Spear, PDAC or MCOPS9 syndrome are alternative names used to refer to combinations of microphthalmia/anophthalmia, malformative cardiac defects, pulmonary dysgenesis, and diaphragmatic hernia. Recently, mutations in STRA6, encoding a membrane receptor for vitamin A-bearing plasma retinol binding protein, have been identified in such patients. We performed STRA6 molecular analysis in three fetuses and one child diagnosed with Matthew-Wood syndrome and in three siblings where two adult living brothers are affected with combinations of clinical anophthalmia, tetralogy of Fallot, and mental retardation. Among these patients, six novel mutations were identified, bringing the current total of known STRA6 mutations to seventeen. We extensively reviewed clinical data pertaining to all twenty-one reported patients with STRA6 mutations (the seven of this report and fourteen described elsewhere) and discuss additional features that may be part of the syndrome. The clinical spectrum associated with STRA6 deficiency is even more variable than initially described. Copyright 2009 Wiley-Liss, Inc.

Large-scale identification of chemically induced mutations in Drosophila melanogaster

PubMed Central

Haelterman, Nele A.; Jiang, Lichun; Li, Yumei; Bayat, Vafa; Sandoval, Hector; Ugur, Berrak; Tan, Kai Li; Zhang, Ke; Bei, Danqing; Xiong, Bo; Charng, Wu-Lin; Busby, Theodore; Jawaid, Adeel; David, Gabriela; Jaiswal, Manish; Venken, Koen J.T.; Yamamoto, Shinya

2014-01-01

Forward genetic screens using chemical mutagens have been successful in defining the function of thousands of genes in eukaryotic model organisms. The main drawback of this strategy is the time-consuming identification of the molecular lesions causative of the phenotypes of interest. With whole-genome sequencing (WGS), it is now possible to sequence hundreds of strains, but determining which mutations are causative among thousands of polymorphisms remains challenging. We have sequenced 394 mutant strains, generated in a chemical mutagenesis screen, for essential genes on the Drosophila X chromosome and describe strategies to reduce the number of candidate mutations from an average of ∼3500 to 35 single-nucleotide variants per chromosome. By combining WGS with a rough mapping method based on large duplications, we were able to map 274 (∼70%) mutations. We show that these mutations are causative, using small 80-kb duplications that rescue lethality. Hence, our findings demonstrate that combining rough mapping with WGS dramatically expands the toolkit necessary for assigning function to genes. PMID:25258387

Complementation between polymerase- and exonuclease-deficient mitochondrial DNA polymerase mutants in genomically engineered flies

PubMed Central

Bratic, Ana; Kauppila, Timo E. S.; Macao, Bertil; Grönke, Sebastian; Siibak, Triinu; Stewart, James B.; Baggio, Francesca; Dols, Jacqueline; Partridge, Linda; Falkenberg, Maria; Wredenberg, Anna; Larsson, Nils-Göran

2015-01-01

Replication errors are the main cause of mitochondrial DNA (mtDNA) mutations and a compelling approach to decrease mutation levels would therefore be to increase the fidelity of the catalytic subunit (POLγA) of the mtDNA polymerase. Here we genomically engineer the tamas locus, encoding fly POLγA, and introduce alleles expressing exonuclease- (exo−) and polymerase-deficient (pol−) POLγA versions. The exo− mutant leads to accumulation of point mutations and linear deletions of mtDNA, whereas pol− mutants cause mtDNA depletion. The mutant tamas alleles are developmentally lethal but can complement each other in trans resulting in viable flies with clonally expanded mtDNA mutations. Reconstitution of human mtDNA replication in vitro confirms that replication is a highly dynamic process where POLγA goes on and off the template to allow complementation during proofreading and elongation. The created fly models are valuable tools to study germ line transmission of mtDNA and the pathophysiology of POLγA mutation disease. PMID:26554610

Recently Identified Mutations in the Ebola Virus-Makona Genome Do Not Alter Pathogenicity in Animal Models.

PubMed

Marzi, Andrea; Chadinah, Spencer; Haddock, Elaine; Feldmann, Friederike; Arndt, Nicolette; Martellaro, Cynthia; Scott, Dana P; Hanley, Patrick W; Nyenswah, Tolbert G; Sow, Samba; Massaquoi, Moses; Feldmann, Heinz

2018-05-08

Ebola virus (EBOV), isolate Makona, the causative agent of the West African EBOV epidemic, has been the subject of numerous investigations to determine the genetic diversity and its potential implication for virus biology, pathogenicity, and transmissibility. Despite various mutations that have emerged over time through multiple human-to-human transmission chains, their biological relevance remains questionable. Recently, mutations in the glycoprotein GP and polymerase L, which emerged and stabilized early during the outbreak, have been associated with improved viral fitness in cell culture. Here, we infected mice and rhesus macaques with EBOV-Makona isolates carrying or lacking those mutations. Surprisingly, all isolates behaved very similarly independent of the genotype, causing severe or lethal disease in mice and macaques, respectively. Likewise, we could not detect any evidence for differences in virus shedding. Thus, no specific biological phenotype could be associated with these EBOV-Makona mutations in two animal models. Published by Elsevier Inc.

Nematode radiobiology and development in space. Results from IML-1

NASA Technical Reports Server (NTRS)

Nelson, Gregory A.; Schubert, W. W.; Kazarians, G. A.; Richards, G. F.; Benton, E. V.; Benton, E. R.; Henke, R.

1994-01-01

The Radiat experiment was one of 17 investigations which used the ESA Biorack on IML-1 (International Microgravity Laboratory) and it had two objectives. The first objective was to isolate and characterize mutations induced by cosmic rays; the second was to assess the fidelity of development in 0-gravity over two consecutive generations. Two strategies were used to isolate mutations in a set of essential genes or a specific gene and to correlate the genetic events with the passage of charged particles. The results were isolation of 60 lethal mutations whose phenotypes are related to the local pattern of energy deposition. 12 mutations in the unc-22 gene include large deletions as characterized by DNA hybridization studies. Development of nematodes proceeded through two consecutive generations with no obvious defects. Cytoplasmic determinants in embryos, nuclear location and symmetry of cellular anatomy were normal as were Mendelian segregation and recombination of genetic markers.

Calmodulin point mutations affect Drosophila development and behavior.

PubMed

Nelson, H B; Heiman, R G; Bolduc, C; Kovalick, G E; Whitley, P; Stern, M; Beckingham, K

1997-12-01

Calmodulin (CAM) is recognized as a major intermediary in intracellular calcium signaling, but as yet little is known of its role in developmental and behavioral processes. We have generated and studied mutations to the endogenous Cam gene of Drosophila melanogaster that change single amino acids within the protein coding region. One of these mutations produces a striking pupal lethal phenotype involving failure of head eversion. Various mutant combinations produce specific patterns of ectopic wing vein formation or melanotic scabs on the cuticle. Anaphase chromosome bridging is also seen as a maternal effect during the early embryonic nuclear divisions. In addition, specific behavioral defects such as poor climbing and flightlessness are detected among these mutants. Comparisons with other Drosophila mutant phenotypes suggests potential CAM targets that may mediate these developmental and behavioral effects, and analysis of the CAM crystal structure suggests the structural consequences of the individual mutations.

Calmodulin Point Mutations Affect Drosophila Development and Behavior

PubMed Central

Nelson, H. B.; Heiman, R. G.; Bolduc, C.; Kovalick, G. E.; Whitley, P.; Stern, M.; Beckingham, K.

1997-01-01

Calmodulin (CAM) is recognized as a major intermediary in intracellular calcium signaling, but as yet little is known of its role in developmental and behavioral processes. We have generated and studied mutations to the endogenous Cam gene of Drosophila melanogaster that change single amino acids within the protein coding region. One of these mutations produces a striking pupal lethal phenotype involving failure of head eversion. Various mutant combinations produce specific patterns of ectopic wing vein formation or melanotic scabs on the cuticle. Anaphase chromosome bridging is also seen as a maternal effect during the early embryonic nuclear divisions. In addition, specific behavioral defects such as poor climbing and flightlessness are detected among these mutants. Comparisons with other Drosophila mutant phenotypes suggests potential CAM targets that may mediate these developmental and behavioral effects, and analysis of the CAM crystal structure suggests the structural consequences of the individual mutations. PMID:9409836

Dissecting the Mutational Landscape of Cutaneous Melanoma: An Omic Analysis Based on Patients from Greece

PubMed Central

Piroti, Georgia; Papadodima, Olga

2018-01-01

Melanoma is a lethal type of skin cancer, unless it is diagnosed early. Formalin-fixed, paraffin-embedded (FFPE) tissue is a valuable source for molecular assays after diagnostic examination, but isolated nucleic acids often suffer from degradation. Here, for the first time, we examine primary melanomas from Greek patients, using whole exome sequencing, so as to derive their mutational profile. Application of a bioinformatic framework revealed a total of 10,030 somatic mutations. Regarding the genes containing putative protein-altering mutations, 73 were common in at least three patients. Sixty-five of these 73 top common genes have been previously identified in melanoma cases. Biological processes related to melanoma were affected by varied genes in each patient, suggesting differences in the components of a pathway possibly contributing to pathogenesis. We performed a multi-level analysis highlighting a short list of candidate genes with a probable causative role in melanoma. PMID:29596374

Loss of the tumor suppressor BAP1 causes myeloid transformation

PubMed Central

Dey, Anwesha; Seshasayee, Dhaya; Noubade, Rajkumar; French, Dorothy M.; Liu, Jinfeng; Chaurushiya, Mira S.; Kirkpatrick, Donald S.; Pham, Victoria C.; Lill, Jennie R.; Bakalarski, Corey E.; Wu, Jiansheng; Phu, Lilian; Katavolos, Paula; Saunders, Lindsay M.; Abdel-Wahab, Omar; Modrusan, Zora; Seshagiri, Somasekar; Dong, Ken; Lin, Zhonghua; Balazs, Mercedesz; Suriben, Rowena; Newton, Kim; Hymowitz, Sarah; Garcia-Manero, Guillermo; Martin, Flavius; Levine, Ross L.; Dixit, Vishva M.

2016-01-01

Deubiquitinating enzyme BAP1 is mutated in a hereditary cancer syndrome with increased risk of mesothelioma and uveal melanoma. Somatic BAP1 mutations occur in various malignancies. We show that mouse Bap1 gene deletion is lethal during embryogenesis, but systemic or hematopoietic-restricted deletion in adults recapitulates features of human myelodysplastic syndrome (MDS). Knock-in mice expressing BAP1 with a 3xFlag tag revealed that BAP1 interacts with HCF-1, OGT, and the polycomb group proteins ASXL1 and ASXL2 in vivo. OGT and HCF-1 levels were decreased by Bap1 deletion, indicating a critical role for BAP1 in stabilizing these epigenetic regulators. Human ASXL1 is mutated frequently in chronic myelomonocytic leukemia (CMML) so an ASXL/BAP1 complex may suppress CMML. A novel BAP1 catalytic mutation found in a MDS patient implies that BAP1 loss of function has similar consequences in mouse and man. PMID:22878500

Detection of COPB2 as a KRAS synthetic lethal partner through integration of functional genomics screens

PubMed Central

Christodoulou, Eleni G.; Yang, Hai; Lademann, Franziska; Pilarsky, Christian; Beyer, Andreas; Schroeder, Michael

2017-01-01

Mutated KRAS plays an important role in many cancers. Although targeting KRAS directly is difficult, indirect inactivation via synthetic lethal partners (SLPs) is promising. Yet to date, there are no SLPs from high-throughput RNAi screening, which are supported by multiple screens. Here, we address this problem by aggregating and ranking data over three independent high-throughput screens. We integrate rankings by minimizing the displacement and by considering established methods such as RIGER and RSA. Our meta analysis reveals COPB2 as a potential SLP of KRAS with good support from all three screens. COPB2 is a coatomer subunit and its knock down has already been linked to disabled autophagy and reduced tumor growth. We confirm COPB2 as SLP in knock down experiments on pancreas and colorectal cancer cell lines. Overall, consistent integration of high throughput data can generate candidate synthetic lethal partners, which individual screens do not uncover. Concretely, we reveal and confirm that COPB2 is a synthetic lethal partner of KRAS and hence a promising cancer target. Ligands inhibiting COPB2 may, therefore, be promising new cancer drugs. PMID:28415695

Epistatic Interactions within the Influenza A Virus Polymerase Complex Mediate Mutagen Resistance and Replication Fidelity

PubMed Central

Pauly, Matthew D.; Lyons, Daniel M.; Fitzsimmons, William J.

2017-01-01

ABSTRACT Lethal mutagenesis is a broad-spectrum antiviral strategy that employs mutagenic nucleoside analogs to exploit the high mutation rate and low mutational tolerance of many RNA viruses. Studies of mutagen-resistant viruses have identified determinants of replicative fidelity and the importance of mutation rate to viral population dynamics. We have previously demonstrated the effective lethal mutagenesis of influenza A virus using three nucleoside analogs as well as the virus’s high genetic barrier to mutagen resistance. Here, we investigate the mutagen-resistant phenotypes of mutations that were enriched in drug-treated populations. We find that PB1 T123A has higher replicative fitness than the wild type, PR8, and maintains its level of genome production during 5-fluorouracil (2,4-dihydroxy-5-fluoropyrimidine) treatment. Surprisingly, this mutagen-resistant variant also has an increased baseline rate of C-to-U and G-to-A mutations. A second drug-selected mutation, PA T97I, interacts epistatically with PB1 T123A to mediate high-level mutagen resistance, predominantly by limiting the inhibitory effect of nucleosides on polymerase activity. Consistent with the importance of epistatic interactions in the influenza virus polymerase, our data suggest that nucleoside analog resistance and replication fidelity are strain dependent. Two previously identified ribavirin {1-[(2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-1H-1,2,4-triazole-3-carboxamide} resistance mutations, PB1 V43I and PB1 D27N, do not confer drug resistance in the PR8 background, and the PR8-PB1 V43I polymerase exhibits a normal baseline mutation rate. Our results highlight the genetic complexity of the influenza A virus polymerase and demonstrate that increased replicative capacity is a mechanism by which an RNA virus can counter the negative effects of elevated mutation rates. IMPORTANCE RNA viruses exist as genetically diverse populations. This standing genetic diversity gives them the potential to adapt rapidly, evolve resistance to antiviral therapeutics, and evade immune responses. Viral mutants with altered mutation rates or mutational tolerance have provided insights into how genetic diversity arises and how it affects the behavior of RNA viruses. To this end, we identified variants within the polymerase complex of influenza virus that are able to tolerate drug-mediated increases in viral mutation rates. We find that drug resistance is highly dependent on interactions among mutations in the polymerase complex. In contrast to other viruses, influenza virus counters the effect of higher mutation rates primarily by maintaining high levels of genome replication. These findings suggest the importance of maintaining large population sizes for viruses with high mutation rates and show that multiple proteins can affect both mutation rate and genome synthesis. PMID:28815216

Inactivation of CDK2 is synthetically lethal to MYCN over-expressing cancer cells

PubMed Central

Molenaar, Jan J.; Ebus, Marli E.; Geerts, Dirk; Koster, Jan; Lamers, Fieke; Valentijn, Linda J.; Westerhout, Ellen M.; Versteeg, Rogier; Caron, Huib N.

2009-01-01

Two genes have a synthetically lethal relationship when the silencing or inhibiting of 1 gene is only lethal in the context of a mutation or activation of the second gene. This situation offers an attractive therapeutic strategy, as inhibition of such a gene will only trigger cell death in tumor cells with an activated second oncogene but spare normal cells without activation of the second oncogene. Here we present evidence that CDK2 is synthetically lethal to neuroblastoma cells with MYCN amplification and over-expression. Neuroblastomas are childhood tumors with an often lethal outcome. Twenty percent of the tumors have MYCN amplification, and these tumors are ultimately refractory to any therapy. Targeted silencing of CDK2 by 3 RNA interference techniques induced apoptosis in MYCN-amplified neuroblastoma cell lines, but not in MYCN single copy cells. Silencing of MYCN abrogated this apoptotic response in MYCN-amplified cells. Inversely, silencing of CDK2 in MYCN single copy cells did not trigger apoptosis, unless a MYCN transgene was activated. The MYCN induced apoptosis after CDK2 silencing was accompanied by nuclear stabilization of P53, and mRNA profiling showed up-regulation of P53 target genes. Silencing of P53 rescued the cells from MYCN-driven apoptosis. The synthetic lethality of CDK2 silencing in MYCN activated neuroblastoma cells can also be triggered by inhibition of CDK2 with a small molecule drug. Treatment of neuroblastoma cells with roscovitine, a CDK inhibitor, at clinically achievable concentrations induced MYCN-dependent apoptosis. The synthetically lethal relationship between CDK2 and MYCN indicates CDK2 inhibitors as potential MYCN-selective cancer therapeutics. PMID:19525400

In silico Analysis of Conformational Changes Induced by Mutation of Aromatic Binding Residues: Consequences for Drug Binding in the hERG K+ Channel

PubMed Central

Knape, Kirsten; Linder, Tobias; Wolschann, Peter; Beyer, Anton; Stary-Weinzinger, Anna

2011-01-01

Pharmacological inhibition of cardiac hERG K+ channels is associated with increased risk of lethal arrhythmias. Many drugs reduce hERG current by directly binding to the channel, thereby blocking ion conduction. Mutation of two aromatic residues (F656 and Y652) substantially decreases the potency of numerous structurally diverse compounds. Nevertheless, some drugs are only weakly affected by mutation Y652A. In this study we utilize molecular dynamics simulations and docking studies to analyze the different effects of mutation Y652A on a selected number of hERG blockers. MD simulations reveal conformational changes in the binding site induced by mutation Y652A. Loss of π-π-stacking between the two aromatic residues induces a conformational change of the F656 side chain from a cavity facing to cavity lining orientation. Docking studies and MD simulations qualitatively reproduce the diverse experimentally observed modulatory effects of mutation Y652A and provide a new structural interpretation for the sensitivity differences. PMID:22194911

Genetics of pancreatic cancer and implications for therapy.

PubMed

Bhosale, Priya; Cox, Veronica; Faria, Silvana; Javadi, Sanaz; Viswanathan, Chitra; Koay, Eugene; Tamm, Eric

2018-02-01

Pancreatic cancer is a highly lethal disease with a dismal 5-year prognosis. Knowledge of its genetics may help in identifying new methods for patient screening, and cancer treatment. In this review, we will describe the most common mutations responsible for the genesis of pancreatic cancer and their impact on screening, patterns of disease progression, and therapy.

Synthetic Lethal Therapeutic Approaches for ARID1A-Mutated Ovarian Cancer

DTIC Science & Technology

2017-10-01

formation by the indicated cells (c). (d-f) ARID1A protein expression in parental and ARID1A CRISPR OVCA429 cells (d). Colony formation assay using...ovarian tumor cultures with (VOA4841) and without (XVOA295) ARID1A expression. n=3 independent experiments. (f) Control and ARID1A CRISPR OVCA429 cells

Mating compatibility and competitiveness between wild and laboratory strains of Eldana saccharina (Lepidoptera: Pyralidae) after radiation treatment

USDA-ARS?s Scientific Manuscript database

The efficacy of the sterile insect technique (SIT) applied as part of area-wide integrated pest management (AW-IPM) programmes depends on the efficient transfer of sperm carrying dominant lethal mutations from sterile males to wild females. The success or failure of this strategy is therefore critic...

Standard Operating Procedure for Mutagenicity Testing Using the Drosophila melanogaster Sex-Linked Recessive Lethal Assay.

DTIC Science & Technology

1982-01-01

60025, 22 December 1978 10. ALDERSON, T. Chemically induced delayed germinal mutation in Drosophila. Nature 207:164-167, 1965 11. BLUM, A. and B.N...Superintendent Commander Academy of Health Sciences US Army Institute of Dental Research ATTN: AHS-COM Washington DC 20012 Fort Sam Houston TX 78234 Assistant

Correlation between In Vitro Cytotoxicity and In Vivo Lethal Activity in Mice of Epsilon Toxin Mutants from Clostridium perfringens

PubMed Central

Dorca-Arévalo, Jonatan; Pauillac, Serge; Díaz-Hidalgo, Laura; Martín-Satué, Mireia; Popoff, Michel R.; Blasi, Juan

2014-01-01

Epsilon toxin (Etx) from Clostridium perfringens is a pore-forming protein with a lethal effect on livestock, producing severe enterotoxemia characterized by general edema and neurological alterations. Site-specific mutations of the toxin are valuable tools to study the cellular and molecular mechanism of the toxin activity. In particular, mutants with paired cysteine substitutions that affect the membrane insertion domain behaved as dominant-negative inhibitors of toxin activity in MDCK cells. We produced similar mutants, together with a well-known non-toxic mutant (Etx-H106P), as green fluorescent protein (GFP) fusion proteins to perform in vivo studies in an acutely intoxicated mouse model. The mutant (GFP-Etx-I51C/A114C) had a lethal effect with generalized edema, and accumulated in the brain parenchyma due to its ability to cross the blood-brain barrier (BBB). In the renal system, this mutant had a cytotoxic effect on distal tubule epithelial cells. The other mutants studied (GFP-Etx-V56C/F118C and GFP-Etx-H106P) did not have a lethal effect or cross the BBB, and failed to induce a cytotoxic effect on renal epithelial cells. These data suggest a direct correlation between the lethal effect of the toxin, with its cytotoxic effect on the kidney distal tubule cells, and the ability to cross the BBB. PMID:25013927

Correlation between in vitro cytotoxicity and in vivo lethal activity in mice of epsilon toxin mutants from Clostridium perfringens.

PubMed

Dorca-Arévalo, Jonatan; Pauillac, Serge; Díaz-Hidalgo, Laura; Martín-Satué, Mireia; Popoff, Michel R; Blasi, Juan

2014-01-01

Epsilon toxin (Etx) from Clostridium perfringens is a pore-forming protein with a lethal effect on livestock, producing severe enterotoxemia characterized by general edema and neurological alterations. Site-specific mutations of the toxin are valuable tools to study the cellular and molecular mechanism of the toxin activity. In particular, mutants with paired cysteine substitutions that affect the membrane insertion domain behaved as dominant-negative inhibitors of toxin activity in MDCK cells. We produced similar mutants, together with a well-known non-toxic mutant (Etx-H106P), as green fluorescent protein (GFP) fusion proteins to perform in vivo studies in an acutely intoxicated mouse model. The mutant (GFP-Etx-I51C/A114C) had a lethal effect with generalized edema, and accumulated in the brain parenchyma due to its ability to cross the blood-brain barrier (BBB). In the renal system, this mutant had a cytotoxic effect on distal tubule epithelial cells. The other mutants studied (GFP-Etx-V56C/F118C and GFP-Etx-H106P) did not have a lethal effect or cross the BBB, and failed to induce a cytotoxic effect on renal epithelial cells. These data suggest a direct correlation between the lethal effect of the toxin, with its cytotoxic effect on the kidney distal tubule cells, and the ability to cross the BBB.

Delineation of the Marfan phenotype associated with mutations in exons 23-32 of the FBN1 gene

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

Putnam, E.A.; Cho, M.; Milewicz, D.M.

Marfan syndrome is a dominantly inherited connective tissue disorder with a wide range of phenotypic severity. The condition is the result of mutations in FBN1, a large gene composed of 65 exons encoding the fibrillin-1 protein. While mutations causing classic manifestations of Marfan syndrome have been identified throughout the FBN1 gene, the six previously characterized mutations resulting in the severe, perinatal lethal form of Marfan syndrome have clustered in exons 24-32 of the gene. We screened 8 patients with either neonatal Marfan syndrome or severe cardiovascular complications of Marfan syndrome for mutations in this region of the gene. Using intron-basedmore » exon-specific primers, we amplified exons 23-32 from genomic DNAs, screened these fragments by single-stranded conformational polymorphism analysis, and sequenced indicated exons. This analysis documented mutations in exons 25-27 of the FBN1 mutations in 6 of these patients. These results, taken together with previously published FBN1 mutations in this region, further define the phenotype associated with mutations in exons 24-32 of the FBN1 gene, information important for the development of possible diagnostic tests and genetic counseling. 49 refs., 4 figs., 2 tabs.« less

Different rates of spontaneous mutation of chloroplastic and nuclear viroids as determined by high-fidelity ultra-deep sequencing.

PubMed

López-Carrasco, Amparo; Ballesteros, Cristina; Sentandreu, Vicente; Delgado, Sonia; Gago-Zachert, Selma; Flores, Ricardo; Sanjuán, Rafael

2017-09-01

Mutation rates vary by orders of magnitude across biological systems, being higher for simpler genomes. The simplest known genomes correspond to viroids, subviral plant replicons constituted by circular non-coding RNAs of few hundred bases. Previous work has revealed an extremely high mutation rate for chrysanthemum chlorotic mottle viroid, a chloroplast-replicating viroid. However, whether this is a general feature of viroids remains unclear. Here, we have used high-fidelity ultra-deep sequencing to determine the mutation rate in a common host (eggplant) of two viroids, each representative of one family: the chloroplastic eggplant latent viroid (ELVd, Avsunviroidae) and the nuclear potato spindle tuber viroid (PSTVd, Pospiviroidae). This revealed higher mutation frequencies in ELVd than in PSTVd, as well as marked differences in the types of mutations produced. Rates of spontaneous mutation, quantified in vivo using the lethal mutation method, ranged from 1/1000 to 1/800 for ELVd and from 1/7000 to 1/3800 for PSTVd depending on sequencing run. These results suggest that extremely high mutability is a common feature of chloroplastic viroids, whereas the mutation rates of PSTVd and potentially other nuclear viroids appear significantly lower and closer to those of some RNA viruses.

Different rates of spontaneous mutation of chloroplastic and nuclear viroids as determined by high-fidelity ultra-deep sequencing

PubMed Central

Ballesteros, Cristina; Sentandreu, Vicente; Gago-Zachert, Selma

2017-01-01

Mutation rates vary by orders of magnitude across biological systems, being higher for simpler genomes. The simplest known genomes correspond to viroids, subviral plant replicons constituted by circular non-coding RNAs of few hundred bases. Previous work has revealed an extremely high mutation rate for chrysanthemum chlorotic mottle viroid, a chloroplast-replicating viroid. However, whether this is a general feature of viroids remains unclear. Here, we have used high-fidelity ultra-deep sequencing to determine the mutation rate in a common host (eggplant) of two viroids, each representative of one family: the chloroplastic eggplant latent viroid (ELVd, Avsunviroidae) and the nuclear potato spindle tuber viroid (PSTVd, Pospiviroidae). This revealed higher mutation frequencies in ELVd than in PSTVd, as well as marked differences in the types of mutations produced. Rates of spontaneous mutation, quantified in vivo using the lethal mutation method, ranged from 1/1000 to 1/800 for ELVd and from 1/7000 to 1/3800 for PSTVd depending on sequencing run. These results suggest that extremely high mutability is a common feature of chloroplastic viroids, whereas the mutation rates of PSTVd and potentially other nuclear viroids appear significantly lower and closer to those of some RNA viruses. PMID:28910391

P-Element Insertion Alleles of Essential Genes on the Third Chromosome of Drosophila Melanogaster: Correlation of Physical and Cytogenetic Maps in Chromosomal Region 86e-87f

PubMed Central

Deak, P.; Omar, M. M.; Saunders, RDC.; Pal, M.; Komonyi, O.; Szidonya, J.; Maroy, P.; Zhang, Y.; Ashburner, M.; Benos, P.; Savakis, C.; Siden-Kiamos, I.; Louis, C.; Bolshakov, V. N.; Kafatos, F. C.; Madueno, E.; Modolell, J.; Glover, D. M.

1997-01-01

We have established a collection of 2460 lethal or semi-lethal mutant lines using a procedure thought to insert single P elements into vital genes on the third chromosome of Drosophila melanogaster. More than 1200 randomly selected lines were examined by in situ hybridization and 90% found to contain single insertions at sites that mark 89% of all lettered subdivisions of the Bridges' map. A set of chromosomal deficiencies that collectively uncover ~25% of the euchromatin of chromosome 3 reveal lethal mutations in 468 lines corresponding to 145 complementation groups. We undertook a detailed analysis of the cytogenetic interval 86E-87F and identified 87 P-element-induced mutations falling into 38 complementation groups, 16 of which correspond to previously known genes. Twenty-one of these 38 complementation groups have at least one allele that has a P-element insertion at a position consistent with the cytogenetics of the locus. We have rescued P elements and flanking chromosomal sequences from the 86E-87F region in 35 lines with either lethal or genetically silent P insertions, and used these as probes to identify cosmids and P1 clones from the Drosophila genome projects. This has tied together the physical and genetic maps and has linked 44 previously identified cosmid contigs into seven ``supercontigs'' that span the interval. STS data for sequences flanking one side of the P-element insertions in 49 lines has identified insertions in the αγ element at 87C, two known transposable elements, and the open reading frames of seven putative single copy genes. These correspond to five known genes in this interval, and two genes identified by the homology of their predicted products to known proteins from other organisms. PMID:9409831

Cloning and Characterization of the Scalloped Region of Drosophila Melanogaster

PubMed Central

Campbell, S. D.; Duttaroy, A.; Katzen, A. L.; Chovnick, A.

1991-01-01

Viable mutants of the scalloped gene (sd) of Drosophila melanogaster exhibit defects that can include gapping of the wing margin and ectopic bristle formation on the wing. Lethal sd alleles characterized in the present study now implicate this gene in a genetic function essential for normal development. In order to further characterize the developmental role of this gene, we have undertaken to clone and characterize the region where sd maps. A P[ry(+)] transposon insertion at 13F associated with sd([ry+2216]) served as the starting point for a 42-kb chromosomal walk. Molecular lesions associated with viable and lethal sd alleles were characterized by genomic hybridization analysis as a means of defining the extent of the gene. DNA rearrangements associated with 11 viable sd alleles map to a 2-kb interval which appears to be a ``hot spot'' for P element activity. Four of five recessive lethal sd mutations were mapped by denaturing gradient gel electrophoresis to a region 12-14 kb away from the region of viable lesions. In a sd(+) genotype, at least two structurally related and developmentally regulated transcripts hybridize to the genomic region where several sd lethal alleles have been localized. A viable mutation, sd(58), used for comparison in the transcript analysis, makes at least two slightly smaller transcripts that also hybridize to this region. Preliminary analysis of cDNA clones has identified three structurally related transcripts that hybridize to this genomic region. The 5' end of these transcripts extends into the 2-kb genomic region wherein DNA rearrangements were seen in the P element rearrangements. We favor the view that the transcripts represented by these cDNA clones are products of the sd gene. If this is true, the sd gene would include genomic sequences extending over at least 14 kb of the described chromosomal walk, and would appear to be subject to alternative splicing. PMID:1706292

Defining essential elements and genetic interactions of the yeast Lsm2-8 ring and demonstration that essentiality of Lsm2-8 is bypassed via overexpression of U6 snRNA or the U6 snRNP subunit Prp24.

PubMed

Roth, Allen J; Shuman, Stewart; Schwer, Beate

2018-06-01

A seven-subunit Lsm2-8 protein ring assembles on the U-rich 3' end of the U6 snRNA. A structure-guided mutational analysis of the Saccharomyces cerevisiae Lsm2-8 ring affords new insights to structure-function relations and genetic interactions of the Lsm subunits. Alanine scanning of 39 amino acids comprising the RNA-binding sites or intersubunit interfaces of Lsm2, Lsm3, Lsm4, Lsm5, and Lsm8 identified only one instance of lethality (Lsm3-R69A) and one severe growth defect (Lsm2-R63A), both involving amino acids that bind the 3'-terminal UUU trinucleotide. All other Ala mutations were benign with respect to vegetative growth. Tests of 235 pairwise combinations of benign Lsm mutants identified six instances of inter-Lsm synthetic lethality and 45 cases of nonlethal synthetic growth defects. Thus, Lsm2-8 ring function is buffered by a network of internal genetic redundancies. A salient finding was that otherwise lethal single-gene deletions lsm2 Δ, lsm3 Δ, lsm4 Δ, lsm5 , and lsm8 Δ were rescued by overexpression of U6 snRNA from a high-copy plasmid. Moreover, U6 overexpression rescued myriad lsm Δ lsm Δ double-deletions and lsm Δ lsm Δ lsm Δ triple-deletions. We find that U6 overexpression also rescues a lethal deletion of the U6 snRNP protein subunit Prp24 and that Prp24 overexpression bypasses the essentiality of the U6-associated Lsm subunits. Our results indicate that abetting U6 snRNA is the only essential function of the yeast Lsm2-8 proteins. © 2018 Roth et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

RIPK3 Mediates Necroptosis during Embryonic Development and Postnatal Inflammation in Fadd-Deficient Mice.

PubMed

Zhao, Qun; Yu, XianJun; Zhang, HaiWei; Liu, YongBo; Zhang, XiXi; Wu, XiaoXia; Xie, Qun; Li, Ming; Ying, Hao; Zhang, Haibing

2017-04-25

RIPK3 mediates cell death and regulates inflammatory responses. Although genetic studies have suggested that RIPK3-MLKL-mediated necroptosis leads to embryonic lethality in Fadd or Caspase-8-deficient mice, the exact mechanisms are not fully understood. Here, we generated Ripk3 mutant mice by altering the RIPK3 kinase domain (Ripk3 Δ/Δ mice), thus abolishing its kinase activity. Ripk3 Δ/Δ cells were resistant to necroptosis stimulation in vitro, and Ripk3 Δ/Δ mice were protected from necroptotic diseases. Although the Ripk3 Δ/Δ mutation rescued embryonic lethality in Fadd -/- embryos, Fadd -/- Ripk3 Δ/Δ mice died within 1 day after birth due to massive inflammation. These results indicate that Ripk3 ablation rescues embryonic lethality in Fadd-deficient mice by suppressing two RIPK3-mediating processes: necroptosis during embryogenesis and inflammation during postnatal development in Fadd -/- mice. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Mutations in the C-terminal fragment of DnaK affecting peptide binding.

PubMed Central

Burkholder, W F; Zhao, X; Zhu, X; Hendrickson, W A; Gragerov, A; Gottesman, M E

1996-01-01

Escherichia coli DnaK acts as a molecular chaperone through its ATP-regulated binding and release of polypeptide substrates. Overexpressing a C-terminal fragment (CTF) of DnaK (Gly-384 to Lys-638) containing the polypeptide substrate binding domain is lethal in wild-type E. coli. This dominant-negative phenotype may result from the nonproductive binding of CTF to cellular polypeptide targets of DnaK. Mutations affecting DnaK substrate binding were identified by selecting noncytotoxic CTF mutants followed by in vitro screening. The clustering of such mutations in the three-dimensional structure of CTF suggests the model that loops L1,2 and L4,5 form a rigid core structure critical for interactions with substrate. Images Fig. 1 Fig. 2 Fig. 3 PMID:8855230

Loss of Nephrocystin-3 Function Can Cause Embryonic Lethality, Meckel-Gruber-like Syndrome, Situs Inversus, and Renal-Hepatic-Pancreatic Dysplasia

PubMed Central

Bergmann, Carsten; Fliegauf, Manfred; Brüchle, Nadina Ortiz; Frank, Valeska; Olbrich, Heike; Kirschner, Jan; Schermer, Bernhard; Schmedding, Ingolf; Kispert, Andreas; Kränzlin, Bettina; Nürnberg, Gudrun; Becker, Christian; Grimm, Tiemo; Girschick, Gundula; Lynch, Sally A.; Kelehan, Peter; Senderek, Jan; Neuhaus, Thomas J.; Stallmach, Thomas; Zentgraf, Hanswalter; Nürnberg, Peter; Gretz, Norbert; Lo, Cecilia; Lienkamp, Soeren; Schäfer, Tobias; Walz, Gerd; Benzing, Thomas; Zerres, Klaus; Omran, Heymut

2008-01-01

Many genetic diseases have been linked to the dysfunction of primary cilia, which occur nearly ubiquitously in the body and act as solitary cellular mechanosensory organelles. The list of clinical manifestations and affected tissues in cilia-related disorders (ciliopathies) such as nephronophthisis is broad and has been attributed to the wide expression pattern of ciliary proteins. However, little is known about the molecular mechanisms leading to this dramatic diversity of phenotypes. We recently reported hypomorphic NPHP3 mutations in children and young adults with isolated nephronophthisis and associated hepatic fibrosis or tapetoretinal degeneration. Here, we chose a combinatorial approach in mice and humans to define the phenotypic spectrum of NPHP3/Nphp3 mutations and the role of the nephrocystin-3 protein. We demonstrate that the pcy mutation generates a hypomorphic Nphp3 allele that is responsible for the cystic kidney disease phenotype, whereas complete loss of Nphp3 function results in situs inversus, congenital heart defects, and embryonic lethality in mice. In humans, we show that NPHP3 mutations can cause a broad clinical spectrum of early embryonic patterning defects comprising situs inversus, polydactyly, central nervous system malformations, structural heart defects, preauricular fistulas, and a wide range of congenital anomalies of the kidney and urinary tract (CAKUT). On the functional level, we show that nephrocystin-3 directly interacts with inversin and can inhibit like inversin canonical Wnt signaling, whereas nephrocystin-3 deficiency leads in Xenopus laevis to typical planar cell polarity defects, suggesting a role in the control of canonical and noncanonical (planar cell polarity) Wnt signaling. PMID:18371931

5-Azacytidine Can Induce Lethal Mutagenesis in Human Immunodeficiency Virus Type 1▿ †

PubMed Central

Dapp, Michael J.; Clouser, Christine L.; Patterson, Steven; Mansky, Louis M.

2009-01-01

Ribonucleosides inhibit human immunodeficiency virus type 1 (HIV-1) replication by mechanisms that have not been fully elucidated. Here, we report the antiviral mechanism for the ribonucleoside analog 5-azacytidine (5-AZC). We hypothesized that the anti-HIV-1 activity of 5-AZC was due to an increase in the HIV-1 mutation rate following its incorporation into viral RNA during transcription. However, we demonstrate that 5-AZC's primary antiviral activity can be attributed to its effect on the early phase of HIV-1 replication. Furthermore, the antiviral activity was associated with an increase in the frequency of viral mutants, suggesting that 5-AZC's primary target is reverse transcription. Sequencing analysis showed an enrichment in G-to-C transversion mutations and further supports the idea that reverse transcription is an antiviral target of 5-AZC. These results indicate that 5-AZC is incorporated into viral DNA following reduction to 5-aza-2′-deoxycytidine. Incorporation into the viral DNA leads to an increase in mutant frequency that is consistent with lethal mutagenesis during reverse transcription as the primary antiviral mechanism of 5-AZC. Antiviral activity and increased mutation frequency were also associated with the late phase of HIV-1 replication; however, 5-AZC's effect on the late phase was less robust. These results reveal that the primary antiviral mechanism of 5-AZC can be attributed to its ability to increase the HIV-1 mutation frequency through viral-DNA incorporation during reverse transcription. Our observations indicate that 5-AZC can affect two steps in HIV-1 replication (i.e., transcription and reverse transcription) but that its primary antiviral activity is due to incorporation during reverse transcription. PMID:19726509

Primary Sex Determination in Drosophila melanogaster Does Not Rely on the Male-Specific Lethal Complex.

PubMed

Erickson, James W

2016-02-01

It has been proposed that the Male Specific Lethal (MSL) complex is active in Drosophila melanogaster embryos of both sexes prior to the maternal-to-zygotic transition. Elevated gene expression from the two X chromosomes of female embryos is proposed to facilitate the stable establishment of Sex-lethal (Sxl) expression, which determines sex and represses further activity of the MSL complex, leaving it active only in males. Important supporting data included female-lethal genetic interactions between the seven msl genes and either Sxl or scute and sisterlessA, two of the X-signal elements (XSE) that regulate early Sxl expression. Here I report contrary findings that there are no female-lethal genetic interactions between the msl genes and Sxl or its XSE regulators. Fly stocks containing the msl3(1) allele were found to exhibit a maternal-effect interaction with Sxl, scute, and sisterlessA mutations, but genetic complementation experiments showed that msl3 is neither necessary nor sufficient for the female-lethal interactions, which appear to be due to an unidentified maternal regulator of Sxl. Published data cited as evidence for an early function of the MSL complex in females, including a maternal effect of msl2, have been reevaluated and found not to support a maternal, or other effect, of the MSL complex in sex determination. These findings suggest that the MSL complex is not involved in primary sex determination or in X chromosome dosage compensation prior to the maternal-to-zygotic transition. Copyright © 2016 by the Genetics Society of America.

DPY-17 and MUA-3 Interact for Connective Tissue-Like Tissue Integrity in Caenorhabditis elegans: A Model for Marfan Syndrome.

PubMed

Fotopoulos, Pauline; Kim, Jeongho; Hyun, Moonjung; Qamari, Waiss; Lee, Inhwan; You, Young-Jai

2015-04-27

mua-3 is a Caenorhabditis elegans homolog of the mammalian fibrillin1, a monogenic cause of Marfan syndrome. We identified a new mutation of mua-3 that carries an in-frame deletion of 131 amino acids in the extracellular domain, which allows the mutants to survive in a temperature-dependent manner; at the permissive temperature, the mutants grow normally without obvious phenotypes, but at the nonpermissive temperature, more than 90% die during the L4 molt due to internal organ detachment. Using the temperature-sensitive lethality, we performed unbiased genetic screens to isolate suppressors to find genetic interactors of MUA-3. From two independent screens, we isolated mutations in dpy-17 as a suppressor. RNAi of dpy-17 in mua-3 rescued the lethality, confirming dpy-17 is a suppressor. dpy-17 encodes a collagen known to genetically interact with dpy-31, a BMP-1/Tolloid-like metalloprotease required for TGFβ activation in mammals. Human fibrillin1 mutants fail to sequester TGFβ2 leading to excess TGFβ signaling, which in turn contributes to Marfan syndrome or Marfan-related syndrome. Consistent with that, RNAi of dbl-1, a TGFβ homolog, modestly rescued the lethality of mua-3 mutants, suggesting a potentially conserved interaction between MUA-3 and a TGFβ pathway in C. elegans. Our work provides genetic evidence of the interaction between TGFβ and a fibrillin homolog, and thus provides a simple yet powerful genetic model to study TGFβ function in development of Marfan pathology. Copyright © 2015 Fotopoulos et al.

Comparative studies on the lethal, mutagenic, and recombinogenic effects of ultraviolet -A, -B, -C, and visible light with and without 8-methoxypsoralen in Saccharomyces cerevisiae.

PubMed

Mondon, P; Shahin, M M

1992-05-01

Genetic effects of UV-A, UV-B, UV-C, and the combination of 8-methoxypsoralen (8-MOP) with UV-A or visible light were studied in the haploid strain XV185-14C and diploid strain D5 of Saccharomyces cerevisiae. The induction of his+, lys+, and hom+ reverse mutations was measured in strain XV185-14C. In strain D5 we measured the induction of genetically altered colonies, particularly twin spot colonies arising from a mitotic crossing-over. UV-C and UV-B induced point mutations at the three loci in the haploid strain and mitotic crossing-over and other genetic alterations in the diploid strain. UV-C was more mutagenic and recombinogenic than UV-B. UV-A or visible light alone did not induce genotoxic effects at the doses tested. However, UV-A plus 8-MOP produced lethal and mutagenic effects in the haploid strain XV185-14C, although mutagenic activity was less than that of UV-B. Visible light plus 8-MOP also induced genotoxic effects in strain XV185-14C. In the diploid strain D5, UV-A plus 8-MOP induced a higher frequency of genetic alterations than UV-B at comparative doses. Visible light plus 8-MOP was also genetically active in strain D5. The haploid strain was more sensitive to the lethal effects of UV-C, UV-B, UV-A, and impure visible light plus 8-MOP than the diploid strain.

Targeted Disruption of Mouse Yin Yang 1 Transcription Factor Results in Peri-Implantation Lethality

PubMed Central

Donohoe, Mary E.; Zhang, Xiaolin; McGinnis, Lynda; Biggers, John; Li, En; Shi, Yang

1999-01-01

Yin Yang 1 (YY1) is a zinc finger-containing transcription factor and a target of viral oncoproteins. To determine the biological role of YY1 in mammalian development, we generated mice deficient for YY1 by gene targeting. Homozygosity for the mutated YY1 allele results in embryonic lethality in the mouse. YY1 mutants undergo implantation and induce uterine decidualization but rapidly degenerate around the time of implantation. A subset of YY1 heterozygote embryos are developmentally retarded and exhibit neurulation defects, suggesting that YY1 may have additional roles during later stages of mouse embryogenesis. Our studies demonstrate an essential function for YY1 in the development of the mouse embryo. PMID:10490658

Ex-vivo assessment and non-invasive in vivo imaging of internal hemorrhages in Aga2/+ mutant mice

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

Ermolayev, Vladimir; Cohrs, Christian M.; Mohajerani, Pouyan

Highlights: ► Aga2/+ mice, model for Osteogenesis imperfecta, have type I collagen mutation. ► Aga2/+ mice display both moderate and severe phenotypes lethal 6–11th postnatal. ► Internal hemorrhages studied in Aga2/+ vs. control mice at 6 and 9 days postnatal. ► Anatomical and functional findings in-vivo contrasted to the ex-vivo appearance. -- Abstract: Mutations in type I collagen genes (COL1A1/2) typically lead to Osteogenesis imperfecta, the most common heritable cause of skeletal fractures and bone deformation in humans. Heterozygous Col1a1{sup Aga2/+}, animals with a dominant mutation in the terminal C-propeptide domain of type I collagen develop typical skeletal hallmarks andmore » internal hemorrhages starting from 6 day after birth. The disease progression for Aga2/+ mice, however, is not uniform differing between severe phenotype lethal at the 6–11th day of life, and moderate-to-severe one with survival to adulthood. Herein we investigated whether a new modality that combines X-ray computer tomography with fluorescence tomography in one hybrid system can be employed to study internal bleedings in relation to bone fractures and obtain insights into disease progression. The disease phenotype was characterized on Aga2/+ vs. wild type mice between 6 and 9 days postnatal. Anatomical and functional findings obtained in-vivo were contrasted to the ex-vivo appearance of the same tissues under cryo-slicing.« less

R-LOCUS DELETERIOUS FACTORS IN MORMONIELLA

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

Whiting, P.W.

1962-01-01

New data are presented on 37 R-locus mutant genes containing deleterious factors or crossover suppressors. Twenty-seven of these genes are among the 206 recognizable eye-color mutants previously found by others in experiments in which wild-type males were irradiated and mated, siring 11062 daughters examined, mutation rate 1.86%. With the addition of eight mutants from later simdlar tests there were 38 mutants failing to breed, probably being dominant steriles, and seven immature, probably dominant lethals. Of the l60 mutants given successful breeding test, 80 were normal and 80 contained delcterious factors of different types - lethals, near-steriles, femalesteriles, and male-stertles. Ratemore » of deleterious factor productdon differs according to the factor mutating to produce the eye-color marker. Among the l07 genes changed in factor S alone, 68 were also deleterious (63.6%) but for the 45 in O, there were only nine (20.0%), suggesting a more sensitive region near S. More than one deleterious factor may be produced simultaneously with an eye-color change and one defeet may mask others. The gene which forms a temporary unit of segregation in heterozygotes is of a higher order of magnitude than units of heredity (gene elements, cistrons) which may be permanently present dn the germ plasm. Because of the high mutation rate to the marker eye colors scarlet and oyster white, the genetical structure of the R region may be easily studied. (auth)« less

A new deletion refines the boundaries of the murine Prader–Willi syndrome imprinting center

PubMed Central

DuBose, Amanda J.; Smith, Emily Y.; Yang, Thomas P.; Johnstone, Karen A.; Resnick, James L.

2011-01-01

The human chromosomal 15q11–15q13 region is subject to both maternal and paternal genomic imprinting. Absence of paternal gene expression from this region results in Prader–Willi syndrome (PWS), while absence of maternal gene expression leads to Angelman syndrome. Transcription of paternally expressed genes in the region depends upon an imprinting center termed the PWS-IC. Imprinting defects in PWS can be caused by microdeletions and the smallest commonly deleted region indicates that the PWS-IC lies within a region of 4.3 kb. The function and location of the PWS-IC is evolutionarily conserved, but delineation of the PWS-IC in mouse has proven difficult. The first targeted mutation of the PWS-IC, a deletion of 35 kb spanning Snrpn exon 1, exhibited a complete PWS-IC deletion phenotype. Pups inheriting this mutation paternally showed a complete loss of paternal gene expression and died neonatally. A reported deletion of 4.8 kb showed only a reduction in paternal gene expression and incomplete penetrance of neonatal lethality, suggesting that some PWS-IC function had been retained. Here, we report that a 6 kb deletion spanning Snrpn exon 1 exhibits a complete PWS-IC deletion phenotype. Pups inheriting this mutation paternally lack detectable expression of all PWS genes and paternal silencing of Ube3a, exhibit maternal DNA methylation imprints at Ndn and Mkrn3 and suffer failure to thrive leading to a fully penetrant neonatal lethality. PMID:21659337

A new deletion refines the boundaries of the murine Prader-Willi syndrome imprinting center.

PubMed

Dubose, Amanda J; Smith, Emily Y; Yang, Thomas P; Johnstone, Karen A; Resnick, James L

2011-09-01

The human chromosomal 15q11-15q13 region is subject to both maternal and paternal genomic imprinting. Absence of paternal gene expression from this region results in Prader-Willi syndrome (PWS), while absence of maternal gene expression leads to Angelman syndrome. Transcription of paternally expressed genes in the region depends upon an imprinting center termed the PWS-IC. Imprinting defects in PWS can be caused by microdeletions and the smallest commonly deleted region indicates that the PWS-IC lies within a region of 4.3 kb. The function and location of the PWS-IC is evolutionarily conserved, but delineation of the PWS-IC in mouse has proven difficult. The first targeted mutation of the PWS-IC, a deletion of 35 kb spanning Snrpn exon 1, exhibited a complete PWS-IC deletion phenotype. Pups inheriting this mutation paternally showed a complete loss of paternal gene expression and died neonatally. A reported deletion of 4.8 kb showed only a reduction in paternal gene expression and incomplete penetrance of neonatal lethality, suggesting that some PWS-IC function had been retained. Here, we report that a 6 kb deletion spanning Snrpn exon 1 exhibits a complete PWS-IC deletion phenotype. Pups inheriting this mutation paternally lack detectable expression of all PWS genes and paternal silencing of Ube3a, exhibit maternal DNA methylation imprints at Ndn and Mkrn3 and suffer failure to thrive leading to a fully penetrant neonatal lethality.

Congenital Heart Disease–Causing Gata4 Mutation Displays Functional Deficits In Vivo

PubMed Central

Misra, Chaitali; Sachan, Nita; McNally, Caryn Rothrock; Koenig, Sara N.; Nichols, Haley A.; Guggilam, Anuradha; Lucchesi, Pamela A.; Pu, William T.; Srivastava, Deepak; Garg, Vidu

2012-01-01

Defects of atrial and ventricular septation are the most frequent form of congenital heart disease, accounting for almost 50% of all cases. We previously reported that a heterozygous G296S missense mutation of GATA4 caused atrial and ventricular septal defects and pulmonary valve stenosis in humans. GATA4 encodes a cardiac transcription factor, and when deleted in mice it results in cardiac bifida and lethality by embryonic day (E)9.5. In vitro, the mutant GATA4 protein has a reduced DNA binding affinity and transcriptional activity and abolishes a physical interaction with TBX5, a transcription factor critical for normal heart formation. To characterize the mutation in vivo, we generated mice harboring the same mutation, Gata4 G295S. Mice homozygous for the Gata4 G295S mutant allele have normal ventral body patterning and heart looping, but have a thin ventricular myocardium, single ventricular chamber, and lethality by E11.5. While heterozygous Gata4 G295S mutant mice are viable, a subset of these mice have semilunar valve stenosis and small defects of the atrial septum. Gene expression studies of homozygous mutant mice suggest the G295S protein can sufficiently activate downstream targets of Gata4 in the endoderm but not in the developing heart. Cardiomyocyte proliferation deficits and decreased cardiac expression of CCND2, a member of the cyclin family and a direct target of Gata4, were found in embryos both homozygous and heterozygous for the Gata4 G295S allele. To further define functions of the Gata4 G295S mutation in vivo, compound mutant mice were generated in which specific cell lineages harbored both the Gata4 G295S mutant and Gata4 null alleles. Examination of these mice demonstrated that the Gata4 G295S protein has functional deficits in early myocardial development. In summary, the Gata4 G295S mutation functions as a hypomorph in vivo and leads to defects in cardiomyocyte proliferation during embryogenesis, which may contribute to the development of congenital heart defects in humans. PMID:22589735

Identification and Characterization of Genes That Interact with Lin-12 in Caenorhabditis Elegans

PubMed Central

Tax, F. E.; Thomas, J. H.; Ferguson, E. L.; Horvitz, H. R.

1997-01-01

We identified and characterized 14 extragenic mutations that suppressed the dominant egg-laying defect of certain lin-12 gain-of-function mutations. These suppressors defined seven genes: sup-17, lag-2, sel-4, sel-5, sel-6, sel-7 and sel-8. Mutations in six of the genes are recessive suppressors, whereas the two mutations that define the seventh gene, lag-2, are semi-dominant suppressors. These suppressor mutations were able to suppress other lin-12 gain-of-function mutations. The suppressor mutations arose at a very low frequency per gene, 10-50 times below the typical loss-of-function mutation frequency. The suppressor mutations in sup-17 and lag-2 were shown to be rare non-null alleles, and we present evidence that null mutations in these two genes cause lethality. Temperature-shift studies for two suppressor genes, sup-17 and lag-2, suggest that both genes act at approximately the same time as lin-12 in specifying a cell fate. Suppressor alleles of six of these genes enhanced a temperature-sensitive loss-of-function allele of glp-1, a gene related to lin-12 in structure and function. Our analysis of these suppressors suggests that the majority of these genes are part of a shared lin-12/glp-1 signal transduction pathway, or act to regulate the expression or stability of lin-12 and glp-1. PMID:9409830

Mutations Allow JC Polyomaviruses to Elude Antibody Recognition | Center for Cancer Research

Cancer.gov

JC polyomavirus (JCV) infects the urinary tract of most adults. In healthy individuals, JCV infection does not cause noticeable symptoms. However, in those with compromised immune systems, JCV can cause a lethal brain disease called progressive multifocal leukoencephalopathy (PML). Data from a recently approved assay to detect serum antibodies specific for the JCV protein VP1

Radiation-induced genomic instability

NASA Technical Reports Server (NTRS)

Kronenberg, A.

1994-01-01

Quantitative assessment of the heritable somatic effects of ionizing radiation exposures has relied upon the assumption that radiation-induced lesions were 'fixed' in the DNA prior to the first postirradiation mitosis. Lesion conversion was thought to occur during the initial round of DNA replication or as a consequence of error-prone enzymatic processing of lesions. The standard experimental protocols for the assessment of a variety of radiation-induced endpoints (cell death, specific locus mutations, neoplastic transformation and chromosome aberrations) evaluate these various endpoints at a single snapshot in time. In contrast with the aforementioned approaches, some studies have specifically assessed radiation effects as a function of time following exposure. Evidence has accumulated in support of the hypothesis that radiation exposure induces a persistent destabilization of the genome. This instability has been observed as a delayed expression of lethal mutations, as an enhanced rate of accumulation of non-lethal heritable alterations, and as a progressive intraclonal chromosomal heterogeneity. The genetic controls and biochemical mechanisms underlying radiation-induced genomic instability have not yet been delineated. The aim is to integrate the accumulated evidence that suggests that radiation exposure has a persistent effect on the stability of the mammalian genome.

Mutations in the Paxillin-binding Site of Integrin-linked Kinase (ILK) Destabilize the Pseudokinase Domain and Cause Embryonic Lethality in Mice*

PubMed Central

Moik, Daniel; Böttcher, Anika; Makhina, Tatiana; Grashoff, Carsten; Bulus, Nada; Zent, Roy; Fässler, Reinhard

2013-01-01

Integrin-linked kinase (ILK) localizes to focal adhesions (FAs) where it regulates cell spreading, migration, and growth factor receptor signaling. Previous reports showed that overexpressed ILK in which Val386 and Thr387 were substituted with glycine residues (ILK-VT/GG) could neither interact with paxillin nor localize to FA in cells expressing endogenous wild-type ILK, implying that paxillin binding to ILK is required for its localization to FAs. Here, we show that introducing this mutation into the germ line of mice (ILK-VT/GG) caused vasculogenesis defects, resulting in a general developmental delay and death at around embryonic day 12.5. Fibroblasts isolated from ILK-VT/GG mice contained mutant ILK in FAs, showed normal adhesion to and spreading on extracellular matrix substrates but displayed impaired migration. Biochemical analysis revealed that VT/GG substitutions decreased ILK protein stability leading to decreased ILK levels and reduced binding to paxillin and α-parvin. Because paxillin depletion did not affect ILK localization to FAs, the embryonic lethality and the in vitro migration defects are likely due to the reduced levels of ILK-VT/GG and diminished binding to parvins. PMID:23658024

Disruption of TTDA Results in Complete Nucleotide Excision Repair Deficiency and Embryonic Lethality

PubMed Central

Theil, Arjan F.; Nonnekens, Julie; Steurer, Barbara; Mari, Pierre-Olivier; de Wit, Jan; Lemaitre, Charlène; Marteijn, Jurgen A.; Raams, Anja; Maas, Alex; Vermeij, Marcel; Essers, Jeroen; Hoeijmakers, Jan H. J.; Giglia-Mari, Giuseppina; Vermeulen, Wim

2013-01-01

The ten-subunit transcription factor IIH (TFIIH) plays a crucial role in transcription and nucleotide excision repair (NER). Inactivating mutations in the smallest 8-kDa TFB5/TTDA subunit cause the neurodevelopmental progeroid repair syndrome trichothiodystrophy A (TTD-A). Previous studies have shown that TTDA is the only TFIIH subunit that appears not to be essential for NER, transcription, or viability. We studied the consequences of TTDA inactivation by generating a Ttda knock-out (Ttda−/−) mouse-model resembling TTD-A patients. Unexpectedly, Ttda−/− mice were embryonic lethal. However, in contrast to full disruption of all other TFIIH subunits, viability of Ttda−/− cells was not affected. Surprisingly, Ttda−/− cells were completely NER deficient, contrary to the incomplete NER deficiency of TTD-A patient-derived cells. We further showed that TTD-A patient mutations only partially inactivate TTDA function, explaining the relatively mild repair phenotype of TTD-A cells. Moreover, Ttda−/− cells were also highly sensitive to oxidizing agents. These findings reveal an essential role of TTDA for life, nucleotide excision repair, and oxidative DNA damage repair and identify Ttda−/− cells as a unique class of TFIIH mutants. PMID:23637614

An allelic series reveals essential roles for FY in plant development in addition to flowering-time control.

PubMed

Henderson, Ian R; Liu, Fuquan; Drea, Sinead; Simpson, Gordon G; Dean, Caroline

2005-08-01

The autonomous pathway functions to promote flowering in Arabidopsis by limiting the accumulation of the floral repressor FLOWERING LOCUS C (FLC). Within this pathway FCA is a plant-specific, nuclear RNA-binding protein, which interacts with FY, a highly conserved eukaryotic polyadenylation factor. FCA and FY function to control polyadenylation site choice during processing of the FCA transcript. Null mutations in the yeast FY homologue Pfs2p are lethal. This raises the question as to whether these essential RNA processing functions are conserved in plants. Characterisation of an allelic series of fy mutations reveals that null alleles are embryo lethal. Furthermore, silencing of FY, but not FCA, is deleterious to growth in Nicotiana. The late-flowering fy alleles are hypomorphic and indicate a requirement for both intact FY WD repeats and the C-terminal domain in repression of FLC. The FY C-terminal domain binds FCA and in vitro assays demonstrate a requirement for both C-terminal FY-PPLPP repeats during this interaction. The expression domain of FY supports its roles in essential and flowering-time functions. Hence, FY may mediate both regulated and constitutive RNA 3'-end processing.

Crystal structure of APOBEC3A bound to single-stranded DNA reveals structural basis for cytidine deamination and specificity

PubMed Central

Kouno, Takahide; Silvas, Tania V.; Hilbert, Brendan J.; Shandilya, Shivender M. D.; Bohn, Markus F.; Kelch, Brian A.; Royer, William E.; Somasundaran, Mohan; Kurt Yilmaz, Nese; Matsuo, Hiroshi; Schiffer, Celia A.

2017-01-01

Nucleic acid editing enzymes are essential components of the immune system that lethally mutate viral pathogens and somatically mutate immunoglobulins, and contribute to the diversification and lethality of cancers. Among these enzymes are the seven human APOBEC3 deoxycytidine deaminases, each with unique target sequence specificity and subcellular localization. While the enzymology and biological consequences have been extensively studied, the mechanism by which APOBEC3s recognize and edit DNA remains elusive. Here we present the crystal structure of a complex of a cytidine deaminase with ssDNA bound in the active site at 2.2 Å. This structure not only visualizes the active site poised for catalysis of APOBEC3A, but pinpoints the residues that confer specificity towards CC/TC motifs. The APOBEC3A–ssDNA complex defines the 5′–3′ directionality and subtle conformational changes that clench the ssDNA within the binding groove, revealing the architecture and mechanism of ssDNA recognition that is likely conserved among all polynucleotide deaminases, thereby opening the door for the design of mechanistic-based therapeutics. PMID:28452355

RADH, a gene of Saccharomyces cerevisiae encoding a putative DNA helicase involved in DNA repair. Characteristics of radH mutants and sequence of the gene.

PubMed

Aboussekhra, A; Chanet, R; Zgaga, Z; Cassier-Chauvat, C; Heude, M; Fabre, F

1989-09-25

A new type of radiation-sensitive mutant of S. cerevisiae is described. The recessive radH mutation sensitizes to the lethal effect of UV radiations haploids in the G1 but not in the G2 mitotic phase. Homozygous diploids are as sensitive as G1 haploids. The UV-induced mutagenesis is depressed, while the induction of gene conversion is increased. The mutation is believed to channel the repair of lesions engaged in the mutagenic pathway into a recombination process, successful if the events involve sister-chromatids but lethal if they involve homologous chromosomes. The sequence of the RADH gene reveals that it may code for a DNA helicase, with a Mr of 134 kDa. All the consensus domains of known DNA helicases are present. Besides these consensus regions, strong homologies with the Rep and UvrD helicases of E. coli were found. The RadH putative helicase appears to belong to the set of proteins involved in the error-prone repair mechanism, at least for UV-induced lesions, and could act in coordination with the Rev3 error-prone DNA polymerase.

Mutator dynamics in sexual and asexual experimental populations of yeast.

PubMed

Raynes, Yevgeniy; Gazzara, Matthew R; Sniegowski, Paul D

2011-06-07

In asexual populations, mutators may be expected to hitchhike with associated beneficial mutations. In sexual populations, recombination is predicted to erode such associations, inhibiting mutator hitchhiking. To investigate the effect of recombination on mutators experimentally, we compared the frequency dynamics of a mutator allele (msh2Δ) in sexual and asexual populations of Saccharomyces cerevisiae. Mutator strains increased in frequency at the expense of wild-type strains in all asexual diploid populations, with some approaching fixation in 150 generations of propagation. Over the same period of time, mutators declined toward loss in all corresponding sexual diploid populations as well as in haploid populations propagated asexually. We report the first experimental investigation of mutator dynamics in sexual populations. We show that a strong mutator quickly declines in sexual populations while hitchhiking to high frequency in asexual diploid populations, as predicted by theory. We also show that the msh2Δ mutator has a high and immediate realized cost that is alone sufficient to explain its decline in sexual populations. We postulate that this cost is indirect; namely, that it is due to a very high rate of recessive lethal or strongly deleterious mutation. However, we cannot rule out the possibility that msh2Δ also has unknown directly deleterious effects on fitness, and that these effects may differ between haploid asexual and sexual populations. Despite these reservations, our results prompt us to speculate that the short-term cost of highly deleterious recessive mutations can be as important as recombination in preventing mutator hitchhiking in sexual populations.

Decreased Expression of Stable RNA Can Alleviate the Lethality Associated with RNase E Deficiency in Escherichia coli.

PubMed

Himabindu, P; Anupama, K

2017-04-15

The endoribonuclease RNase E participates in mRNA degradation, rRNA processing, and tRNA maturation in Escherichia coli , but the precise reasons for its essentiality are unclear and much debated. The enzyme is most active on RNA substrates with a 5'-terminal monophosphate, which is sensed by a domain in the enzyme that includes residue R169; E. coli also possesses a 5'-pyrophosphohydrolase, RppH, that catalyzes conversion of 5'-terminal triphosphate to 5'-terminal monophosphate on RNAs. Although the C-terminal half (CTH), beyond residue approximately 500, of RNase E is dispensable for viability, deletion of the CTH is lethal when combined with an R169Q mutation or with deletion of rppH In this work, we show that both these lethalities can be rescued in derivatives in which four or five of the seven rrn operons in the genome have been deleted. We hypothesize that the reduced stable RNA levels under these conditions minimize the need of RNase E to process them, thereby allowing for its diversion for mRNA degradation. In support of this hypothesis, we have found that other conditions that are known to reduce stable RNA levels also suppress one or both lethalities: (i) alterations in relA and spoT , which are expected to lead to increased basal ppGpp levels; (ii) stringent rpoB mutations, which mimic high intracellular ppGpp levels; and (iii) overexpression of DksA. Lethality suppression by these perturbations was RNase R dependent. Our work therefore suggests that its actions on the various substrates (mRNA, rRNA, and tRNA) jointly contribute to the essentiality of RNase E in E. coli IMPORTANCE The endoribonuclease RNase E is essential for viability in many Gram-negative bacteria, including Escherichia coli Different explanations have been offered for its essentiality, including its roles in global mRNA degradation or in the processing of several tRNA and rRNA species. Our work suggests that, rather than its role in the processing of any one particular substrate, its distributed functions on all the different substrates (mRNA, rRNA, and tRNA) are responsible for the essentiality of RNase E in E. coli . Copyright © 2017 American Society for Microbiology.

Drosophila model of Meier-Gorlin syndrome based on the mutation in a conserved C-Terminal domain of Orc6.

PubMed

Balasov, Maxim; Akhmetova, Katarina; Chesnokov, Igor

2015-11-01

Meier-Gorlin syndrome (MGS) is an autosomal recessive disorder characterized by microtia, primordial dwarfism, small ears, and skeletal abnormalities. Patients with MGS often carry mutations in the genes encoding the components of the pre-replicative complex such as Origin Recognition Complex (ORC) subunits Orc1, Orc4, Orc6, and helicase loaders Cdt1 and Cdc6. Orc6 is an important component of ORC and has functions in both DNA replication and cytokinesis. Mutation in conserved C-terminal motif of Orc6 associated with MGS impedes the interaction of Orc6 with core ORC. In order to study the effects of MGS mutation in an animal model system we introduced MGS mutation in Orc6 and established Drosophila model of MGS. Mutant flies die at third instar larval stage with abnormal chromosomes and DNA replication defects. The lethality can be rescued by elevated expression of mutant Orc6 protein. Rescued MGS flies are unable to fly and display multiple planar cell polarity defects. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.

TALPID3 controls centrosome and cell polarity and the human ortholog KIAA0586 is mutated in Joubert syndrome (JBTS23)

PubMed Central

Stephen, Louise A; Tawamie, Hasan; Davis, Gemma M; Tebbe, Lars; Nürnberg, Peter; Nürnberg, Gudrun; Thiele, Holger; Thoenes, Michaela; Boltshauser, Eugen; Uebe, Steffen; Rompel, Oliver; Reis, André; Ekici, Arif B; McTeir, Lynn; Fraser, Amy M; Hall, Emma A; Mill, Pleasantine; Daudet, Nicolas; Cross, Courtney; Wolfrum, Uwe; Jamra, Rami Abou; Davey, Megan G; Bolz, Hanno J

2015-01-01

Joubert syndrome (JBTS) is a severe recessive neurodevelopmental ciliopathy which can affect several organ systems. Mutations in known JBTS genes account for approximately half of the cases. By homozygosity mapping and whole-exome sequencing, we identified a novel locus, JBTS23, with a homozygous splice site mutation in KIAA0586 (alias TALPID3), a known lethal ciliopathy locus in model organisms. Truncating KIAA0586 mutations were identified in two additional patients with JBTS. One mutation, c.428delG (p.Arg143Lysfs*4), is unexpectedly common in the general population and may be a major contributor to JBTS. We demonstrate KIAA0586 protein localization at the basal body in human and mouse photoreceptors, as is common for JBTS proteins, and also in pericentriolar locations. We show that loss of TALPID3 (KIAA0586) function in animal models causes abnormal tissue polarity, centrosome length and orientation, and centriolar satellites. We propose that JBTS and other ciliopathies may in part result from cell polarity defects. DOI: http://dx.doi.org/10.7554/eLife.08077.001 PMID:26386247

Identification of a novel insertion mutation in FGFR3 that causes thanatophoric dysplasia type 1.

PubMed

Lindy, Amanda S; Basehore, Monica J; Munisha, Mumingjiang; Williams, Aimee Leanne; Friez, Michael J; Writzl, Karin; Willems, Patrick; Dougan, Scott T

2016-06-01

Thanatophoric dysplasia is a type of short-limbed neonatal dwarfism that is usually lethal in the perinatal period. It is characterized by short limbs, a narrow, bell-shaped thorax, macrocephaly with a prominent forehead, and flattened vertebral bodies. These malformations result from autosomal dominant mutations in the fibroblast growth factor receptor 3 (FGFR3) gene. In this report, we describe a novel FGFR3 insertion mutation in a fetus with shortened limbs, curved femurs, and a narrow thorax. The diagnosis of thanatophoric dysplasia type 1 was suspected clinically, and FGFR3 sequencing showed a c.742_743insTGT variant, which predicts p.R248delinsLC. In vivo studies in zebrafish demonstrated that this mutation resulted in the overexpression of zebrafish Fgfr3, leading to the over-activation of downstream signaling and dorsalized embryos. To date, no insertions or deletions in FGFR3 have been reported to cause thanatophoric dysplasia types 1 or 2; therefore, this represents the first report to describe such a mutation. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Atelosteogenesis type II is caused by mutations in the diastrophic dysplasia sulfate-transporter gene (DTDST): Evidence for a phenotypic series involving three chondrodysplasias

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

Haestbacka, J.; Lander, E.S.; Superti-Furga, A.

1996-02-01

Atelosteogenesis type II (AO II) is a neonatally lethal chondrodysplasia whose clinical and histological characteristics resemble those of another chondrodysplasia, the much less severe diastrophic dysplasia (DTD). The similarity suggests a shared pathogenesis involving lesions in the same biochemical pathway and perhaps the same gene. DTD is caused by mutations in the recently identified diastrophic dysplasia sulfate-transporter gene (DTDST). Here, we report that AOII patients also have DTDST mutations, which lead to defective uptake of inorganic sulfate and insufficient sulfation of macromolecules by patient mesenchymal cells in vitro. Together with our recent observation that a third even more severe chondrodysplasia,more » achondrogenesis type IB, is also caused by mutations in DTDST, these results demonstrate a phenotypic series of three chondrodysplasias of increasing severity caused by lesions in a single sulfate-transporter gene. The severity of the phenotype appears to be correlated with the predicted effect of the mutations on the residual activity of the DTDST protein. 24 refs., 6 figs., 1 tab.« less

ON A CHANGE IN THE SPECTRUM OF SOMATIC MUTATIONS IN EPHESTIA KUEHNIELLA Z. BY TEMPERATURE TREATMENT BEFORE IRRADITION

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

Loebbecke, E.; Oltmanns, O.

1961-01-01

Pupae were maintained at different temperatures (-7, doses of x rays. The incidence of 4 types of scale mutations (ES 1, ES 2, ES 3, ES 4) in the butterflies was studied. It was found to vary significantly according to temperature. The ratio of ES 1/ES 2 mutations was lowest (5: 1) at -7 un. Concent 85% and highest (11: 1) at 35 un. Concent 85% . The ES 1 mutant showed highest frequency at 25 un. Concent 85% and fell to the lowest value after preincubation at 40 un. Concent 85% . The ES 2 mutant reached its lowestmore » incidence at 35 un. Concent 85% . The ES 3 mutant varied inconsistentiy and ES 4 frequency was oniy slightly dependent on temperature. The preincubation temperature of 40 un. Concent 85% , the lethal limit for the species, generally depressed mutation frequency. The precise reason for the effect of temperature on mutation frequency is unknown but it was previously found that chromosome fragmentation and translocation were reduced at elevated temperatures. (H.H.D.)« less

The role of deleterious mutations in the stability of hybridogenetic water frog complexes

PubMed Central

2014-01-01

Background Some species of water frogs originated from hybridization between different species. Such hybrid populations have a particular reproduction system called hybridogenesis. In this paper we consider the two species Pelophylax ridibundus and Pelophylax lessonae, and their hybrids Pelophylax esculentus. P. lessonae and P. esculentus form stable complexes (L-E complexes) in which P. esculentus are hemiclonal. In L-E complexes all the transmitted genomes by P. esculentus carry deleterious mutations which are lethal in homozygosity. Results We analyze, by means of an individual based computational model, L-E complexes. The results of simulations based on the model show that, by eliminating deleterious mutations, L-E complexes collapse. In addition, simulations show that particular female preferences can contribute to the diffusion of deleterious mutations among all P. esculentus frogs. Finally, simulations show how L-E complexes react to the introduction of translocated P. ridibundus. Conclusions The conclusions are the following: (i) deleterious mutations (combined with sexual preferences) strongly contribute to the stability of L-E complexes; (ii) female sexual choice can contribute to the diffusion of deleterious mutations; and (iii) the introduction of P. ridibundus can destabilize L-E complexes. PMID:24885008

Novel Compound Heterozygous Mutations Expand the Recognized Phenotypes of FARS2-Linked Disease.

PubMed

Walker, Melissa A; Mohler, Kyle P; Hopkins, Kyle W; Oakley, Derek H; Sweetser, David A; Ibba, Michael; Frosch, Matthew P; Thibert, Ronald L

2016-08-01

Mutations in mitochondrial aminoacyl-tRNA synthetases are an increasingly recognized cause of human diseases, often arising in individuals with compound heterozygous mutations and presenting with system-specific phenotypes, frequently neurologic. FARS2 encodes mitochondrial phenylalanyl transfer ribonucleic acid (RNA) synthetase (mtPheRS), perturbations of which have been reported in 6 cases of an infantile, lethal disease with refractory epilepsy and progressive myoclonus. Here the authors report the case of juvenile onset refractory epilepsy and progressive myoclonus with compound heterozygous FARS2 mutations. The authors describe the clinical course over 6 years of care at their institution and diagnostic studies including electroencephalogram (EEG), brain magnetic resonance imaging (MRI), serum and cerebrospinal fluid analyses, skeletal muscle biopsy histology, and autopsy gross and histologic findings, which include features shared with Alpers-Huttenlocher syndrome, Leigh syndrome, and a previously published case of FARS2 mutation associated infantile onset disease. The authors also present structure-guided analysis of the relevant mutations based on published mitochondrial phenylalanyl transfer RNA synthetase and related protein crystal structures as well as biochemical analysis of the corresponding recombinant mutant proteins. © The Author(s) 2016.

Ascorbate, added after irradiation, reduces the mutant yield and alters the spectrum of CD59- mutations in A(L) cells irradiated with high LET carbon ions

NASA Technical Reports Server (NTRS)

Ueno, Akiko; Vannais, Diane; Lenarczyk, Marek; Waldren, Charles A.; Chatterjee, A. (Principal Investigator)

2002-01-01

It has been reported that X-ray induced HPRT- mutation in cultured human cells is prevented by ascorbate added after irradiation. Mutation extinction is attributed to neutralization by ascorbate, of radiation-induced long-lived radicals (LLR) with half-lives of several hours. We here show that post-irradiation treatment with ascorbate (5 mM added 30 min after radiation) reduces, but does not eliminate, the induction of CD59- mutants in human-hamster hybrid A(L) cells exposed to high-LET carbon ions (LET of 100 KeV/microm). RibCys, [2(R,S)-D-ribo-1',2',3',4'-Tetrahydroxybutyl]-thiazolidene-4(R)-ca riboxylic acid] (4 mM) gave a similar but lesser effect. The lethality of the carbon ions was not altered by these chemicals. Preliminary data are presented that ascorbate also alters the spectrum of CD59- mutations induced by the carbon beam, mainly by reducing the incidence of small mutations and mutants displaying transmissible genomic instability (TGI), while large mutations are unaffected. Our results suggest that LLR are important in initiating TGI.

KRAS: Reasons for optimism in lung cancer.

PubMed

Lindsay, C R; Jamal-Hanjani, M; Forster, M; Blackhall, F

2018-06-09

Despite being the most frequent gain-of-function genetic alteration in human cancer, KRAS mutation has to date offered only limited potential as a prognostic and predictive biomarker. Results from the phase III SELECT-1 trial in non-small cell lung cancer (NSCLC) recently added to a number of historical and more contemporary disappointments in targeting KRAS mutant disease, including farnesyl transferase inhibition and synthetic lethality partners such as STK33. This narrative review uses the context of these previous failures to demonstrate how the knowledge gained from these experiences can be used as a platform for exciting advances in NSCLC on the horizon. It now seems clear that mutational subtype (most commonly G12C) of individual mutations is of greater relevance than the categorical evaluation of KRAS mutation presence or otherwise. A number of direct small molecules targeted to these subtypes are in development and have shown promising biological activity, with some in the late stages of preclinical validation. Copyright © 2018 Elsevier Ltd. All rights reserved.

Generation of muscular dystrophy model rats with a CRISPR/Cas system.

PubMed

Nakamura, Katsuyuki; Fujii, Wataru; Tsuboi, Masaya; Tanihata, Jun; Teramoto, Naomi; Takeuchi, Shiho; Naito, Kunihiko; Yamanouchi, Keitaro; Nishihara, Masugi

2014-07-09

Duchenne muscular dystrophy (DMD) is an X-linked lethal muscle disorder caused by mutations in the Dmd gene encoding Dystrophin. DMD model animals, such as mdx mice and canine X-linked muscular dystrophy dogs, have been widely utilized in the development of a treatment for DMD. Here, we demonstrate the generation of Dmd-mutated rats using a clustered interspaced short palindromic repeats (CRISPR)/Cas system, an RNA-based genome engineering technique that is also adaptive to rats. We simultaneously targeted two exons in the rat Dmd gene, which resulted in the absence of Dystrophin expression in the F0 generation. Dmd-mutated rats exhibited a decline in muscle strength, and the emergence of degenerative/regenerative phenotypes in the skeletal muscle, heart, and diaphragm. These mutations were heritable by the next generation, and F1 male rats exhibited similar phenotypes in their skeletal muscles. These model rats should prove to be useful for developing therapeutic methods to treat DMD.

Identification of Mutations Causing Aberrant Termination and Deficient Splice Donor Site on the HBA1 Gene.

PubMed

Farashi, Samaneh; Vakili, Shadi; Garous, Negin F; Ashki, Mehri; Forouzesh Pour, Fatemeh; Zeinali, Fatemeh; Rad, Fariba; Imanian, Hashem; Azarkeivan, Azita; Najmabadi, Hossein

2016-01-01

α-Thalassemia (α-thal) is a common genetic disorder in Iran and many parts of the world. Genetic defects on the α-globin gene cluster can result in α-thal that may develop a clinical phenotype varying from almost asymptomatic to a lethal hemolytic anemia. In the present study, four Iranian individuals with hypochromic microcytic anemia, who revealed none of the known mutations responsible for α-thal, were subjected for further investigations. The thalassemic phenotype of these patients resulted from abnormal RNA splicing sites owing to a missense at the splice donor site, a truncated protein or hemoglobin (Hb) variants as a result of two different substitutions on the α1-globin gene. The clinical presentation of mild anemia in these individuals showed the contribution of these novel mutations in α-thal in spite of the dominantly expressed α2-globin gene. This study describes hematological manifestations of subjects carrying some novel mutations comparable to the reported phenotype of α(+)-thal trait.

A de novo mutation in KIT causes white spotting in a subpopulation of German Shepherd dogs.

PubMed

Wong, A K; Ruhe, A L; Robertson, K R; Loew, E R; Williams, D C; Neff, M W

2013-06-01

Although variation in the KIT gene is a common cause of white spotting among domesticated animals, KIT has not been implicated in the diverse white spotting observed in the dog. Here, we show that a loss-of-function mutation in KIT recapitulates the coat color phenotypes observed in other species. A spontaneous white spotting observed in a pedigree of German Shepherd dogs was mapped by linkage analysis to a single locus on CFA13 containing KIT (pairwise LOD = 15). DNA sequence analysis identified a novel 1-bp insertion in the second exon that co-segregated with the phenotype. The expected frameshift and resulting premature stop codons predicted a severely truncated c-Kit receptor with presumably abolished activity. No dogs homozygous for the mutation were recovered from multiple intercrosses (P = 0.01), suggesting the mutation is recessively embryonic lethal. These observations are consistent with the effects of null alleles of KIT in other species. © 2012 The Authors, Animal Genetics © 2012 Stichting International Foundation for Animal Genetics.

Chromosomal Fragmentation in "Escherichia Coli": Its Absence in "mutT" Mutants and Its Mechanisms in "seqA" Mutants

ERIC Educational Resources Information Center

Rotman, Ella Rose

2009-01-01

Chromosomal fragmentation in "Escherichia coli" is a lethal event for the cell unless mended by the recombinational repair proteins RecA, RecBCD, and RuvABC. Certain mutations exacerbate problems that cause the cell to be dependent on the recombinational repair proteins for viability. We tested whether the absence of the MutT protein caused…

Identification of Lethal Mutations in Yeast Threonyl-tRNA Synthetase Revealing Critical Residues in Its Human Homolog*

PubMed Central

Ruan, Zhi-Rong; Fang, Zhi-Peng; Ye, Qing; Lei, Hui-Yan; Eriani, Gilbert; Zhou, Xiao-Long; Wang, En-Duo

2015-01-01

Aminoacyl-tRNA synthetases (aaRSs) are a group of ancient enzymes catalyzing aminoacylation and editing reactions for protein biosynthesis. Increasing evidence suggests that these critical enzymes are often associated with mammalian disorders. Therefore, complete determination of the enzymes functions is essential for informed diagnosis and treatment. Here, we show that a yeast knock-out strain for the threonyl-tRNA synthetase (ThrRS) gene is an excellent platform for such an investigation. Saccharomyces cerevisiae ThrRS has a unique modular structure containing four structural domains and a eukaryote-specific N-terminal extension. Using randomly mutated libraries of the ThrRS gene (thrS) and a genetic screen, a set of loss-of-function mutants were identified. The mutations affected the synthetic and editing activities and influenced the dimer interface. The results also highlighted the role of the N-terminal extension for enzymatic activity and protein stability. To gain insights into the pathological mechanisms induced by mutated aaRSs, we systematically introduced the loss-of-function mutations into the human cytoplasmic ThrRS gene. All mutations induced similar detrimental effects, showing that the yeast model could be used to study pathology-associated point mutations in mammalian aaRSs. PMID:25416776

INTERACTION OF X AND ULTRAVIOLET RADIATION IN PRODUCTION OF RECESSIVE LETHALS IN DROSOPHILA MELANOGASTER (in Italian)

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

Nicoletti, B.; Olivieri, G.

1962-01-01

The possibility that uv rays given to different biological systems before or after x rays could modify genetic or cytological effects is reviewed and discussed. Kaufmann and Hollaender's conclusions about the recovering effect of uv rays on chromosomal damage induced in Drosophila sperms by a pre-treatment of x rays are discussed and analyzed taking into accourt some general considerations. Preliminary results of similar experiments on the frequency of sex-linked recessive lethals induced after single and combined x + uv treatments in Drosophila sperms are reported. All our experiments indicate no effect of the uv treatment (at the given wave lengthsmore » and doses) in lowering the frequency of the x-ray-induced recessive lethals. On the contrary, there are some indications for a synergistic action between the two radiations. These results not in agreement with the generally accepted theory that uv rays do recover X-ray- induced chromosomal damages, could be expiained With the well established correlation between chromosomal rejoined breaks and genic mutations. (auth)« less

Interferon-induced TRAIL-independent cell death in DNase II-/- embryos.

PubMed

Kitahara, Yusuke; Kawane, Kohki; Nagata, Shigekazu

2010-09-01

The chromosomal DNA of apoptotic cells and the nuclear DNA expelled from erythroid precursors is cleaved by DNase II in lysosomes after the cells or nuclei are engulfed by macrophages. DNase II(-/-) embryos suffer from lethal anemia due to IFN-beta produced in the macrophages carrying undigested DNA. Here, we show that Type I IFN induced a caspase-dependent cell death in human epithelial cells that were transformed to express a high level of IFN type I receptor. During this death process, a set of genes was strongly activated, one of which encoded TRAIL, a death ligand. A high level of TRAIL mRNA was also found in the fetal liver of the lethally anemic DNase II(-/-) embryos, and a lack of IFN type I receptor in the DNase II(-/-) IFN-IR(-/-) embryos blocked the expression of TRAIL mRNA. However, a null mutation in TRAIL did not rescue the lethal anemia of the DNase II(-/-) embryos, indicating that TRAIL is dispensable for inducing the apoptosis of erythroid cells in DNase II(-/-) embryos, and therefore, that there is a TRAIL-independent mechanism for the IFN-induced apoptosis.

A spontaneous mutation of the Wwox gene and audiogenic seizures in rats with lethal dwarfism and epilepsy.

PubMed

Suzuki, H; Katayama, K; Takenaka, M; Amakasu, K; Saito, K; Suzuki, K

2009-10-01

The lde/lde rat is characterized by dwarfism, postnatal lethality, male hypogonadism, a high incidence of epilepsy and many vacuoles in the hippocampus and amygdala. We used a candidate approach to identify the gene responsible for the lde phenotype and assessed the susceptibility of lde/lde rats for audiogenic seizures. Following backcross breeding of lethal dwarfism with epilepsy (LDE) to Brown Norway rats, the lde/lde rats with an altered genetic background showed all pleiotropic phenotypes. The lde locus was mapped to a 1.5-Mbp region on rat chromosome 19 that included the latter half of the Wwox gene. Sequencing of the full-length Wwox transcript identified a 13-bp deletion in exon 9 in lde/lde rats. This mutation causes a frame shift, resulting in aberrant amino acid sequences at the C-terminal. Western blotting showed that both the full-length products of the Wwox gene and its isoform were present in normal testes and hippocampi, whereas both products were undetectable in the testes and hippocampi of lde/lde rats. Sound stimulation induced epileptic seizures in 95% of lde/lde rats, with starting as wild running (WR), sometimes progressing to tonic-clonic convulsions. Electroencephalogram (EEG) analysis showed interictal spikes, fast waves during WR and burst of spikes during clonic phases. The Wwox protein is expressed in the central nervous system (CNS), indicating that abnormal neuronal excitability in lde/lde rats may be because of a lack of Wwox function. The lde/lde rat is not only useful for understanding the multiple functions of Wwox but is also a unique model for studying the physiological function of Wwox in CNS.

Adaptation and Preadaptation of Salmonella enterica to Bile

PubMed Central

Hernández, Sara B.; Cota, Ignacio; Ducret, Adrien; Aussel, Laurent; Casadesús, Josep

2012-01-01

Bile possesses antibacterial activity because bile salts disrupt membranes, denature proteins, and damage DNA. This study describes mechanisms employed by the bacterium Salmonella enterica to survive bile. Sublethal concentrations of the bile salt sodium deoxycholate (DOC) adapt Salmonella to survive lethal concentrations of bile. Adaptation seems to be associated to multiple changes in gene expression, which include upregulation of the RpoS-dependent general stress response and other stress responses. The crucial role of the general stress response in adaptation to bile is supported by the observation that RpoS− mutants are bile-sensitive. While adaptation to bile involves a response by the bacterial population, individual cells can become bile-resistant without adaptation: plating of a non-adapted S. enterica culture on medium containing a lethal concentration of bile yields bile-resistant colonies at frequencies between 10−6 and 10−7 per cell and generation. Fluctuation analysis indicates that such colonies derive from bile-resistant cells present in the previous culture. A fraction of such isolates are stable, indicating that bile resistance can be acquired by mutation. Full genome sequencing of bile-resistant mutants shows that alteration of the lipopolysaccharide transport machinery is a frequent cause of mutational bile resistance. However, selection on lethal concentrations of bile also provides bile-resistant isolates that are not mutants. We propose that such isolates derive from rare cells whose physiological state permitted survival upon encountering bile. This view is supported by single cell analysis of gene expression using a microscope fluidic system: batch cultures of Salmonella contain cells that activate stress response genes in the absence of DOC. This phenomenon underscores the existence of phenotypic heterogeneity in clonal populations of bacteria and may illustrate the adaptive value of gene expression fluctuations. PMID:22275872

The Hmr and Lhr Hybrid Incompatibility Genes Suppress a Broad Range of Heterochromatic Repeats

PubMed Central

Satyaki, P. R. V.; Cuykendall, Tawny N.; Wei, Kevin H-C.; Brideau, Nicholas J.; Kwak, Hojoong; Aruna, S.; Ferree, Patrick M.; Ji, Shuqing; Barbash, Daniel A.

2014-01-01

Hybrid incompatibilities (HIs) cause reproductive isolation between species and thus contribute to speciation. Several HI genes encode adaptively evolving proteins that localize to or interact with heterochromatin, suggesting that HIs may result from co-evolution with rapidly evolving heterochromatic DNA. Little is known, however, about the intraspecific function of these HI genes, the specific sequences they interact with, or the evolutionary forces that drive their divergence. The genes Hmr and Lhr genetically interact to cause hybrid lethality between Drosophila melanogaster and D. simulans, yet mutations in both genes are viable. Here, we report that Hmr and Lhr encode proteins that form a heterochromatic complex with Heterochromatin Protein 1 (HP1a). Using RNA-Seq analyses we discovered that Hmr and Lhr are required to repress transcripts from satellite DNAs and many families of transposable elements (TEs). By comparing Hmr and Lhr function between D. melanogaster and D. simulans we identify several satellite DNAs and TEs that are differentially regulated between the species. Hmr and Lhr mutations also cause massive overexpression of telomeric TEs and significant telomere lengthening. Hmr and Lhr therefore regulate three types of heterochromatic sequences that are responsible for the significant differences in genome size and structure between D. melanogaster and D. simulans and have high potential to cause genetic conflicts with host fitness. We further find that many TEs are overexpressed in hybrids but that those specifically mis-expressed in lethal hybrids do not closely correlate with Hmr function. Our results therefore argue that adaptive divergence of heterochromatin proteins in response to repetitive DNAs is an important underlying force driving the evolution of hybrid incompatibility genes, but that hybrid lethality likely results from novel epistatic genetic interactions that are distinct to the hybrid background. PMID:24651406

Monoketone analogs of curcumin, a new class of Fanconi anemia pathway inhibitors.

PubMed

Landais, Igor; Hiddingh, Sanne; McCarroll, Matthew; Yang, Chao; Sun, Aiming; Turker, Mitchell S; Snyder, James P; Hoatlin, Maureen E

2009-12-31

The Fanconi anemia (FA) pathway is a multigene DNA damage response network implicated in the repair of DNA lesions that arise during replication or after exogenous DNA damage. The FA pathway displays synthetic lethal relationship with certain DNA repair genes such as ATM (Ataxia Telangectasia Mutated) that are frequently mutated in tumors. Thus, inhibition of FANCD2 monoubiquitylation (FANCD2-Ub), a key step in the FA pathway, might target tumor cells defective in ATM through synthetic lethal interaction. Curcumin was previously identified as a weak inhibitor of FANCD2-Ub. The aim of this study is to identify derivatives of curcumin with better activity and specificity. Using a replication-free assay in Xenopus extracts, we screened monoketone analogs of curcumin for inhibition of FANCD2-Ub and identified analog EF24 as a strong inhibitor. Mechanistic studies suggest that EF24 targets the FA pathway through inhibition of the NF-kB pathway kinase IKK. In HeLa cells, nanomolar concentrations of EF24 inhibited hydroxyurea (HU)-induced FANCD2-Ub and foci in a cell-cycle independent manner. Survival assays revealed that EF24 specifically sensitizes FA-competent cells to the DNA crosslinking agent mitomycin C (MMC). In addition, in contrast with curcumin, ATM-deficient cells are twofold more sensitive to EF24 than matched wild-type cells, consistent with a synthetic lethal effect between FA pathway inhibition and ATM deficiency. An independent screen identified 4H-TTD, a compound structurally related to EF24 that displays similar activity in egg extracts and in cells. These results suggest that monoketone analogs of curcumin are potent inhibitors of the FA pathway and constitute a promising new class of targeted anticancer compounds.

Monoketone analogs of curcumin, a new class of Fanconi anemia pathway inhibitors

PubMed Central

2009-01-01

Background The Fanconi anemia (FA) pathway is a multigene DNA damage response network implicated in the repair of DNA lesions that arise during replication or after exogenous DNA damage. The FA pathway displays synthetic lethal relationship with certain DNA repair genes such as ATM (Ataxia Telangectasia Mutated) that are frequently mutated in tumors. Thus, inhibition of FANCD2 monoubiquitylation (FANCD2-Ub), a key step in the FA pathway, might target tumor cells defective in ATM through synthetic lethal interaction. Curcumin was previously identified as a weak inhibitor of FANCD2-Ub. The aim of this study is to identify derivatives of curcumin with better activity and specificity. Results Using a replication-free assay in Xenopus extracts, we screened monoketone analogs of curcumin for inhibition of FANCD2-Ub and identified analog EF24 as a strong inhibitor. Mechanistic studies suggest that EF24 targets the FA pathway through inhibition of the NF-kB pathway kinase IKK. In HeLa cells, nanomolar concentrations of EF24 inhibited hydroxyurea (HU)-induced FANCD2-Ub and foci in a cell-cycle independent manner. Survival assays revealed that EF24 specifically sensitizes FA-competent cells to the DNA crosslinking agent mitomycin C (MMC). In addition, in contrast with curcumin, ATM-deficient cells are twofold more sensitive to EF24 than matched wild-type cells, consistent with a synthetic lethal effect between FA pathway inhibition and ATM deficiency. An independent screen identified 4H-TTD, a compound structurally related to EF24 that displays similar activity in egg extracts and in cells. Conclusions These results suggest that monoketone analogs of curcumin are potent inhibitors of the FA pathway and constitute a promising new class of targeted anticancer compounds. PMID:20043851

Experimental Determination and Prediction of the Fitness Effects of Random Point Mutations in the Biosynthetic Enzyme HisA

PubMed Central

Lundin, Erik; Tang, Po-Cheng; Guy, Lionel; Näsvall, Joakim; Andersson, Dan I

2018-01-01

Abstract The distribution of fitness effects of mutations is a factor of fundamental importance in evolutionary biology. We determined the distribution of fitness effects of 510 mutants that each carried between 1 and 10 mutations (synonymous and nonsynonymous) in the hisA gene, encoding an essential enzyme in the l-histidine biosynthesis pathway of Salmonella enterica. For the full set of mutants, the distribution was bimodal with many apparently neutral mutations and many lethal mutations. For a subset of 81 single, nonsynonymous mutants most mutations appeared neutral at high expression levels, whereas at low expression levels only a few mutations were neutral. Furthermore, we examined how the magnitude of the observed fitness effects was correlated to several measures of biophysical properties and phylogenetic conservation.We conclude that for HisA: (i) The effect of mutations can be masked by high expression levels, such that mutations that are deleterious to the function of the protein can still be neutral with regard to organism fitness if the protein is expressed at a sufficiently high level; (ii) the shape of the fitness distribution is dependent on the extent to which the protein is rate-limiting for growth; (iii) negative epistatic interactions, on an average, amplified the combined effect of nonsynonymous mutations; and (iv) no single sequence-based predictor could confidently predict the fitness effects of mutations in HisA, but a combination of multiple predictors could predict the effect with a SD of 0.04 resulting in 80% of the mutations predicted within 12% of their observed selection coefficients. PMID:29294020

Introduction of translation stop codons into the viral glycoprotein gene in a fish DNA vaccine eliminates induction of protective immunity

USGS Publications Warehouse

Garver, K.A.; Conway, C.M.; Kurath, G.

2006-01-01

A highly efficacious DNA vaccine against a fish rhabdovirus, infectious hematopoietic necrosis virus (IHNV), was mutated to introduce two stop codons to prevent glycoprotein translation while maintaining the plasmid DNA integrity and RNA transcription ability. The mutated plasmid vaccine, denoted pIHNw-G2stop, when injected intramuscularly into fish at high doses, lacked detectable glycoprotein expression in the injection site muscle, and did not provide protection against lethal virus challenge 7 days post-vaccination. These results suggest that the G-protein itself is required to stimulate the early protective antiviral response observed after vaccination with the nonmutated parental DNA vaccine. ?? Springer Science+Business Media, Inc. 2006.

Introduction of translation stop condons into the viral glycoprotein gene in a fish DNA vaccine eliminates induction of protective immunity

USGS Publications Warehouse

Garver, Kyle A.; Conway, Carla M.; Kurath, Gael

2006-01-01

A highly efficacious DNA vaccine against a fish rhabdovirus, infectious hematopoietic necrosis virus (IHNV), was mutated to introduce two stop codons to prevent glycoprotein translation while maintaining the plasmid DNA integrity and RNA transcription ability. The mutated plasmid vaccine, denoted pIHNw-G2stop, when injected intramuscularly into fish at high doses, lacked detectable glycoprotein expression in the injection site muscle, and did not provide protection against lethal virus challenge 7 days post-vaccination. These results suggest that the G-protein itself is required to stimulate the early protective antiviral response observed after vaccination with the nonmutated parental DNA vaccine.

Three patients with Schaaf-Yang syndrome exhibiting arthrogryposis and endocrinological abnormalities.

PubMed

Enya, Takuji; Okamoto, Nobuhiko; Iba, Yoshinori; Miyazawa, Tomoki; Okada, Mitsuru; Ida, Shinobu; Naruto, Takuya; Imoto, Issei; Fujita, Atsushi; Miyake, Noriko; Matsumoto, Naomichi; Sugimoto, Keisuke; Takemura, Tsukasa

2018-03-01

MAGEL2 is the paternally expressed gene within Prader-Willi syndrome critical region at 15q11.2. We encountered three individuals in whom truncating mutations of MAGEL2 were identified. Patients 1 and 2, siblings born to healthy, non-consanguineous Japanese parents, showed generalized hypotonia, lethargy, severe respiratory difficulty, poor feeding, and multiple anomalies including arthrogryposis soon after birth. We carried out whole-exome sequencing, which detected a MAGEL2 mutation (c.1912C>T, p.Gln638*, heterozygous). The patients' father was heterozygous for the mutation. Patient 3 was a female infant, showed respiratory difficulty reflecting pulmonary hypoplasia, generalized hypotonia, feeding difficulty and multiple anomalies soon after birth. Targeted next-generation sequencing detected a novel heterozygous mutation in MAGEL2 (c.3131C>A, p.Ser1044*). This mutation was not found in the parents. MAGEL2 mutations, first reported to be the cause of the Prader-Willi like syndrome with autism by Schaaf et al. (2013) Nature Genetics, 45: 1405-1408 show the wide range of phenotypic spectrum from lethal arthrogryposis multiplex congenital to autism spectrum disorder (ASD) and mild intellectual disability (ID). Our results indicate that MAGEL2 mutations cause multiple congenital anomalies and intellectual disability accompanied by arthrogryposis multiplex congenita and various endocrinologic abnormalities, supporting that the view that clinical phenotypes of MAGEL2 mutations are variable. © 2018 Wiley Periodicals, Inc.

The role of PARP inhibition in triple-negative breast cancer: Unraveling the wide spectrum of synthetic lethality.

PubMed

Papadimitriou, Marios; Mountzios, Giannis; Papadimitriou, Christos A

2018-05-02

Triple-negative breast cancer (TNBC) accounts for approximately 15-20% of all breast cancers and is characterized by a lack of immunohistochemical expression of estrogen receptors (ER), progesterone receptors (PR) and HER2. TNBC is associated with poor long-term outcomes compared with other breast cancer subtypes. Many of these tumors are also basal-like cancers which are characterized by an aggressive biological behavior with a distant recurrence peak observed early at 3 years following diagnosis. Furthermore, metastatic TNBC bears a dismal prognosis with an average survival of 12 months. Although the prevalence of genetic alterations among women with TNBC differs significantly by ethnicity, race and age, BRCA mutations (including both germline mutations and somatic genetic aberrations) are found in up to 20-25% of unselected patients and especially in those of the basal-like immunophenotype. Therefore, defects in the DNA repair pathway could represent a promising therapeutic target for this subgroup of TNBC patients. Poly(ADP-ribose) polymerase (PARP) inhibitors exploit this deficiency through synthetic lethality and have emerged as promising anticancer therapies, especially in BRCA1 or BRCA2 mutation carriers. Several PARP inhibitors are currently being evaluated in the adjuvant, neo-adjuvant, and metastatic setting for the treatment of breast cancer patients with a deficient homologous recombination pathway. In this article, we review the major molecular characteristics of TNBC, the mechanisms of homologous recombination, and the role of PARP inhibition as an emerging therapeutic strategy. Copyright © 2018 Elsevier Ltd. All rights reserved.

The Yeast Gene, MDM20, Is Necessary for Mitochondrial Inheritance and Organization of the Actin Cytoskeleton

PubMed Central

Hermann, Greg J.; King, Edward J.; Shaw, Janet M.

1997-01-01

In Saccharomyces cerevisiae, the growing bud inherits a portion of the mitochondrial network from the mother cell soon after it emerges. Although this polarized transport of mitochondria is thought to require functions of the cytoskeleton, there are conflicting reports concerning the nature of the cytoskeletal element involved. Here we report the isolation of a yeast mutant, mdm20, in which both mitochondrial inheritance and actin cables (bundles of actin filaments) are disrupted. The MDM20 gene encodes a 93-kD polypeptide with no homology to other characterized proteins. Extra copies of TPM1, a gene encoding the actin filament–binding protein tropomyosin, suppress mitochondrial inheritance defects and partially restore actin cables in mdm20Δ cells. Synthetic lethality is also observed between mdm20 and tpm1 mutant strains. Overexpression of a second yeast tropomyosin, Tpm2p, rescues mutant phenotypes in the mdm20 strain to a lesser extent. Together, these results provide compelling evidence that mitochondrial inheritance in yeast is an actin-mediated process. MDM20 and TPM1 also exhibit the same pattern of genetic interactions; mutations in MDM20 are synthetically lethal with mutations in BEM2 and MYO2 but not SAC6. Although MDM20 and TPM1 are both required for the formation and/or stabilization of actin cables, mutations in these genes disrupt mitochondrial inheritance and nuclear segregation to different extents. Thus, Mdm20p and Tpm1p may act in vivo to establish molecular and functional heterogeneity of the actin cytoskeleton. PMID:9105043

C-terminal phenylalanine of bacteriophage T7 single-stranded DNA-binding protein is essential for strand displacement synthesis by T7 DNA polymerase at a nick in DNA.

PubMed

Ghosh, Sharmistha; Marintcheva, Boriana; Takahashi, Masateru; Richardson, Charles C

2009-10-30

Single-stranded DNA-binding protein (gp2.5), encoded by gene 2.5 of bacteriophage T7, plays an essential role in DNA replication. Not only does it remove impediments of secondary structure in the DNA, it also modulates the activities of the other replication proteins. The acidic C-terminal tail of gp2.5, bearing a C-terminal phenylalanine, physically and functionally interacts with the helicase and DNA polymerase. Deletion of the phenylalanine or substitution with a nonaromatic amino acid gives rise to a dominant lethal phenotype, and the altered gp2.5 has reduced affinity for T7 DNA polymerase. Suppressors of the dominant lethal phenotype have led to the identification of mutations in gene 5 that encodes the T7 DNA polymerase. The altered residues in the polymerase are solvent-exposed and lie in regions that are adjacent to the bound DNA. gp2.5 lacking the C-terminal phenylalanine has a lower affinity for gp5-thioredoxin relative to the wild-type gp2.5, and this affinity is partially restored by the suppressor mutations in DNA polymerase. gp2.5 enables T7 DNA polymerase to catalyze strand displacement DNA synthesis at a nick in DNA. The resulting 5'-single-stranded DNA tail provides a loading site for T7 DNA helicase. gp2.5 lacking the C-terminal phenylalanine does not support this event with wild-type DNA polymerase but does to a limited extent with T7 DNA polymerase harboring the suppressor mutations.

A rare complex DNA rearrangement in the murine Steel gene results in exon duplication and a lethal phenotype.

PubMed

Chandra, Saurabh; Kapur, Reuben; Chuzhanova, Nadia; Summey, Victoria; Prentice, David; Barker, Jane; Cooper, David N; Williams, David A

2003-11-15

Kit ligand (Kitl), encoded by the Steel (Sl) locus, plays an essential role in hematopoiesis, gametogenesis, and melanogenesis during both embryonic and adult life. We have characterized a new spontaneous mutant of the Sl locus in mice designated KitlSl-20J that arose in the breeding colony at Jackson Laboratories. Heterozygous KitlSl-20J mice display a white belly spot and intercrossing results in an embryonic lethal phenotype in the homozygous state. Analysis of homozygous embryos demonstrated a significant reduction in fetal liver cellularity, colony forming unit-erythroid (CFU-E) progenitors, and a total absence of germ cells. Although expressed in vivo, recombinant mutant protein demonstrated loss of bioactivity that was correlated with lack of receptor binding. Analysis of the Sl gene transcripts in heterozygous KitlSl-20J mice revealed an in-frame tandem duplication of exon 3. A long-range polymerase chain reaction (PCR) strategy using overlapping primers in exon 3 amplified an approximately 7-kilobase (kb) product from DNA isolated from heterozygous KitlSl-20J mice but not from wild-type DNA that contained sequences from both introns 2 and 3 and an inverted intron 2 sequence, suggesting a complex rearrangement as the mechanism of the mutation. "Complexity analysis" of the sequence of the amplified product strongly suggests that local DNA motifs may have contributed to the generation of this spontaneous KitlSl-20J allele, likely mediated by a 2-step process. The KitlSl-20J mutation is a unique KitlSl allele and represents an unusual mechanism of mutation.

Generation of the first Autosomal Dominant Osteopetrosis Type II (ADO2) disease models

PubMed Central

Alam, Imranul; Gray, Amie K.; Chu, Kang; Ichikawa, Shoji; Mohammad, Khalid S.; Capannolo, Marta; Capulli, Mattia; Maurizi, Antonio; Muraca, Maurizio; Teti, Anna; Econs, Michael J.; Fattore, Andrea Del

2013-01-01

Autosomal Dominant Osteopetrosis Type II (ADO2) is a heritable osteosclerotic disorder dependent on osteoclast impairment. In most patients it results from heterozygous missense mutations in the chloride channel 7 (CLCN7) gene, encoding for a 2Cl−/1H+ antiporter. By a knock-in strategy inserting a missense mutation in the Clcn7 gene, our two research groups independently generated mouse models of ADO2 on different genetic backgrounds carrying the homolog of the most frequent heterozygous mutation (p.G213R) in the Clcn7 gene found in humans. Our results demonstrate that the heterozygous model holds true presenting with higher bone mass, increased numbers of poorly resorbing osteoclasts and a lethal phenotype in the homozygous state. Considerable variability is observed in the heterozygous mice according with the mouse background, suggesting that modifier genes could influence the penetrance of the disease gene. PMID:24185277

Spontaneous Generation of Infectious Prion Disease in Transgenic Mice

PubMed Central

Castilla, Joaquín; Pintado, Belén; Gutiérrez-Adan, Alfonso; Andréoletti, Olivier; Aguilar-Calvo, Patricia; Arroba, Ana-Isabel; Parra-Arrondo, Beatriz; Ferrer, Isidro; Manzanares, Jorge; Espinosa, Juan-Carlos

2013-01-01

We generated transgenic mice expressing bovine cellular prion protein (PrPC) with a leucine substitution at codon 113 (113L). This protein is homologous to human protein with mutation 102L, and its genetic link with Gerstmann–Sträussler–Scheinker syndrome has been established. This mutation in bovine PrPC causes a fully penetrant, lethal, spongiform encephalopathy. This genetic disease was transmitted by intracerebral inoculation of brain homogenate from ill mice expressing mutant bovine PrP to mice expressing wild-type bovine PrP, which indicated de novo generation of infectious prions. Our findings demonstrate that a single amino acid change in the PrPC sequence can induce spontaneous generation of an infectious prion disease that differs from all others identified in hosts expressing the same PrPC sequence. These observations support the view that a variety of infectious prion strains might spontaneously emerge in hosts displaying random genetic PrPC mutations. PMID:24274622

Characterization of a Novel MMS-Sensitive Allele of Schizosaccharomyces pombe mcm4+

PubMed Central

Ranatunga, Nimna S.; Forsburg, Susan L.

2016-01-01

The minichromosome maintenance (MCM) complex is the conserved helicase motor of the eukaryotic replication fork. Mutations in the Mcm4 subunit are associated with replication stress and double strand breaks in multiple systems. In this work, we characterize a new temperature-sensitive allele of Schizosaccharomyces pombe mcm4+. Uniquely among known mcm4 alleles, this mutation causes sensitivity to the alkylation damaging agent methyl methanesulfonate (MMS). Even in the absence of treatment or temperature shift, mcm4-c106 cells show increased repair foci of RPA and Rad52, and require the damage checkpoint for viability, indicating genome stress. The mcm4-c106 mutant is synthetically lethal with mutations disrupting fork protection complex (FPC) proteins Swi1 and Swi3. Surprisingly, we found that the deletion of rif1+ suppressed the MMS-sensitive phenotype without affecting temperature sensitivity. Together, these data suggest that mcm4-c106 destabilizes replisome structure. PMID:27473316

Radiation effects in nematodes: Results from IML-1 experiments

NASA Technical Reports Server (NTRS)

Nelson, G. A.; Schubert, W. W.; Kazarians, G. A.; Richards, G. F.; Benton, E. V.; Benton, E. R.; Henke, R.

1994-01-01

The nematode Caenorhabditis elegans was exposed to natural space radiation using the ESA biorack facility aboard Spacelab on International Microgravity Laboratory 1, STS-42. For the major experimental objective dormant animals were suspended in buffer or on agar or immobilized next to CR-39 plastic nuclear track detectors to correlate fluence of HZE particles with genetic events. This configuration was used to isolate mutations in a set of 350 essential genes as well as in the unc-22 structural gene. From flight samples 13 mutants in the unc-22 gene were isolated along with 53 lethal mutations from autosomal regions balanced by a translocation eT1(III;V). Preliminary analysis suggests that mutants from worms correlated with specific cosmic ray tracks may have a higher proportion of rearrangements than those isolated from tube cultures on a randomly sampled basis. Flight sample mutation rate was approximately 8-fold higher than ground controls which exhibited laboratory spontaneous frequencies.

The nonreceptor protein tyrosine phosphatase corkscrew functions in multiple receptor tyrosine kinase pathways in Drosophila.

PubMed

Perkins, L A; Johnson, M R; Melnick, M B; Perrimon, N

1996-11-25

Corkscrew (csw) encodes a nonreceptor protein tyrosine phosphatase (PTPase) that has been implicated in signaling from the Torso receptor tyrosine kinase (RTK). csw mutations, unlike tor mutations, are associated with zygotic lethality, indicating that Csw plays additional roles during development. We have conducted a detailed phenotypic analysis of csw mutations to identify these additional functions of Csw. Our results indicate that Csw operates positively downstream of other Drosophila RTKs such as the Drosophila epidermal growth factor receptor (DER), the fibroblast growth factor receptor (Breathless), and likely other RTKs. This model is substantiated by specific dosage interactions between csw and DER. It is proposed that Csw is part of the evolutionarily conserved "signaling cassette" that operates downstream of all RTKs. In support of this hypothesis, we demonstrate that SHP-2, a vertebrate PTPase similar to Csw and previously implicated in RTK signaling, encodes the functional vertebrate homologue of Csw.

Synthetic Lethal Screens Identify Vulnerabilities in GPCR Signaling and Cytoskeletal Organization in E-Cadherin-Deficient Cells.

PubMed

Telford, Bryony J; Chen, Augustine; Beetham, Henry; Frick, James; Brew, Tom P; Gould, Cathryn M; Single, Andrew; Godwin, Tanis; Simpson, Kaylene J; Guilford, Parry

2015-05-01

The CDH1 gene, which encodes the cell-to-cell adhesion protein E-cadherin, is frequently mutated in lobular breast cancer (LBC) and diffuse gastric cancer (DGC). However, because E-cadherin is a tumor suppressor protein and lost from the cancer cell, it is not a conventional drug target. To overcome this, we have taken a synthetic lethal approach to determine whether the loss of E-cadherin creates druggable vulnerabilities. We first conducted a genome-wide siRNA screen of isogenic MCF10A cells with and without CDH1 expression. Gene ontology analysis demonstrated that G-protein-coupled receptor (GPCR) signaling proteins were highly enriched among the synthetic lethal candidates. Diverse families of cytoskeletal proteins were also frequently represented. These broad classes of E-cadherin synthetic lethal hits were validated using both lentiviral-mediated shRNA knockdown and specific antagonists, including the JAK inhibitor LY2784544, Pertussis toxin, and the aurora kinase inhibitors alisertib and danusertib. Next, we conducted a 4,057 known drug screen and time course studies on the CDH1 isogenic MCF10A cell lines and identified additional drug classes with linkages to GPCR signaling and cytoskeletal function that showed evidence of E-cadherin synthetic lethality. These included multiple histone deacetylase inhibitors, including vorinostat and entinostat, PI3K inhibitors, and the tyrosine kinase inhibitors crizotinib and saracatinib. Together, these results demonstrate that E-cadherin loss creates druggable vulnerabilities that have the potential to improve the management of both sporadic and familial LBC and DGC. ©2015 American Association for Cancer Research.

Systematic screening of isogenic cancer cells identifies DUSP6 as context-specific synthetic lethal target in melanoma

PubMed Central

Wittig-Blaich, Stephanie; Wittig, Rainer; Schmidt, Steffen; Lyer, Stefan; Bewerunge-Hudler, Melanie; Gronert-Sum, Sabine; Strobel-Freidekind, Olga; Müller, Carolin; List, Markus; Jaskot, Aleksandra; Christiansen, Helle; Hafner, Mathias; Schadendorf, Dirk; Block, Ines; Mollenhauer, Jan

2017-01-01

Next-generation sequencing has dramatically increased genome-wide profiling options and conceptually initiates the possibility for personalized cancer therapy. State-of-the-art sequencing studies yield large candidate gene sets comprising dozens or hundreds of mutated genes. However, few technologies are available for the systematic downstream evaluation of these results to identify novel starting points of future cancer therapies. We improved and extended a site-specific recombination-based system for systematic analysis of the individual functions of a large number of candidate genes. This was facilitated by a novel system for the construction of isogenic constitutive and inducible gain- and loss-of-function cell lines. Additionally, we demonstrate the construction of isogenic cell lines with combinations of the traits for advanced functional in vitro analyses. In a proof-of-concept experiment, a library of 108 isogenic melanoma cell lines was constructed and 8 genes were identified that significantly reduced viability in a discovery screen and in an independent validation screen. Here, we demonstrate the broad applicability of this recombination-based method and we proved its potential to identify new drug targets via the identification of the tumor suppressor DUSP6 as potential synthetic lethal target in melanoma cell lines with BRAF V600E mutations and high DUSP6 expression. PMID:28423600

TP53 mutations, expression and interaction networks in human cancers

PubMed Central

Wang, Xiaosheng; Sun, Qingrong

2017-01-01

Although the associations of p53 dysfunction, p53 interaction networks and oncogenesis have been widely explored, a systematic analysis of TP53 mutations and its related interaction networks in various types of human cancers is lacking. Our study explored the associations of TP53 mutations, gene expression, clinical outcomes, and TP53 interaction networks across 33 cancer types using data from The Cancer Genome Atlas (TCGA). We show that TP53 is the most frequently mutated gene in a number of cancers, and its mutations appear to be early events in cancer initiation. We identified genes potentially repressed by p53, and genes whose expression correlates significantly with TP53 expression. These gene products may be especially important nodes in p53 interaction networks in human cancers. This study shows that while TP53-truncating mutations often result in decreased TP53 expression, other non-truncating TP53 mutations result in increased TP53 expression in some cancers. Survival analyses in a number of cancers show that patients with TP53 mutations are more likely to have worse prognoses than TP53-wildtype patients, and that elevated TP53 expression often leads to poor clinical outcomes. We identified a set of candidate synthetic lethal (SL) genes for TP53, and validated some of these SL interactions using data from the Cancer Cell Line Project. These predicted SL genes are promising candidates for experimental validation and the development of personalized therapeutics for patients with TP53-mutated cancers. PMID:27880943

TP53 mutations, expression and interaction networks in human cancers.

PubMed

Wang, Xiaosheng; Sun, Qingrong

2017-01-03

Although the associations of p53 dysfunction, p53 interaction networks and oncogenesis have been widely explored, a systematic analysis of TP53 mutations and its related interaction networks in various types of human cancers is lacking. Our study explored the associations of TP53 mutations, gene expression, clinical outcomes, and TP53 interaction networks across 33 cancer types using data from The Cancer Genome Atlas (TCGA). We show that TP53 is the most frequently mutated gene in a number of cancers, and its mutations appear to be early events in cancer initiation. We identified genes potentially repressed by p53, and genes whose expression correlates significantly with TP53 expression. These gene products may be especially important nodes in p53 interaction networks in human cancers. This study shows that while TP53-truncating mutations often result in decreased TP53 expression, other non-truncating TP53 mutations result in increased TP53 expression in some cancers. Survival analyses in a number of cancers show that patients with TP53 mutations are more likely to have worse prognoses than TP53-wildtype patients, and that elevated TP53 expression often leads to poor clinical outcomes. We identified a set of candidate synthetic lethal (SL) genes for TP53, and validated some of these SL interactions using data from the Cancer Cell Line Project. These predicted SL genes are promising candidates for experimental validation and the development of personalized therapeutics for patients with TP53-mutated cancers.

Measuring the spectrum of mutation induced by nitrogen ions and protons in the human-hamster hybrid cell line A(L)C

NASA Technical Reports Server (NTRS)

Kraemer, S. M.; Kronenberg, A.; Ueno, A.; Waldren, C. A.; Chatterjee, A. (Principal Investigator)

2000-01-01

Astronauts can be exposed to charged particles, including protons, alpha particles and heavier ions, during space flights. Therefore, studying the biological effectiveness of these sparsely and densely ionizing radiations is important to understanding the potential health effects for astronauts. We evaluated the mutagenic effectiveness of sparsely ionizing 55 MeV protons and densely ionizing 32 MeV/nucleon nitrogen ions using cells of two human-hamster cell lines, A(L) and A(L)C. We have previously characterized a spectrum of mutations, including megabase deletions, in human chromosome 11, the sole human chromosome in the human-hamster hybrid cell lines A(L)C and A(L). CD59(-) mutants have lost expression of a human cell surface antigen encoded by the CD59 gene located at 11p13. Deletion of genes located on the tip of the short arm of 11 (11p15.5) is lethal to the A(L) hybrid, so that CD59 mutants that lose the entire chromosome 11 die and escape detection. In contrast, deletion of the 11p15.5 region is not lethal in the hybrid A(L)C, allowing for the detection of chromosome loss or other chromosomal mutations involving 11p15.5. The 55 MeV protons and 32 MeV/nucleon nitrogen ions were each about 10 times more mutagenic per unit dose at the CD59 locus in A(L)C cells than in A(L) cells. In the case of nitrogen ions, the mutations observed in A(L)C cells were predominantly due to chromosome loss events or 11p deletions, often containing a breakpoint in the pericentromeric region. The increase in the CD59(-) mutant fraction for A(L)C cells exposed to protons was associated with either translocation of portions of 11q onto a hamster chromosome, or discontinuous or "skipping" mutations. We demonstrate here that A(L)C cells are a powerful tool that will aid in the understanding of the mutagenic effects of different types of ionizing radiation.

The phenotypic spectrum in patients with arginine to cysteine mutations in the COL2A1 gene

PubMed Central

Hoornaert, K P; Dewinter, C; Vereecke, I; Beemer, F A; Courtens, W; Fryer, A; Fryssira, H; Lees, M; Müllner‐Eidenböck, A; Rimoin, D L; Siderius, L; Superti‐Furga, A; Temple, K; Willems, P J; Zankl, A; Zweier, C; De Paepe, A; Coucke, P; Mortier, G R

2006-01-01

Background The majority of COL2A1 missense mutations are substitutions of obligatory glycine residues in the triple helical domain. Only a few non‐glycine missense mutations have been reported and among these, the arginine to cysteine substitutions predominate. Objective To investigate in more detail the phenotype resulting from arginine to cysteine mutations in the COL2A1 gene. Methods The clinical and radiographic phenotype of all patients in whom an arginine to cysteine mutation in the COL2A1 gene was identified in our laboratory, was studied and correlated with the abnormal genotype. The COL2A1 genotyping involved DHPLC analysis with subsequent sequencing of the abnormal fragments. Results Six different mutations (R75C, R365C, R519C, R704C, R789C, R1076C) were found in 11 unrelated probands. Each mutation resulted in a rather constant and site‐specific phenotype, but a perinatally lethal disorder was never observed. Spondyloarthropathy with normal stature and no ocular involvement were features of patients with the R75C, R519C, or R1076C mutation. Short third and/or fourth toes was a distinguishing feature of the R75C mutation and brachydactyly with enlarged finger joints a key feature of the R1076C substitution. Stickler dysplasia with brachydactyly was observed in patients with the R704C mutation. The R365C and R789C mutations resulted in classic Stickler dysplasia and spondyloepiphyseal dysplasia congenita (SEDC), respectively. Conclusions Arginine to cysteine mutations are rather infrequent COL2A1 mutations which cause a spectrum of phenotypes including classic SEDC and Stickler dysplasia, but also some unusual entities that have not yet been recognised and described as type II collagenopathies. PMID:16155195

Mutations in PPIB (cyclophilin B) delay type I procollagen chain association and result in perinatal lethal to moderate osteogenesis imperfecta phenotypes.

PubMed

Pyott, Shawna M; Schwarze, Ulrike; Christiansen, Helena E; Pepin, Melanie G; Leistritz, Dru F; Dineen, Richard; Harris, Catharine; Burton, Barbara K; Angle, Brad; Kim, Katherine; Sussman, Michael D; Weis, Maryann; Eyre, David R; Russell, David W; McCarthy, Kevin J; Steiner, Robert D; Byers, Peter H

2011-04-15

Recessive mutations in the cartilage-associated protein (CRTAP), leucine proline-enriched proteoglycan 1 (LEPRE1) and peptidyl prolyl cis-trans isomerase B (PPIB) genes result in phenotypes that range from lethal in the perinatal period to severe deforming osteogenesis imperfecta (OI). These genes encode CRTAP (encoded by CRTAP), prolyl 3-hydroxylase 1 (P3H1; encoded by LEPRE1) and cyclophilin B (CYPB; encoded by PPIB), which reside in the rough endoplasmic reticulum (RER) and can form a complex involved in prolyl 3-hydroxylation in type I procollagen. CYPB, a prolyl cis-trans isomerase, has been thought to drive the prolyl-containing peptide bonds to the trans configuration needed for triple helix formation. Here, we describe mutations in PPIB identified in cells from three individuals with OI. Cultured dermal fibroblasts from the most severely affected infant make some overmodified type I procollagen molecules. Proα1(I) chains are slow to assemble into trimers, and abnormal procollagen molecules concentrate in the RER, and bind to protein disulfide isomerase (PDI) and prolyl 4-hydroxylase 1 (P4H1). These findings suggest that although CYPB plays a role in helix formation another effect is on folding of the C-terminal propeptide and trimer formation. The extent of procollagen accumulation and PDI/P4H1 binding differs among cells with mutations in PPIB, CRTAP and LEPRE1 with the greatest amount in PPIB-deficient cells and the least in LEPRE1-deficient cells. These findings suggest that prolyl cis-trans isomerase may be required to effectively fold the proline-rich regions of the C-terminal propeptide to allow proα chain association and suggest an order of action for CRTAP, P3H1 and CYPB in procollagen biosynthesis and pathogenesis of OI.

Mutations in PPIB (cyclophilin B) delay type I procollagen chain association and result in perinatal lethal to moderate osteogenesis imperfecta phenotypes

PubMed Central

Pyott, Shawna M.; Schwarze, Ulrike; Christiansen, Helena E.; Pepin, Melanie G.; Leistritz, Dru F.; Dineen, Richard; Harris, Catharine; Burton, Barbara K.; Angle, Brad; Kim, Katherine; Sussman, Michael D.; Weis, MaryAnn; Eyre, David R.; Russell, David W.; McCarthy, Kevin J.; Steiner, Robert D.; Byers, Peter H.

2011-01-01

Recessive mutations in the cartilage-associated protein (CRTAP), leucine proline-enriched proteoglycan 1 (LEPRE1) and peptidyl prolyl cis–trans isomerase B (PPIB) genes result in phenotypes that range from lethal in the perinatal period to severe deforming osteogenesis imperfecta (OI). These genes encode CRTAP (encoded by CRTAP), prolyl 3-hydroxylase 1 (P3H1; encoded by LEPRE1) and cyclophilin B (CYPB; encoded by PPIB), which reside in the rough endoplasmic reticulum (RER) and can form a complex involved in prolyl 3-hydroxylation in type I procollagen. CYPB, a prolyl cis–trans isomerase, has been thought to drive the prolyl-containing peptide bonds to the trans configuration needed for triple helix formation. Here, we describe mutations in PPIB identified in cells from three individuals with OI. Cultured dermal fibroblasts from the most severely affected infant make some overmodified type I procollagen molecules. Proα1(I) chains are slow to assemble into trimers, and abnormal procollagen molecules concentrate in the RER, and bind to protein disulfide isomerase (PDI) and prolyl 4-hydroxylase 1 (P4H1). These findings suggest that although CYPB plays a role in helix formation another effect is on folding of the C-terminal propeptide and trimer formation. The extent of procollagen accumulation and PDI/P4H1 binding differs among cells with mutations in PPIB, CRTAP and LEPRE1 with the greatest amount in PPIB-deficient cells and the least in LEPRE1-deficient cells. These findings suggest that prolyl cis–trans isomerase may be required to effectively fold the proline-rich regions of the C-terminal propeptide to allow proα chain association and suggest an order of action for CRTAP, P3H1 and CYPB in procollagen biosynthesis and pathogenesis of OI. PMID:21282188

Mutation induction in bacteria after heavy ion irradiation

NASA Technical Reports Server (NTRS)

Horneck, G.; Kozubek, S.

1994-01-01

From a compilation of experimental data on the mutagenic effects of heavy ions in bacteria, main conclusions have been drawn as follows: (1) The mutagenic efficacy of heavy ions in bacteria depends on physical and biological variables. Physical variables are the radiation dose, energy and charge of the ion; the biological variables are the bacterial strain, the repair genotype of bacteria, and the endpoint investigated (type of mutation, induction of enzymes related to mutagenesis); (2) The responses on dose or fluence are mainly linear or linear quadratic. The quadratic component, if found for low LET radiation, is gradually reduced with increasing LET; (3) At low values of Z and LET the cross section of mutation induction sigma m (as well as SOS response, sigma sos. and lambda phage induction, sigma lambda versus LET curves can be quite consistently described by a common function which increases up to approximately 100 keV/mu m. For higher LET values, the sigma(m) versus LET curves show the so-called 'hooks' observed also for other endpoints; (4) For light ions (Z is less than or equal to 4), the cross sections mostly decrease with increasing ion energy, which is probably related to the decrease of the specific energy departed by the ion inside the sensitive volume (cell). For ions in the range of Z = 10, sigma(m) is nearly independent on the ion energy. For heavier ions (Z is greater than or equal to 16), sigma(m) increases with the energy up to a maximum or saturation around 10 MeV/u. The increment becomes steeper with increasing atomic number of the ion. It correlates with the increasing track radius of the heavy ion; (5) The mutagenic efficiency per lethal event changes slightly with ion energy, if Z is small indicating a rough correlation between cellular lethality and mutation induction, only. For ions of higher Z this relation increases with energy, indicating a change in the 'mode' of radiation action from 'killing-prone' to 'mutation-prone'; and (6) Repair genotype substantially influences the radiation induced mutagenesis. Different mechanisms of mutation induction and/or different types of biologically significant lesions in wild type cells compared to repair deficient strains are a likely explanation.

A family of oculofaciocardiodental syndrome (OFCD) with a novel BCOR mutation and genomic rearrangements involving NHS.

PubMed

Kondo, Yukiko; Saitsu, Hirotomo; Miyamoto, Toshinobu; Nishiyama, Kiyomi; Tsurusaki, Yoshinori; Doi, Hiroshi; Miyake, Noriko; Ryoo, Na-Kyung; Kim, Jeong Hun; Yu, Young Suk; Matsumoto, Naomichi

2012-03-01

Oculofaciocardiodental syndrome (OFCD) is an X-linked dominant disorder associated with male lethality, presenting with congenital cataract, dysmorphic face, dental abnormalities and septal heart defects. Mutations in BCOR (encoding BCL-6-interacting corepressor) cause OFCD. Here, we report on a Korean family with common features of OFCD including bilateral 2nd-3rd toe syndactyly and septal heart defects in three affected females (mother and two daughters). Through the mutation screening and copy number analysis using genomic microarray, we identified a novel heterozygous mutation, c.888delG, in the BCOR gene and two interstitial microduplications at Xp22.2-22.13 and Xp21.3 in all the three affected females. The BCOR mutation may lead to a premature stop codon (p.N297IfsX80). The duplication at Xp22.2-22.13 involved the NHS gene causative for Nance-Horan syndrome, which is an X-linked disorder showing similar clinical features with OFCD in affected males, and in carrier females with milder presentation. Considering the presence of bilateral 2nd-3rd toe syndactyly and septal heart defects, which is unique to OFCD, the mutation in BCOR is likely to be the major determinant for the phenotypes in this family.

Oligosyndactylism Mice Have an Inversion of Chromosome 8

PubMed Central

Wise, Thomas L.; Pravtcheva, Dimitrina D.

2004-01-01

The radiation-induced mutation Oligosyndactylism (Os) is associated with limb and kidney defects in heterozygotes and with mitotic arrest and embryonic lethality in homozygotes. We reported that the cell cycle block in Os and in the 94-A/K transgene-induced mutations is due to disruption of the Anapc10 (Apc10/Doc1) gene. To understand the genetic basis of the limb and kidney abnormalities in Os mice we characterized the structural changes of chromosome 8 associated with this mutation. We demonstrate that the Os chromosome 8 has suffered two breaks that are 5 cM (∼10 Mb) apart and the internal fragment delineated by the breaks is in an inverted orientation on the mutant chromosome. While sequences in proximity to the distal break are present in an abnormal Os-specific Anapc10 hybrid transcript, transcription of these sequences in normal mice is low and difficult to detect. Transfer of the Os mutation onto an FVB/N background indicated that the absence of dominant effects in 94-A/K mice is not due to strain background effects on the mutation. Further analysis of this mutation will determine if a gene interrupted by the break or a long-range effect of the rearrangement on neighboring genes is responsible for the dominant effects of Os. PMID:15611179

The human Cx26-D50A and Cx26-A88V mutations causing keratitis-ichthyosis-deafness syndrome display increased hemichannel activity

PubMed Central

Mhaske, Pallavi V.; Levit, Noah A.; Li, Leping; Wang, Hong-Zhan; Lee, Jack R.; Shuja, Zunaira; Brink, Peter R.

2013-01-01

Mutations in the human gene encoding connexin 26 (Cx26 or GJB2) cause either nonsyndromic deafness or syndromic deafness associated with skin diseases. That distinct clinical disorders can be caused by different mutations within the same gene suggests that different channel activities influence the ear and skin. Here we use three different expression systems to examine the functional characteristics of two Cx26 mutations causing either mild (Cx26-D50A) or lethal (Cx26-A88V) keratitis-ichthyosis-deafness (KID) syndrome. In either cRNA-injected Xenopus oocytes, transfected HeLa cells, or transfected primary human keratinocytes, we show that both Cx26-D50A and Cx26-A88V form active hemichannels that significantly increase membrane current flow compared with wild-type Cx26. This increased membrane current accelerated cell death in low extracellular calcium solutions and was not due to increased mutant protein expression. Elevated mutant hemichannel currents could be blocked by increased extracellular calcium concentration. These results show that these two mutations exhibit a shared gain of functional activity and support the hypothesis that increased hemichannel activity is a common feature of human Cx26 mutations responsible for KID syndrome. PMID:23447037

Processing closely spaced lesions during Nucleotide Excision Repair triggers mutagenesis in E. coli

PubMed Central

Isogawa, Asako; Fujii, Shingo

2017-01-01

It is generally assumed that most point mutations are fixed when damage containing template DNA undergoes replication, either right at the fork or behind the fork during gap filling. Here we provide genetic evidence for a pathway, dependent on Nucleotide Excision Repair, that induces mutations when processing closely spaced lesions. This pathway, referred to as Nucleotide Excision Repair-induced Mutagenesis (NERiM), exhibits several characteristics distinct from mutations that occur within the course of replication: i) following UV irradiation, NER-induced mutations are fixed much more rapidly (t ½ ≈ 30 min) than replication dependent mutations (t ½ ≈ 80–100 min) ii) NERiM specifically requires DNA Pol IV in addition to Pol V iii) NERiM exhibits a two-hit dose-response curve that suggests processing of closely spaced lesions. A mathematical model let us define the geometry (infer the structure) of the toxic intermediate as being formed when NER incises a lesion that resides in close proximity of another lesion in the complementary strand. This critical NER intermediate requires Pol IV / Pol II for repair, it is either lethal if left unrepaired or mutation-prone when repaired. Finally, NERiM is found to operate in stationary phase cells providing an intriguing possibility for ongoing evolution in the absence of replication. PMID:28686598

Nonlethal sec71-1 and sec72-1 mutations eliminate proteins associated with the Sec63p-BiP complex from S. cerevisiae.

PubMed Central

Fang, H; Green, N

1994-01-01

The sec71-1 and sec72-1 mutations were identified by a genetic assay that monitored membrane protein integration into the endoplasmic reticulum (ER) membrane of the yeast Saccharomyces cerevisiae. The mutations inhibited integration of various chimeric membrane proteins and translocation of a subset of water soluble proteins. In this paper we show that SEC71 encodes the 31.5-kDa transmembrane glycoprotein (p31.5) and SEC72 encodes the 23-kDa protein (p23) of the Sec63p-BiP complex. SEC71 is therefore identical to SEC66 (HSS1), which was previously shown to encode p31.5. DNA sequence analyses reveal that sec71-1 cells contain a nonsense mutation that removes approximately two-thirds of the cytoplasmic C-terminal domain of p31.5. The sec72-1 mutation shifts the reading frame of the gene encoding p23. Unexpectedly, the sec71-1 mutant lacks p31.5 and p23. Neither mutation is lethal, although sec71-1 cells exhibit a growth defect at 37 degrees C. These results show that p31.5 and p23 are important for the trafficking of a subset of proteins to the ER membrane. Images PMID:7841522

Genetic and orthopedic aspects of collagen disorders.

PubMed

Carter, Erin M; Raggio, Cathleen L

2009-02-01

'Collagens' are a family of structurally related proteins that play a wide variety of roles in the extracellular matrix. To date, there are at least 29 known types of collagen. Accordingly, abnormality in the various collagens produces a large category of diseases with heterogeneous symptoms. This review presents genetic and orthopedic aspects of type II, IX, and XI collagen disorders. Although a diverse group of conditions, mutation of collagens affecting the articular cartilage typically produces an epiphyseal skeletal dysplasia phenotype. Often, the ocular or auditory systems or both are also involved. Treatment of these collagenopathies is symptomatic and individualized. Study of tissue from animal models allows examination of mutation effects on the abnormal protein structure and function. The collagen superfamily comprises an important structural protein in mammalian connective tissue. Mutation of collagens produces a wide variety of genetic disorders, and those mutations affecting types II, IX, and XI collagens produce an overlapping spectrum of skeletal dysplasias. Findings range from lethal to mild, depending on the mutation of the collagen gene and its subsequent effect on the structure and/or metabolism of the resultant procollagen and/or collagen protein and its function in the body.

A comprehensive characterization of rare mitochondrial DNA variants in neuroblastoma.

PubMed

Calabrese, Francesco Maria; Clima, Rosanna; Pignataro, Piero; Lasorsa, Vito Alessandro; Hogarty, Michael D; Castellano, Aurora; Conte, Massimo; Tonini, Gian Paolo; Iolascon, Achille; Gasparre, Giuseppe; Capasso, Mario

2016-08-02

Neuroblastoma, a tumor of the developing sympathetic nervous system, is a common childhood neoplasm that is often lethal. Mitochondrial DNA (mtDNA) mutations have been found in most tumors including neuroblastoma. We extracted mtDNA data from a cohort of neuroblastoma samples that had undergone Whole Exome Sequencing (WES) and also used snap-frozen samples in which mtDNA was entirely sequenced by Sanger technology. We next undertook the challenge of determining those mutations that are relevant to, or arisen during tumor development. The bioinformatics pipeline used to extract mitochondrial variants from matched tumor/blood samples was enriched by a set of filters inclusive of heteroplasmic fraction, nucleotide variability, and in silico prediction of pathogenicity. Our in silico multistep workflow applied both on WES and Sanger-sequenced neuroblastoma samples, allowed us to identify a limited burden of somatic and germline mitochondrial mutations with a potential pathogenic impact. The few singleton germline and somatic mitochondrial mutations emerged, according to our in silico analysis, do not appear to impact on the development of neuroblastoma. Our findings are consistent with the hypothesis that most mitochondrial somatic mutations can be considered as 'passengers' and consequently have no discernible effect in this type of cancer.

Modeling the dynamics of chromosomal alteration progression in cervical cancer: A computational model

PubMed Central

2017-01-01

Computational modeling has been applied to simulate the heterogeneity of cancer behavior. The development of Cervical Cancer (CC) is a process in which the cell acquires dynamic behavior from non-deleterious and deleterious mutations, exhibiting chromosomal alterations as a manifestation of this dynamic. To further determine the progression of chromosomal alterations in precursor lesions and CC, we introduce a computational model to study the dynamics of deleterious and non-deleterious mutations as an outcome of tumor progression. The analysis of chromosomal alterations mediated by our model reveals that multiple deleterious mutations are more frequent in precursor lesions than in CC. Cells with lethal deleterious mutations would be eliminated, which would mitigate cancer progression; on the other hand, cells with non-deleterious mutations would become dominant, which could predispose them to cancer progression. The study of somatic alterations through computer simulations of cancer progression provides a feasible pathway for insights into the transformation of cell mechanisms in humans. During cancer progression, tumors may acquire new phenotype traits, such as the ability to invade and metastasize or to become clinically important when they develop drug resistance. Non-deleterious chromosomal alterations contribute to this progression. PMID:28723940

a/alpha-specific effect on the mms3 mutation on ultraviolet mutagenesis in Saccharomyces cerevisiae.

PubMed

Martin, P; Prakash, L; Prakash, S

1981-05-01

A new gene involved in error-prone repair of ultraviolet (UV) damage has been identified in Saccharomyces cerevisiae by the mms3-1 mutation. UV-induced reversion is reduced in diploids that are homozygous for mms3-1, only if they are also heterozygous (MATa/MAT alpha) at the mating type locus. The mms3-1 mutation has no effect on UV-induced reversion either in haploids or MATa/MATa or MAT alpha/MAT alpha diploids. The mutation confers sensitivity to UV and methyl methane sulfonate in both haploids and diploids. Even though mutation induction by UV is restored to wild-type levels in MATa/MATa mms3-1/mms3-1 or MAT alpha/MAT alpha mms3-1/mms3-1 diploids, such strains still retain sensitivity to the lethal effects of UV. Survival after UV irradiation in mms3-1 rad double mutant combinations indicates that mms3-1 is epistatic to rad6-1 whereas non-epistatic interactions are observed with rad3 and rad52 mutants. When present in the homozygous state in MATa/MAT alpha his1-1/his1-315 heteroallelic diploids, mms3-1 was found to lower UV-induced mitotic recombination.

Neoplastic cell transformation by high-LET radiation - Molecular mechanisms

NASA Technical Reports Server (NTRS)

Yang, Tracy Chui-Hsu; Craise, Laurie M.; Tobias, Cornelius A.; Mei, Man-Tong

1989-01-01

Quantitative data were collected on dose-response curves of cultured mouse-embryo cells (C3H10T1/2) irradiated with heavy ions of various charges and energies. Results suggests that two breaks formed on DNA within 80 A may cause cell transformation and that two DNA breaks formed within 20 A may be lethal. From results of experiments with restriction enzymes which produce DNA damages at specific sites, it was found that DNA double strand breaks are important primary lesions for radiogenic cell transformation and that blunt-ended double-strand breaks can form lethal as well as transformational damages due to misrepair or incomplete repair in the cell. The RBE-LET relationship for high-LET radiation is similar to that for HGPRT locus mutation, chromosomal deletion, and cell transformation, indicating that common lesions may be involved in these radiation effects.

Exposure to Glycolytic Carbon Sources Reveals a Novel Layer of Regulation for the MalT Regulon

PubMed Central

Reimann, Sylvia A.; Wolfe, Alan J.

2011-01-01

Bacteria adapt to changing environments by means of tightly coordinated regulatory circuits. The use of synthetic lethality, a genetic phenomenon in which the combination of two nonlethal mutations causes cell death, facilitates identification and study of such circuitry. In this study, we show that the E. coli ompR malT con double mutant exhibits a synthetic lethal phenotype that is environmentally conditional. MalTcon, the constitutively active form of the maltose system regulator MalT, causes elevated expression of the outer membrane porin LamB, which leads to death in the absence of the osmoregulator OmpR. However, the presence and metabolism of glycolytic carbon sources, such as sorbitol, promotes viability and unveils a novel layer of regulation within the complex circuitry that controls maltose transport and metabolism. PMID:21912549

Exposure to Glycolytic Carbon Sources Reveals a Novel Layer of Regulation for the MalT Regulon.

PubMed

Reimann, Sylvia A; Wolfe, Alan J

2011-01-01

Bacteria adapt to changing environments by means of tightly coordinated regulatory circuits. The use of synthetic lethality, a genetic phenomenon in which the combination of two nonlethal mutations causes cell death, facilitates identification and study of such circuitry. In this study, we show that the E. coli ompR malT(con) double mutant exhibits a synthetic lethal phenotype that is environmentally conditional. MalT(con), the constitutively active form of the maltose system regulator MalT, causes elevated expression of the outer membrane porin LamB, which leads to death in the absence of the osmoregulator OmpR. However, the presence and metabolism of glycolytic carbon sources, such as sorbitol, promotes viability and unveils a novel layer of regulation within the complex circuitry that controls maltose transport and metabolism.

The Genetics of a Small Autosomal Region of DROSOPHILA MELANOGASTER Containing the Structural Gene for Alcohol Dehydrogenase. III. Hypomorphic and Hypermorphic Mutations Affecting the Expression of Hairless

PubMed Central

Ashburner, Michael

1982-01-01

A lethal locus (l(2)br7;35B6-10), near Adh on chromosome arm 2L of D. melanogaster, is identified with Plunkett's dominant suppressor of Hairless (H). Of eight new alleles, seven act as dominant suppressors of H, the eighth is a dominant enhancer of H. One of the suppressor alleles is both a leaky lethal and a weak suppressor of H. Confirming Nash (1970), deletions of l(2)br7 are dominant suppressors, and duplications are dominant enhancers of H. A simple model is proposed to account for the interaction of l(2)br7 and H, assuming that amorphic (or hypomorphic) alleles of l(2)br7 suppress H and that hypermorphic alleles enhance H. PMID:6816670

Myosin-driven rescue of contractile reserve and energetics in mouse hearts bearing familial hypertrophic cardiomyopathy-associated mutant troponin T is mutation-specific.

PubMed

He, Huamei; Hoyer, Kirsten; Tao, Hai; Rice, Ronald; Jimenez, Jesus; Tardiff, Jil C; Ingwall, Joanne S

2012-11-01

The thin filament protein troponin T (TnT) is a regulator of sarcomere function. Whole heart energetics and contractile reserve are compromised in transgenic mice bearing missense mutations at R92 within the tropomyosin-binding domain of cTnT, despite being distal to the ATP hydrolysis domain of myosin. These mutations are associated with familial hypertrophic cardiomyopathy (FHC). Here we test the hypothesis that genetically replacing murine αα-MyHC with murine ββ-MyHC in hearts bearing the R92Q cTnT mutation, a particularly lethal FHC-associated mutation, leads to sufficiently large perturbations in sarcomere function to rescue whole heart energetics and decrease the cost of contraction. By comparing R92Q cTnT and R92L cTnT mutant hearts, we also test whether any rescue is mutation-specific. We defined the energetic state of the isolated perfused heart using (31)P-NMR spectroscopy while simultaneously measuring contractile performance at four work states. We found that the cost of increasing contraction in intact mouse hearts with R92Q cTnT depends on the type of myosin present in the thick filament. We also found that the salutary effect of this manoeuvre is mutation-specific, demonstrating the major regulatory role of cTnT on sarcomere function at the whole heart level.

Woot, an Active Gypsy-Class Retrotransposon in the Flour Beetle, Tribolium Castaneum, Is Associated with a Recent Mutation

PubMed Central

Beeman, R. W.; Thomson, M. S.; Clark, J. M.; DeCamillis, M. A.; Brown, S. J.; Denell, R. E.

1996-01-01

A recently isolated, lethal mutation of the homeotic Abdominal gene of the red flour beetle Tribolium castaneum is associated with an insertion of a novel retrotransposon into an intron. Sequence analysis indicates that this retrotransposon, named Woot, is a member of the gypsy family of mobile elements. Most strains of T. castaneum appear to harbor ~25-35 copies of Woot per genome. Woot is composed of long terminal repeats of unprecedented length (3.6 kb each), flanking an internal coding region 5.0 kb in length. For most copies of Woot, the internal region includes two open reading frames (ORFs) that correspond to the gag and pol genes of previously described retrotransposons and retroviruses. The copy of Woot inserted into Abdominal bears an apparent single frameshift mutation that separates the normal second ORF into two. Woot does not appear to generate infectious virions by the criterion that no envelop gene is discernible. The association of Woot with a recent mutation suggests that this retroelement is currently transpositionally active in at least some strains. PMID:8722793

Three endocrine neoplasms: an unusual combination of pheochromocytoma, pituitary adenoma, and papillary thyroid carcinoma.

PubMed

Sisson, James C; Giordano, Thomas J; Avram, Anca M

2012-04-01

Three endocrine neoplasms-bilateral pheochromocytomas, somatotrophic pituitary adenoma inducing acromegaly, and papillary carcinoma of the thyroid-occurred concurrently in a patient. A genetic mutation was hypothesized. Possible previously described genetic mutations were explored. Clinical assessments, laboratory data, images of tumors, histopathology, and immunohistochemistry of excised tissues documented the three neoplasms. Clinical assessment of the patient, family history, and a review of the literature sought a familial basis for the disorders. The methods confirmed the presence of three endocrine neoplasms. Each neoplasm was surgically excised and histologically verified. Surgical and (131)I treatments reduced the papillary carcinoma, but eventually this tumor progressed to a lethal degree. History, including that of nine siblings, uncovered no familial neoplasms. No similar case was found in the literature, but possible associations with germline mutations were considered. The concurrent development of pheochromocytomas, pituitary somatotrophic adenoma, and papillary thyroid carcinoma appears to be unique. Nevertheless, such tumors, particularly bilateral pheochromocytomas, strongly suggest a de novo germline mutation in a gene not previously associated with multiple endocrine neoplasia syndromes.

Early onset hearing loss in autosomal recessive hypophosphatemic rickets caused by loss of function mutation in ENPP1.

PubMed

Steichen-Gersdorf, Elisabeth; Lorenz-Depiereux, Bettina; Strom, Tim Matthias; Shaw, Nicholas J

2015-07-01

Autosomal recessive hypophosphatemic rickets 2 (ARHR2) is a rare form of renal tubular phosphate wasting disorder. Loss of function mutations of the ecto-nucleotide pyrophosphatase/pyrophosphodiesterase 1 gene (ENPP1) causes a wide spectrum of phenotypes, ranging from lethal generalized arterial calcification of infancy to hypophosphatemic rickets with hypertension. Hearing loss was not previously thought to be one of the features of the disease entities and was merely regarded as a complication rather than a part of the disease. We report two children who presented in mid to late childhood with progressive varus deformity of their legs due to hypophosphatemic rickets caused by mutations in the ENPP1 gene. Both children had evidence of progressive hearing loss requiring the use of hearing aids. This report of two unrelated infants with compound heterozygous mutations in ENPP1 and previously published cases confirms that mild to moderate hearing loss is frequently associated with ARHR2. Early onset conductive hearing loss may further distinguish the autosomal recessive ENPP1 related type from other types of hypophosphatemia.

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

Fujimura, Hiroaki

Mating pheromones, a- and {alpha}-factors, arrest the division of cells of opposite mating types, {alpha} and a cells, respectively. The author has isolated a sterile mutant of Saccharomyces cerevisiae using EMS that is defective in division arrest in response to {alpha}-factor but not defective in morphological changes and agglutinin induction. The mutation was designated dac2 for division arrest control by mating pheromones. The dac2 mutation was closely linked to gal1 and was different from the previously identified cell type nonspecific sterile mutations (ste4, ste5, ste7, ste11, ste12, ste18, and dac1). Although dac2 cells had no phenotype in the absence ofmore » pheromones, they showed morphological alterations and divided continuously in the presence of pheromones. As a result, dac2 cells had a mating defect. The dac2 mutation could suppress the lethality caused by the disruption of the GPA1 gene. These results suggest that the DAC2 product may control the signal for G-protein-mediated cell-cycle arrest and indicate that the synchronization of haploid yeast cell cycles by mating pheromones is essential for cell fusion during conjugation.« less

The medaka mutation tintachina sheds light on the evolution of V-ATPase B subunits in vertebrates

NASA Astrophysics Data System (ADS)

Müller, Claudia; Maeso, Ignacio; Wittbrodt, Joachim; Martínez-Morales, Juan R.

2013-11-01

Vacuolar-type H+ ATPases (V-ATPases) are multimeric protein complexes that play a universal role in the acidification of intracellular compartments in eukaryotic cells. We have isolated the recessive medaka mutation tintachina (tch), which carries an inactivating modification of the conserved glycine residue (G75R) of the proton pump subunit atp6v1Ba/vatB1. Mutant embryos show penetrant pigmentation defects, massive brain apoptosis and lethality before hatching. Strikingly, an equivalent mutation in atp6v1B1 (G78R) has been reported in a family of patients suffering from distal renal tubular acidosis (dRTA), a hereditary disease that causes metabolic acidosis due to impaired kidney function. This poses the question as to how molecularly identical mutations result in markedly different phenotypes in two vertebrate species. Our work offers an explanation for this phenomenon. We propose that, after successive rounds of whole-genome duplication, the emergence of paralogous copies allowed the divergence of the atp6v1B cis-regulatory control in different vertebrate groups.

MKS3/TMEM67 mutations are a major cause of COACH Syndrome, a Joubert Syndrome related disorder with liver involvement.

PubMed

Brancati, Francesco; Iannicelli, Miriam; Travaglini, Lorena; Mazzotta, Annalisa; Bertini, Enrico; Boltshauser, Eugen; D'Arrigo, Stefano; Emma, Francesco; Fazzi, Elisa; Gallizzi, Romina; Gentile, Mattia; Loncarevic, Damir; Mejaski-Bosnjak, Vlatka; Pantaleoni, Chiara; Rigoli, Luciana; Salpietro, Carmelo D; Signorini, Sabrina; Stringini, Gilda Rita; Verloes, Alain; Zabloka, Dominika; Dallapiccola, Bruno; Gleeson, Joseph G; Valente, Enza Maria

2009-02-01

The acronym COACH defines an autosomal recessive condition of Cerebellar vermis hypo/aplasia, Oligophrenia, congenital Ataxia, Coloboma and Hepatic fibrosis. Patients present the "molar tooth sign", a midbrain-hindbrain malformation pathognomonic for Joubert Syndrome (JS) and Related Disorders (JSRDs). The main feature of COACH is congenital hepatic fibrosis (CHF), resulting from malformation of the embryonic ductal plate. CHF is invariably found also in Meckel syndrome (MS), a lethal ciliopathy already found to be allelic with JSRDs at the CEP290 and RPGRIP1L genes. Recently, mutations in the MKS3 gene (approved symbol TMEM67), causative of about 7% MS cases, have been detected in few Meckel-like and pure JS patients. Analysis of MKS3 in 14 COACH families identified mutations in 8 (57%). Features such as colobomas and nephronophthisis were found only in a subset of mutated cases. These data confirm COACH as a distinct JSRD subgroup with core features of JS plus CHF, which major gene is MKS3, and further strengthen gene-phenotype correlates in JSRDs. (c) 2008 Wiley-Liss, Inc.

Mutation in a primate-conserved retrotransposon reveals a noncoding RNA as a mediator of infantile encephalopathy

PubMed Central

Cartault, François; Munier, Patrick; Benko, Edgar; Desguerre, Isabelle; Hanein, Sylvain; Boddaert, Nathalie; Bandiera, Simonetta; Vellayoudom, Jeanine; Krejbich-Trotot, Pascale; Bintner, Marc; Hoarau, Jean-Jacques; Girard, Muriel; Génin, Emmanuelle; de Lonlay, Pascale; Fourmaintraux, Alain; Naville, Magali; Rodriguez, Diana; Feingold, Josué; Renouil, Michel; Munnich, Arnold; Westhof, Eric; Fähling, Michael; Lyonnet, Stanislas; Henrion-Caude, Alexandra

2012-01-01

The human genome is densely populated with transposons and transposon-like repetitive elements. Although the impact of these transposons and elements on human genome evolution is recognized, the significance of subtle variations in their sequence remains mostly unexplored. Here we report homozygosity mapping of an infantile neurodegenerative disease locus in a genetic isolate. Complete DNA sequencing of the 400-kb linkage locus revealed a point mutation in a primate-specific retrotransposon that was transcribed as part of a unique noncoding RNA, which was expressed in the brain. In vitro knockdown of this RNA increased neuronal apoptosis, consistent with the inappropriate dosage of this RNA in vivo and with the phenotype. Moreover, structural analysis of the sequence revealed a small RNA-like hairpin that was consistent with the putative gain of a functional site when mutated. We show here that a mutation in a unique transposable element-containing RNA is associated with lethal encephalopathy, and we suggest that RNAs that harbor evolutionarily recent repetitive elements may play important roles in human brain development. PMID:22411793

MKS3/TMEM67 Mutations Are a Major Cause of COACH Syndrome, a Joubert Syndrome Related Disorder with Liver Involvement

PubMed Central

Brancati, Francesco; Iannicelli, Miriam; Travaglini, Lorena; Mazzotta, Annalisa; Bertini, Enrico; Boltshauser, Eugen; D’Arrigo, Stefano; Emma, Francesco; Fazzi, Elisa; Gallizzi, Romina; Gentile, Mattia; Loncarevic, Damir; Mejaski-Bosnjak, Vlatka; Pantaleoni, Chiara; Rigoli, Luciana; Salpietro, Carmelo D.; Signorini, Sabrina; Stringini, Gilda Rita; Verloes, Alain; Zabloka, Dominika; Dallapiccola, Bruno; Gleeson, Joseph G.; Valente, Enza Maria

2008-01-01

The acronym COACH defines an autosomal recessive condition of Cerebellar vermis hypo/aplasia, Oligophrenia, congenital Ataxia, Coloboma and Hepatic fibrosis. Patients present the “molar tooth sign”, a midbrain-hindbrain malformation pathognomonic for Joubert Syndrome (JS) and Related Disorders (JSRDs). The main feature of COACH is congenital hepatic fibrosis (CHF), resulting from malformation of the embryonic ductal plate. CHF is invariably found also in Meckel syndrome (MS), a lethal ciliopathy already found to be allelic with JSRDs at the CEP290 and RPGRIP1L genes. Recently, mutations in the MKS3 gene (approved symbol TMEM67), causative of about 7% MS cases, have been detected in few Meckel-like and pure JS patients. Analysis of MKS3 in 14 COACH families identified mutations in 8 (57%). Features such as colobomas and nephronophthisis were found only in a subset of mutated cases. These data confirm COACH as a distinct JSRD subgroup with core features of JS plus CHF, which major gene is MKS3, and further strengthen gene-phenotype correlates in JSRDs. PMID:19058225

Mitochondrial Genome Integrity Mutations Uncouple the Yeast Saccharomyces cerevisiae ATP Synthase*║

PubMed Central

Wang, Yamin; Singh, Usha; Mueller, David M.

2013-01-01

The mitochondrial ATP synthase is a molecular motor, which couples the flow of rotons with phosphorylation of ADP. Rotation of the central stalk within the core of ATP synthase effects conformational changes in the active sites driving the synthesis of ATP. Mitochondrial genome integrity (mgi) mutations have been previously identified in the α-, β-, and γ-subunits of ATP synthase in yeast Kluyveromyces lactis and trypanosome Trypanosoma brucei. These mutations reverse the lethality of the loss of mitochondrial DNA in petite negative strains. Introduction of the homologous mutations in Saccharomyces cerevisiae results in yeast strains that lose mitochondrial DNA at a high rate and accompanied decreases in the coupling of the ATP synthase. The structure of yeast F1-ATPase reveals that the mgi residues cluster around the γ-subunit and selectively around the collar region of F1. These results indicate that residues within the mgi complementation group are necessary for efficient coupling of ATP synthase, possibly acting as a support to fix the axis of rotation of the central stalk. PMID:17244612

Loss-of-function mutations and inducible RNAi suppression of Arabidopsis LCB2 genes reveal the critical role of sphingolipids in gametophytic and sporophytic cell viability.

PubMed

Dietrich, Charles R; Han, Gongshe; Chen, Ming; Berg, R Howard; Dunn, Teresa M; Cahoon, Edgar B

2008-04-01

Serine palmitoyltransferase (SPT) catalyzes the first step in sphingolipid biosynthesis, and downregulation of this enzyme provides a means for exploring sphingolipid function in cells. We have previously demonstrated that Arabidopsis SPT requires LCB1 and LCB2 subunits for activity, as is the case in other eukaryotes. In this study, we show that Arabidopsis has two genes (AtLCB2a and AtLCB2b) that encode functional isoforms of the LCB2 subunit. No alterations in sphingolipid content or growth were observed in T-DNA mutants for either gene, but homozygous double mutants were not recoverable, suggesting that these genes are functionally redundant. Reciprocal crosses conducted with Atlcb2a and Atlcb2b mutants indicated that lethality is associated primarily with the inability to transmit the lcb2 null genotype through the haploid pollen. Consistent with this, approximately 50% of the pollen obtained from plants homozygous for a mutation in one gene and heterozygous for a mutation in the second gene arrested during transition from uni-nucleate microspore to bicellular pollen. Ultrastructural analyses revealed that these pollen grains contained aberrant endomembranes and lacked an intine layer. To examine sphingolipid function in sporophytic cells, Arabidopsis lines were generated that allowed inducible RNAi silencing of AtLCB2b in an Atlcb2a mutant background. Studies conducted with these lines demonstrated that sphingolipids are essential throughout plant development, and that lethality resulting from LCB2 silencing in seedlings could be partially rescued by supplying exogenous long-chain bases. Overall, these studies provide insights into the genetic and biochemical properties of SPT and sphingolipid function in Arabidopsis.

A Genetic Screen Reveals an Unexpected Role for Yorkie Signaling in JAK/STAT-Dependent Hematopoietic Malignancies in Drosophila melanogaster

PubMed Central

Anderson, Abigail M.; Bailetti, Alessandro A.; Rodkin, Elizabeth; De, Atish; Bach, Erika A.

2017-01-01

A gain-of-function mutation in the tyrosine kinase JAK2 (JAK2V617F) causes human myeloproliferative neoplasms (MPNs). These patients present with high numbers of myeloid lineage cells and have numerous complications. Since current MPN therapies are not curative, there is a need to find new regulators and targets of Janus kinase/Signal transducer and activator of transcription (JAK/STAT) signaling that may represent additional clinical interventions . Drosophila melanogaster offers a low complexity model to study MPNs as JAK/STAT signaling is simplified with only one JAK [Hopscotch (Hop)] and one STAT (Stat92E). hopTumorous-lethal (Tum-l) is a gain-of-function mutation that causes dramatic expansion of myeloid cells, which then form lethal melanotic tumors. Through an F1 deficiency (Df) screen, we identified 11 suppressors and 35 enhancers of melanotic tumors in hopTum-l animals. Dfs that uncover the Hippo (Hpo) pathway genes expanded (ex) and warts (wts) strongly enhanced the hopTum-l tumor burden, as did mutations in ex, wts, and other Hpo pathway genes. Target genes of the Hpo pathway effector Yorkie (Yki) were significantly upregulated in hopTum-l blood cells, indicating that Yki signaling was increased. Ectopic hematopoietic activation of Yki in otherwise wild-type animals increased hemocyte proliferation but did not induce melanotic tumors. However, hematopoietic depletion of Yki significantly reduced the hopTum-l tumor burden, demonstrating that Yki is required for melanotic tumors in this background. These results support a model in which elevated Yki signaling increases the number of hemocytes, which become melanotic tumors as a result of elevated JAK/STAT signaling. PMID:28620086

Adaptive Mutations That Occurred during Circulation in Humans of H1N1 Influenza Virus in the 2009 Pandemic Enhance Virulence in Mice.

PubMed

Otte, A; Sauter, M; Daxer, M A; McHardy, A C; Klingel, K; Gabriel, G

2015-07-01

During the 2009 H1N1 influenza pandemic, infection attack rates were particularly high among young individuals who suffered from pneumonia with occasional death. Moreover, previously reported determinants of mammalian adaptation and pathogenicity were not present in 2009 pandemic H1N1 influenza A viruses. Thus, it was proposed that unknown viral factors might have contributed to disease severity in humans. In this study, we performed a comparative analysis of two clinical 2009 pandemic H1N1 strains that belong to the very early and later phases of the pandemic. We identified mutations in the viral hemagglutinin (HA) and the nucleoprotein (NP) that occurred during pandemic progression and mediate increased virulence in mice. Lethal disease outcome correlated with elevated viral replication in the alveolar epithelium, increased proinflammatory cytokine and chemokine responses, pneumonia, and lymphopenia in mice. These findings show that viral mutations that have occurred during pandemic circulation among humans are associated with severe disease in mice. In this study, novel determinants of 2009 pandemic H1N1 influenza pathogenicity were identified in the viral hemagglutinin (HA) and the nucleoprotein (NP) genes. In contrast to highly pathogenic avian influenza viruses, increased virulence in mice did not correlate with enhanced polymerase activity but with reduced activity. Lethal 2009 pandemic H1N1 infection in mice correlated with lymphopenia and severe pneumonia. These studies suggest that molecular mechanisms that mediate 2009 pandemic H1N1 influenza pathogenicity are distinct from those that mediate avian influenza virus pathogenicity in mice. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

C-terminal Phenylalanine of Bacteriophage T7 Single-stranded DNA-binding Protein Is Essential for Strand Displacement Synthesis by T7 DNA Polymerase at a Nick in DNA*

PubMed Central

Ghosh, Sharmistha; Marintcheva, Boriana; Takahashi, Masateru; Richardson, Charles C.

2009-01-01

Single-stranded DNA-binding protein (gp2.5), encoded by gene 2.5 of bacteriophage T7, plays an essential role in DNA replication. Not only does it remove impediments of secondary structure in the DNA, it also modulates the activities of the other replication proteins. The acidic C-terminal tail of gp2.5, bearing a C-terminal phenylalanine, physically and functionally interacts with the helicase and DNA polymerase. Deletion of the phenylalanine or substitution with a nonaromatic amino acid gives rise to a dominant lethal phenotype, and the altered gp2.5 has reduced affinity for T7 DNA polymerase. Suppressors of the dominant lethal phenotype have led to the identification of mutations in gene 5 that encodes the T7 DNA polymerase. The altered residues in the polymerase are solvent-exposed and lie in regions that are adjacent to the bound DNA. gp2.5 lacking the C-terminal phenylalanine has a lower affinity for gp5-thioredoxin relative to the wild-type gp2.5, and this affinity is partially restored by the suppressor mutations in DNA polymerase. gp2.5 enables T7 DNA polymerase to catalyze strand displacement DNA synthesis at a nick in DNA. The resulting 5′-single-stranded DNA tail provides a loading site for T7 DNA helicase. gp2.5 lacking the C-terminal phenylalanine does not support this event with wild-type DNA polymerase but does to a limited extent with T7 DNA polymerase harboring the suppressor mutations. PMID:19726688

Prevention and treatment of colon cancer by peroral administration of HAMLET (human α-lactalbumin made lethal to tumour cells).

PubMed

Puthia, Manoj; Storm, Petter; Nadeem, Aftab; Hsiung, Sabrina; Svanborg, Catharina

2014-01-01

Most colon cancers start with dysregulated Wnt/β-catenin signalling and remain a major therapeutic challenge. Examining whether HAMLET (human α-lactalbumin made lethal to tumour cells) may be used for colon cancer treatment is logical, based on the properties of the complex and its biological context. To investigate if HAMLET can be used for colon cancer treatment and prevention. Apc(Min)(/+) mice, which carry mutations relevant to hereditary and sporadic human colorectal tumours, were used as a model for human disease. HAMLET was given perorally in therapeutic and prophylactic regimens. Tumour burden and animal survival of HAMLET-treated and sham-fed mice were compared. Tissue analysis focused on Wnt/β-catenin signalling, proliferation markers and gene expression, using microarrays, immunoblotting, immunohistochemistry and ELISA. Confocal microscopy, reporter assay, immunoprecipitation, immunoblotting, ion flux assays and holographic imaging were used to determine effects on colon cancer cells. Peroral HAMLET administration reduced tumour progression and mortality in Apc(Min)(/+) mice. HAMLET accumulated specifically in tumour tissue, reduced β-catenin and related tumour markers. Gene expression analysis detected inhibition of Wnt signalling and a shift to a more differentiated phenotype. In colon cancer cells with APC mutations, HAMLET altered β-catenin integrity and localisation through an ion channel-dependent pathway, defining a new mechanism for controlling β-catenin signalling. Remarkably, supplying HAMLET to the drinking water from the time of weaning also significantly prevented tumour development. These data identify HAMLET as a new, peroral agent for colon cancer prevention and treatment, especially needed in people carrying APC mutations, where colon cancer remains a leading cause of death.

Prevention and treatment of colon cancer by peroral administration of HAMLET (human α-lactalbumin made lethal to tumour cells)

PubMed Central

Puthia, Manoj; Storm, Petter; Nadeem, Aftab; Hsiung, Sabrina; Svanborg, Catharina

2014-01-01

Background Most colon cancers start with dysregulated Wnt/β-catenin signalling and remain a major therapeutic challenge. Examining whether HAMLET (human α-lactalbumin made lethal to tumour cells) may be used for colon cancer treatment is logical, based on the properties of the complex and its biological context. Objective To investigate if HAMLET can be used for colon cancer treatment and prevention. ApcMin/+ mice, which carry mutations relevant to hereditary and sporadic human colorectal tumours, were used as a model for human disease. Method HAMLET was given perorally in therapeutic and prophylactic regimens. Tumour burden and animal survival of HAMLET-treated and sham-fed mice were compared. Tissue analysis focused on Wnt/β-catenin signalling, proliferation markers and gene expression, using microarrays, immunoblotting, immunohistochemistry and ELISA. Confocal microscopy, reporter assay, immunoprecipitation, immunoblotting, ion flux assays and holographic imaging were used to determine effects on colon cancer cells. Results Peroral HAMLET administration reduced tumour progression and mortality in ApcMin/+ mice. HAMLET accumulated specifically in tumour tissue, reduced β-catenin and related tumour markers. Gene expression analysis detected inhibition of Wnt signalling and a shift to a more differentiated phenotype. In colon cancer cells with APC mutations, HAMLET altered β-catenin integrity and localisation through an ion channel-dependent pathway, defining a new mechanism for controlling β-catenin signalling. Remarkably, supplying HAMLET to the drinking water from the time of weaning also significantly prevented tumour development. Conclusions These data identify HAMLET as a new, peroral agent for colon cancer prevention and treatment, especially needed in people carrying APC mutations, where colon cancer remains a leading cause of death. PMID:23348960

Determination of the Inactivating Alterations in Two Mutant Alleles of the Neurospora Crassa Cross-Pathway Control Gene Cpc-1

PubMed Central

Paluh, J. L.; Plamann, M.; Kruger, D.; Barthelmess, I. B.; Yanofsky, C.; Perkins, D. D.

1990-01-01

cpc-1 is the locus specifying what is believed to be the major trans-activating transcription factor that regulates expression of amino acid biosynthetic genes subject to cross-pathway control in Neurospora crassa. Mutants altered at this locus are incapable of the global increase in gene expression normally seen in response to amino acid starvation. Using polymerase chain reaction methodology we have cloned and sequenced the inactive mutant allele, cpc-1 (CD15). The cpc-1 (CD15) mutation was found to be a single base pair deletion in codon 93 of the cpc-1 structural gene. A second, presumed lethal, allele, cpc-1 (j-5), also was investigated. Northern analyses with strains carrying the cpc-1 (j-5) allele revealed that no cpc-1 mRNA is produced. Southern and genetic analyses established that the cpc-1 (j-5) mutation involved a chromosomal rearrangement in which a break occurred within the cpc-1 locus, normally resident on linkage group VI; a small fragment from the left arm of linkage group VI, containing the cpc-1 promoter region and ylo-1, was translocated to the right arm of linkage group I. Other studies indicate that the cpc-1 locus itself is not essential for viability. Lethality previously attributed to the cpc-1 (j-5) mutation is due instead to the production of progeny that are deficient for essential genes in an adjoining segment of linkage group VI. Molecular characterization of cpc-1 (j-5) X ylo-1 pan-2 duplication progeny indicated that cpc-1 is normally transcribed towards the linkage group VI centromere. PMID:2138111

Genome-Wide Mutation Avalanches Induced in Diploid Yeast Cells by a Base Analog or an APOBEC Deaminase

PubMed Central

Lada, Artem G.; Stepchenkova, Elena I.; Waisertreiger, Irina S. R.; Noskov, Vladimir N.; Dhar, Alok; Eudy, James D.; Boissy, Robert J.; Hirano, Masayuki; Rogozin, Igor B.; Pavlov, Youri I.

2013-01-01

Genetic information should be accurately transmitted from cell to cell; conversely, the adaptation in evolution and disease is fueled by mutations. In the case of cancer development, multiple genetic changes happen in somatic diploid cells. Most classic studies of the molecular mechanisms of mutagenesis have been performed in haploids. We demonstrate that the parameters of the mutation process are different in diploid cell populations. The genomes of drug-resistant mutants induced in yeast diploids by base analog 6-hydroxylaminopurine (HAP) or AID/APOBEC cytosine deaminase PmCDA1 from lamprey carried a stunning load of thousands of unselected mutations. Haploid mutants contained almost an order of magnitude fewer mutations. To explain this, we propose that the distribution of induced mutation rates in the cell population is uneven. The mutants in diploids with coincidental mutations in the two copies of the reporter gene arise from a fraction of cells that are transiently hypersensitive to the mutagenic action of a given mutagen. The progeny of such cells were never recovered in haploids due to the lethality caused by the inactivation of single-copy essential genes in cells with too many induced mutations. In diploid cells, the progeny of hypersensitive cells survived, but their genomes were saturated by heterozygous mutations. The reason for the hypermutability of cells could be transient faults of the mutation prevention pathways, like sanitization of nucleotide pools for HAP or an elevated expression of the PmCDA1 gene or the temporary inability of the destruction of the deaminase. The hypothesis on spikes of mutability may explain the sudden acquisition of multiple mutational changes during evolution and carcinogenesis. PMID:24039593

A Founder Mutation in LEPRE1 Carried by 1.5% of West Africans and 0.4% of African Americans Causes Lethal Recessive Osteogenesis Imperfecta

PubMed Central

Cabral, Wayne A.; Barnes, Aileen M.; Adeyemo, Adebowale; Cushing, Kelly; Chitayat, David; Porter, Forbes D.; Panny, Susan R.; Gulamali-Majid, Fizza; Tishkoff, Sarah A.; Rebbeck, Timothy R.; Gueye, Serigne M.; Bailey-Wilson, Joan E.; Brody, Lawrence C.; Rotimi, Charles N.; Marini, Joan C.

2012-01-01

Purpose Deficiency of prolyl 3-hydroxylase 1, encoded by LEPRE1, causes recessive osteogenesis imperfecta. We previously identified a LEPRE1 mutation, exclusively in African Americans and contemporary West Africans. We hypothesized that this allele originated in West Africa and was introduced to the Americas with the Atlantic slave trade. We aimed to determine the frequency of carriers for this mutation among African Americans and West Africans, and the mutation origin and age. Methods Genomic DNA was screened for the mutation using PCR and restriction digestion, and a custom TaqMan genomic SNP assay. The mutation age was estimated using microsatellites and short tandem repeats spanning 4.2 Mb surrounding LEPRE1 in probands and carriers. Results Approximately 0.4% of Mid-Atlantic African Americans carry this mutation, estimating recessive OI in 1/260,000 births in this population. In Nigeria and Ghana, 1.48% of unrelated individuals are heterozygous carriers, predicting 1/18,260 births will be affected with recessive OI, equal to the incidence of de novo dominant OI. The mutation was not detected in Africans from surrounding countries. All carriers shared a haplotype of 63-770 Kb, consistent with a single founder for this mutation. Using linkage disequilibrium analysis, the mutation was estimated to have originated between 650 and 900 years before present (1100-1350 C.E.). Conclusions We identified a West African founder mutation for recessive OI in LEPRE1. Nearly 1.5% of Ghanians and Nigerians are carriers. The age of this allele is consistent with introduction to North America via the Atlantic slave trade (1501 – 1867 C.E). PMID:22281939

A novel class of Saccharomyces cerevisiae mutants specifically UV-sensitive to "petite" induction.

PubMed

Moustacchi, E; Perlman, P S; Mahler, H R

1976-11-17

A mutant of Saccharomyces cerevisiae has been isolated which, though exhibiting a normal response to nuclear genetic damage by ultraviolet light (UV), is more sensitive than its wild type specifically in the production of the cytoplasmic (rho-) mutation by this agent. Some of the features of this mutation which has been designated uvsrho 5 are: i) The mutation is recessive, it exhibits a Mendelian, and hence presumably nuclear, pattern of segregation, but manifests its effects specifically and pleiotropically on mitochondrial functions. ii) Mutant cells resemble their wild type parents in a) growth characteristics on glucose; b) in their UV induced dose response to lethality or nuclear mutation and c) the ability of their mitochondrial genome, upon mating with appropriate testers, of transmitting and recombining various markers, albeit with enhanced efficiency. Similarly, d) they are able to modulate the expression of mitochondrial mutagenesis by ethidium bromide. Thus their mitochondrial DNA appears genetically as competent as that of the wild type. iii) Mutant cells differ from their wild type parents in a) growth characteristics on glycerol; b) susceptibility to induction of the mitochondrial (rho-) mutation by various mutagens, in that the rate of spontaneous mutation is slightly and that by UV is significantly enhanced, whild that by ethidium bromide is greatly diminished. Conversely, c) modulating influences resulting in the repair of initial damage are diminished fro UV and stimulated in the case of Berenil. iv) The amount of mitochondrial DNA per cell appears elevated in the mutant, relative to wild type, and its rate of degradation subsequent to a mutagenic exposure to either UV or ethidium bromide is diminished. v) A self-consistent scheme to account for this and all other information so far available for the induction and modulation of the (rho-) mutation is presented. In a previous study it was shown that some nuclear mutants of Saccharomyces cerevisiae, more sensitive to lethal damage induced by ultraviolet light (rad) than their parent wild type (RAD), also exhibit a concomitant modification in sensitivity to both nuclear and cytoplasmic genetic damage (Moustacchi, 1971). However, another class of rad mutants respond to the induction of the cytoplasmic "petite" also designated as rho- (or rho-) mutation by UV in a manner indistinguishable from that of the RAD strain. One possible interpretation of this last observation is that some of the steps in the expression of the UV damage on mitochondrial (mt)DNA may be governed by other nuclear and cytoplasmic genetic determinants, the products of which may then act specifically on mitochondrial lesions. If this assumption is correct, it should be possible to find mutants with a normal response to nuclear damage but specifically UV-sensitive towards induction of (rho-)...

Defects in cholesterol synthesis genes in mouse and in humans: lessons for drug development and safer treatments.

PubMed

Horvat, Simon; McWhir, Jim; Rozman, Damjana

2011-02-01

This review describes the mouse knockout models of cholesterol synthesis, together with human malformations and drugs that target cholesterogenic enzymes. Generally, the sooner a gene acts in cholesterol synthesis, the earlier the phenotype occurs. Humans with loss of function of early cholesterogenic enzymes have not yet been described, and in the mouse, loss of Hmgcr is preimplantation lethal. Together, these results indicate that the widely prescribed cholesterol-lowering statins are potentially teratogenic. The Mvk knockout is early embryonic lethal in the mouse, the absence of Fdft1 is lethal at E9.5-12.5 dpc, while the Cyp51 knockouts die at 15.0 dpc. Fungal CYP51 inhibitor azoles are teratogenic in humans, potentially leading to symptoms of Antley-Bixler syndrome. The X-linked mutations in Nsdhl and Ebp are embryonic lethal in male mice, while heterozygous females are also affected. Consequently, the anticancer drugs, tamoxifen and toremifene, inhibiting human EBP, may be harmful in early pregnancy. The Dhcr7 and Dhcr24 knockout mice die shortly after birth, while humans survive with Smith-Lemli-Opitz syndrome or desmosterolosis. Since cholesterol is essential for hedgehog signaling, disturbance of this pathway by antipsychotics and -depressants explains some drug side effects. In conclusion, defects in cholesterol synthesis are generally lethal in mice, while humans with impaired later steps of the pathway can survive with severe malformations. Evidence shows that drugs targeting or, by coincidence, inhibiting human cholesterol synthesis are better avoided in early pregnancy. Since some drugs with teratogenic potential still stay on the market, this should be avoided in new cholesterol-related drug development.

[Screening of full human anthrax lethal factor neutralizing antibody in transgenic mice].

PubMed

Wang, Xiaolin; Chi, Xiangyang; Liu, Ju; Liu, Weicen; Liu, Shuling; Qiu, Shunfang; Wen, Zhonghua; Fan, Pengfei; Liu, Kun; Song, Xiaohong; Fu, Ling; Zhang, Jun; Yu, Changming

2016-11-25

Anthrax is a highly lethal infectious disease caused by the spore-forming bacterium Bacillus anthracis. The major virulence factor of B. anthracis consists of protective antigen (PA), lethal factor (LF) and edema factor (EF). PA binds with LF to form lethal toxin (LT), and PA binds with EF to form edema toxin (ET). Antibiotics is hard to work in advanced anthrax infections, because injuries and deaths of the infected are mainly caused by lethal toxin (LT). Thus, the therapeutic neutralizing antibody is the most effective treatment of anthrax. Currently most of the anthrax toxin antibodies are monoclonal antibodies (MAbs) for PA and US FDA has approved ABTHRAX humanized PA monoclonal antibody for the treatment of inhalational anthrax. Once B. anthracis was artificially reconstructed or PA had mutations within recognized neutralization epitopes, anti-PA MAbs would no longer be effective. Therefore, anti-LF MAbs is an important supplement for anthrax treatment. Most of the anti-LF antibodies are murine or chimeric antibodies. By contrast, fully human MAbs can avoid the high immunogenicity of murine antibodies. First, we used LF to immunize the transgenic mice and used fluorescent cell sorting to get antigen-specific memory B cells from transgenic mice spleen lymphocytes. By single cell PCR method, we quickly found two strains of anti-LF MAbs with binding activity, 1D7 and 2B9. Transiently transfected Expi 293F cells to obtain MAbs protein after purification. Both 1D7 and 2B9 efficiently neutralized LT in vitro, and had good synergistic effect when mixed with anti-PA MAbs. In summary, combining the advantages of transgenic mice, fluorescent cell sorting and single-cell PCR methods, this study shows new ideas and methods for the rapid screening of fully human monoclonal antibodies.

Destabilization of the metal site as a hub for the pathogenic mechanism of five ALS-linked mutants of copper, zinc superoxide dismutase.

PubMed

Mera-Adasme, Raúl; Erdmann, Hannes; Bereźniak, Tomasz; Ochsenfeld, Christian

2016-10-01

Amyotrophic lateral sclerosis (ALS) is a lethal neurodegenerative disease, with no effective pharmacological treatment. Its pathogenesis is unknown, although a subset of the cases is linked to genetic mutations. A significant fraction of the mutations occur in one protein, copper, zinc superoxide dismutase (SOD1). The toxic function of mutant SOD1 has not been elucidated, but damage to the metal site of the protein is believed to play a major role. In this work, we study the electrostatic loop of SOD1, which we had previously proposed to work as a "solvent seal" isolating the metal site from water molecules. Out of the five contact points identified between the electrostatic loop and its dock in the rest of the protein, three points were found to be affected by ALS-linked mutations, with a total of five mutations identified. The effect of the five mutations was studied using methods of computational chemistry. We found that four of the mutations destabilize the proposed solvent seal, while the fifth mutation directly affects the metal-site stability. In the two contact points unaffected by ALS-linked mutations, the side chains of the residues were not found to play a stabilizing role. Our results show that the docking of the electrostatic loop to the rest of SOD1 plays a role in ALS pathogenesis, in support of that structure acting as a solvent barrier for the metal site. The results provide a unified pathogenic mechanism for five different ALS-linked mutations of SOD1.

Six post-implantation lethal knockouts of genes for lipophilic MAPK pathway proteins are expressed in preimplantation mouse embryos and trophoblast stem cells.

PubMed

Xie, Yufen; Wang, Yingchun; Sun, Tong; Wang, Fangfei; Trostinskaia, Anna; Puscheck, Elizabeth; Rappolee, Daniel A

2005-05-01

Mitogen-activated protein kinase (MAPK) signaling pathways play an important role in controlling embryonic proliferation and differentiation. It has been demonstrated that sequential lipophilic signal transduction mediators that participate in the MAPK pathway are null post-implantation lethal. It is not clear why the lethality of these null mutants arises after implantation and not before. One hypothesis is that the gene product of these post-implantation lethal null mutants are not present before implantation in normal embryos and do not have function until after implantation. To test this hypothesis, we selected a set of lipophilic genes mediating MAPK signal transduction pathways whose null mutants result in early peri-implantation or placental lethality. These included FRS2alpha, GAB1, GRB2, SOS1, Raf-B, and Raf1. Products of these selected genes were detected and their locations and functions indicated by indirect immunocytochemistry and Western blotting for proteins and RT-polymerase chain reaction (PCR) for mRNA transcription. We report here that all six signal mediators are detected at the protein level in preimplantation mouse embryo, placental trophoblasts, and in cultured trophoblast stem cells (TSC). Proteins are all detected in E3.5 embryos at a time when the first known mitogenic intercellular communication has been documented. mRNA transcripts of two post-implantation null mutant genes are expressed in mouse preimplantation embryos and unfertilized eggs. These mRNA transcripts were detected as maternal mRNA in unfertilized eggs that could delay the lethality of null mutants. All of the proteins were detected in the cytoplasm or in the cell membrane. This study of spatial and temporal expression revealed that all of these six null mutants post-implantation genes in MAPK pathway are expressed and, where tested, phosphorylated/activated proteins are detected in the blastocyst. Studies on RNA expression using RT-PCR suggest that maternal RNA could play an important role in delaying the presence of the lethal phenotype of null mutations. Copyright (c) 2005 Wiley-Liss, Inc.

Clinical presentations of Ehlers Danlos syndrome type IV.

PubMed Central

Pope, F M; Narcisi, P; Nicholls, A C; Liberman, M; Oorthuys, J W

1988-01-01

Ehlers Danlos syndrome type IV is an often lethal disease caused by various mutations of type III collagen genes. It presents in infancy and childhood in several ways, and the symptoms and signs include low birth weight, prematurity, congenital dislocation of the hips, easy inappropriate bruising (sometimes suspected as child battering), and a diagnostic facial phenotype. These features predict a lethal adult disease often complicated by fatal arterial rupture in early or middle adult life. Most affected patients can be diagnosed from radiolabelled collagen protein profiles by polyacrylamide gel electrophoresis. Prenatal diagnosis by specific type III collagen restriction fragment length polymorphisms is possible in some families, and will become increasingly important. Prenatal diagnosis and prevention of the disease in selected families is already possible and will be widely available in the future. Images Fig 1 Fig 2 Fig 3 Fig 4 Fig 5 Fig 6 Fig 7 Fig 8 Fig 9 Fig 10 Fig 11 PMID:3178263

The effect of polyandry on a distorter system with differential viabilities in the sexes.

PubMed

Manser, Andri; Lindholm, Anna K; König, Barbara; Bagheri, Homayoun C

2012-11-01

The presence of selfish genetic elements can have fatal consequences for populations that harbor them. In the well known t haplotype in wild house mice, large proportions of the population die from t/t recessive lethal effects. Due to strong advantages at the gamete level (drive), t haplotypes nevertheless occur at substantial frequencies. The stable presence of a lethal is not the only effect of the t. It also distorts the fate of mutations that differentially affect male and female survival and reproduction (such as in sexual conflict), by giving male selective effects a strong advantage over female selective effects. In a recent study, we proposed polyandry as a potential counterstrategy against t deleterious effects. Here, we show that (1) the efficiency of polyandry in reducing the t frequency strongly depends on the selective context and (2) polyandry helps to reduce male-biased leverage in sex dependent selection.

Identification of two poorly prognosed ovarian carcinoma subtypes associated with CHEK2 germ-line mutation and non-CHEK2 somatic mutation gene signatures.

PubMed

Ow, Ghim Siong; Ivshina, Anna V; Fuentes, Gloria; Kuznetsov, Vladimir A

2014-01-01

High-grade serous ovarian cancer (HG-SOC), a major histologic type of epithelial ovarian cancer (EOC), is a poorly-characterized, heterogeneous and lethal disease where somatic mutations of TP53 are common and inherited loss-of-function mutations in BRCA1/2 predispose to cancer in 9.5-13% of EOC patients. However, the overall burden of disease due to either inherited or sporadic mutations is not known. We performed bioinformatics analyses of mutational and clinical data of 334 HG-SOC tumor samples from The Cancer Genome Atlas to identify novel tumor-driving mutations, survival-significant patient subgroups and tumor subtypes potentially driven by either hereditary or sporadic factors. We identified a sub-cluster of high-frequency mutations in 22 patients and 58 genes associated with DNA damage repair, apoptosis and cell cycle. Mutations of CHEK2, observed with the highest intensity, were associated with poor therapy response and overall survival (OS) of these patients (P = 8.00e-05), possibly due to detrimental effect of mutations at the nuclear localization signal. A 21-gene mutational prognostic signature significantly stratifies patients into relatively low or high-risk subgroups with 5-y OS of 37% or 6%, respectively (P = 7.31e-08). Further analysis of these genes and high-risk subgroup revealed 2 distinct classes of tumors characterized by either germline mutations of genes such as CHEK2, RPS6KA2 and MLL4, or somatic mutations of other genes in the signature. Our results could provide improvement in prediction and clinical management of HG-SOC, facilitate our understanding of this complex disease, guide the design of targeted therapeutics and improve screening efforts to identify women at high-risk of hereditary ovarian cancers distinct from those associated with BRCA1/2 mutations.

Identification of two poorly prognosed ovarian carcinoma subtypes associated with CHEK2 germ-line mutation and non-CHEK2 somatic mutation gene signatures

PubMed Central

Ow, Ghim Siong; Ivshina, Anna V; Fuentes, Gloria; Kuznetsov, Vladimir A

2014-01-01

High-grade serous ovarian cancer (HG-SOC), a major histologic type of epithelial ovarian cancer (EOC), is a poorly-characterized, heterogeneous and lethal disease where somatic mutations of TP53 are common and inherited loss-of-function mutations in BRCA1/2 predispose to cancer in 9.5–13% of EOC patients. However, the overall burden of disease due to either inherited or sporadic mutations is not known.     We performed bioinformatics analyses of mutational and clinical data of 334 HG-SOC tumor samples from The Cancer Genome Atlas to identify novel tumor-driving mutations, survival-significant patient subgroups and tumor subtypes potentially driven by either hereditary or sporadic factors. We identified a sub-cluster of high-frequency mutations in 22 patients and 58 genes associated with DNA damage repair, apoptosis and cell cycle. Mutations of CHEK2, observed with the highest intensity, were associated with poor therapy response and overall survival (OS) of these patients (P = 8.00e-05), possibly due to detrimental effect of mutations at the nuclear localization signal. A 21-gene mutational prognostic signature significantly stratifies patients into relatively low or high-risk subgroups with 5-y OS of 37% or 6%, respectively (P = 7.31e-08). Further analysis of these genes and high-risk subgroup revealed 2 distinct classes of tumors characterized by either germline mutations of genes such as CHEK2, RPS6KA2 and MLL4, or somatic mutations of other genes in the signature. Our results could provide improvement in prediction and clinical management of HG-SOC, facilitate our understanding of this complex disease, guide the design of targeted therapeutics and improve screening efforts to identify women at high-risk of hereditary ovarian cancers distinct from those associated with BRCA1/2 mutations. PMID:24879340

A method distinguishing expressed vs. null mutations of the Col1A1 gene in osteogenesis imperfecta

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

Redford-Badwal, D.A.; Stover, M.L.; McKinstry, M.

Osteogenesis imperfecta (OI) is a heterogeneous group of heritable disorders of bone characterized by increased susceptibility to fracture. Most of the causative mutations were identified in patients with the lethal form of the disease. Attention is now shifting to the milder forms of OI where glycine substitutions and null producing mutations have been found. Single amino acid substitutions can be identified by RT/PCR of total cellular RNA, but this approach does not work well for null mutations since the defective transcript does not accumulate in the cytoplasm. We have altered our RNA extraction method to separate RNA from the nuclearmore » and cytoplasmic compartments of cultured fibroblasts. Standard methods of mutation identification (RT/PCR followed by SSCP) is applied to each RNA fraction. DNA from an abnormal band on the SSCP gel is eluted and amplified by PCR for cloning and sequencing. Using this approach we have identified an Asp to Asn change in exon 50 (type II OI) and a Gly to Arg in exon 11 (type I OI) of the COL1A1 gene. These changes were found in both nuclear and cytoplasmic compartments. These putative mutations are currently being confirmed by protein studies. In contrast, three patients with mild OI associated with reduced {proportional_to}(I)mRNA, had distinguishing SSCP bands present in the nuclear but not the cytoplasmic compartment. In one case a frame shift mutation was observed, while the other two revealed polymorphisms. The compartmentalization of the mutant allele has directed us to look elsewhere in the transcript for the causative mutation. This approach to mutation identification is capable of distinguishing these fundamentally different types of mutations and allows for preferential cloning and sequencing of the abnormal allele.« less

Small Cell Lung Cancer Exhibits Frequent Inactivating Mutations in the Histone Methyltransferase KMT2D/MLL2: CALGB 151111 (Alliance)

PubMed Central

Augert, Arnaud; Zhang, Qing; Bates, Breanna; Cui, Min; Wang, Xiaofei; Wildey, Gary; Dowlati, Afshin; MacPherson, David

2017-01-01

Introduction SCLC is a lethal neuroendocrine tumor type that is highly prone to metastasis. There is an urgency to understand the mutated genes that promote SCLC, as there are no approved targeted therapies yet available. SCLC is rarely resected, limiting the number of samples available for genomic analyses of somatic mutations. Methods To identify potential driver mutations in human SCLC we sequenced the whole exomes of 18 primary SCLCs and seven cell lines along with matched normal controls. We extended these data by resequencing a panel of genes across 40 primary SCLCs and 48 cell lines. Results We report frequent mutations in the lysine methyltransferase 2D gene (KMT2D) (also known as MLL2), a key regulator of transcriptional enhancer function. KMT2D exhibited truncating nonsense/frameshift/splice site mutations in 8% of SCLC tumors and 17% of SCLC cell lines. We found that KMT2D mutation in human SCLC cell lines was associated with reduced lysine methyltransferase 2D protein levels and reduced monomethylation of histone H3 lysine 4, a mark associated with transcriptional enhancers. We also found mutations in other genes associated with transcriptional enhancer control, including CREB binding protein gene (CREBBP), E1A binding protein p300 gene (EP300), and chromodomain helicase DNA binding protein 7 gene (CHD7), and we report mutations in additional chromatin remodeling genes such as polybromo 1 gene (PBRM1). Conclusions These data indicate that KMT2D is one of the major mutated genes in SCLC, and they point to perturbation of transcriptional enhancer control as potentially contributing to SCLC. PMID:28007623

Killed but metabolically active Bacillus anthracis vaccines induce broad and protective immunity against anthrax.

PubMed

Skoble, Justin; Beaber, John W; Gao, Yi; Lovchik, Julie A; Sower, Laurie E; Liu, Weiqun; Luckett, William; Peterson, Johnny W; Calendar, Richard; Portnoy, Daniel A; Lyons, C Rick; Dubensky, Thomas W

2009-04-01

Bacillus anthracis is the causative agent of anthrax. We have developed a novel whole-bacterial-cell anthrax vaccine utilizing B. anthracis that is killed but metabolically active (KBMA). Vaccine strains that are asporogenic and nucleotide excision repair deficient were engineered by deleting the spoIIE and uvrAB genes, rendering B. anthracis extremely sensitive to photochemical inactivation with S-59 psoralen and UV light. We also introduced point mutations into the lef and cya genes, which allowed inactive but immunogenic toxins to be produced. Photochemically inactivated vaccine strains maintained a high degree of metabolic activity and secreted protective antigen (PA), lethal factor, and edema factor. KBMA B. anthracis vaccines were avirulent in mice and induced less injection site inflammation than recombinant PA adsorbed to aluminum hydroxide gel. KBMA B. anthracis-vaccinated animals produced antibodies against numerous anthrax antigens, including high levels of anti-PA and toxin-neutralizing antibodies. Vaccination with KBMA B. anthracis fully protected mice against challenge with lethal doses of toxinogenic unencapsulated Sterne 7702 spores and rabbits against challenge with lethal pneumonic doses of fully virulent Ames strain spores. Guinea pigs vaccinated with KBMA B. anthracis were partially protected against lethal Ames spore challenge, which was comparable to vaccination with the licensed vaccine anthrax vaccine adsorbed. These data demonstrate that KBMA anthrax vaccines are well tolerated and elicit potent protective immune responses. The use of KBMA vaccines may be broadly applicable to bacterial pathogens, especially those for which the correlates of protective immunity are unknown.

Targeting human apurinic/apyrimidinic endonuclease 1 (APE1) in phosphatase and tensin homolog (PTEN) deficient melanoma cells for personalized therapy.

PubMed

Abbotts, Rachel; Jewell, Rosalyn; Nsengimana, Jérémie; Maloney, David J; Simeonov, Anton; Seedhouse, Claire; Elliott, Faye; Laye, Jon; Walker, Christy; Jadhav, Ajit; Grabowska, Anna; Ball, Graham; Patel, Poulam M; Newton-Bishop, Julia; Wilson, David M; Madhusudan, Srinivasan

2014-05-30

Phosphatase and tensin homolog (PTEN) loss is associated with genomic instability. APE1 is a key player in DNA base excision repair (BER) and an emerging drug target in cancer. We have developed small molecule inhibitors against APE1 repair nuclease activity. In the current study we explored a synthetic lethal relationship between PTEN and APE1 in melanoma. Clinicopathological significance of PTEN mRNA and APE1 mRNA expression was investigated in 191 human melanomas. Preclinically, PTEN-deficient BRAF-mutated (UACC62, HT144, and SKMel28), PTEN-proficient BRAF-wildtype (MeWo), and doxycycline-inducible PTEN-knockout BRAF-wildtype MeWo melanoma cells were DNA repair expression profiled and investigated for synthetic lethality using a panel of four prototypical APE1 inhibitors. In human tumours, low PTEN mRNA and high APE1 mRNA was significantly associated with reduced relapse free and overall survival. Pre-clinically, compared to PTEN-proficient cells, PTEN-deficient cells displayed impaired expression of genes involved in DNA double strand break (DSB) repair. Synthetic lethality in PTEN-deficient cells was evidenced by increased sensitivity, accumulation of DSBs and induction of apoptosis following treatment with APE1 inhibitors. We conclude that PTEN deficiency is not only a promising biomarker in melanoma, but can also be targeted by a synthetic lethality strategy using inhibitors of BER, such as those targeting APE1.

Defeat mutant KRAS with synthetic lethality

PubMed Central

Pang, Xiufeng; Liu, Mingyao

2017-01-01

ABSTRACT Ras proteins are considered as the founding members of a large superfamily of small GTPases that control fundamental cellular functions. Mutationally activated RAS genes were discovered in human cancer cells more than 3 decades ago, but intensive efforts on Ras structure, biochemistry, function and signaling continue even now. Because mutant Ras proteins are inherently difficult to inhibit and have yet been therapeutically conquered, it was designated as “the Everest of oncogenes” in the cancer genome landscape, further promoting a “renaissance” in RAS research. Different paths to directly or indirectly targeting mutant Ras signaling are currently under investigation in the hope of finding an efficacious regimen. Inhibitors directly binding to KRASG12C to block its downstream signaling have been revealed, supporting the notion of Ras' druggability. An alternative indirect approach by targeting synthetic lethal interactors of mutant RAS is underway. We recently employed a synthetic lethal drug screen plus a combinatorial strategy using a panel of clinical agents and discovered that KRAS-mutant cancers were fragile to the combined inhibition of polo-like kinase 1 (Plk1) and RhoA/Rho kinase (ROCK). The combined regimen of BI-2536 (a Plk1 inhibitor) and fasudil (a ROCK inhibitor) promoted a significant inhibition of patient-derived lung cancer xenografts and prolonged the survival of LSL-KRASG12D mice. In this commentary, we will summarize the state-of-the art for the direction of synthetic lethality, and also speculate on the future development of this approach. PMID:27463838

Mutation at p53 serine 389 does not rescue the embryonic lethality in mdm2 or mdm4 null mice.

PubMed

Iwakuma, Tomoo; Parant, John M; Fasulo, Mark; Zwart, Edwin; Jacks, Tyler; de Vries, Annemieke; Lozano, Guillermina

2004-10-07

Mdm2 and its homolog Mdm4 inhibit the function of the tumor suppressor p53. Targeted disruption of either mdm2 or mdm4 genes in mice results in embryonic lethality that is completely rescued by concomitant deletion of p53, suggesting that deletion of negative regulators of p53 results in a constitutively active p53. Thus, these mouse models offer a unique in vivo system to assay the functional significance of different p53 modifications. Phosphorylation of serine 389 in murine p53 occurs specifically after ultraviolet-light-induced DNA damage, and phosphorylation of this site enhances p53 activity both in vitro and in vivo. Recently, mice with a serine to alanine substitution at serine 389 (p53S389A) in the endogenous p53 locus were generated. To examine the in vivo significance of serine 389 phosphorylation during embryogenesis, we crossed these mutant mice to mice lacking mdm2 or mdm4. The p53S389A allele did not alter the embryonic lethality of mdm2 or mdm4. Additional crosses to assay the effect of one p53S389A allele with a p53 null allele also did not rescue the lethal phenotypes. In conclusion, the phenotypes due to loss of mdm2 or mdm4 were not even partially rescued by p53S389A, suggesting that p53S389A is functionally wild type during embryogenesis.

K-RasV14I recapitulates Noonan syndrome in mice

PubMed Central

Hernández-Porras, Isabel; Fabbiano, Salvatore; Schuhmacher, Alberto J.; Aicher, Alexandra; Cañamero, Marta; Cámara, Juan Antonio; Cussó, Lorena; Desco, Manuel; Heeschen, Christopher; Mulero, Francisca; Bustelo, Xosé R.; Guerra, Carmen; Barbacid, Mariano

2014-01-01

Noonan syndrome (NS) is an autosomal dominant genetic disorder characterized by short stature, craniofacial dysmorphism, and congenital heart defects. NS also is associated with a risk for developing myeloproliferative disorders (MPD), including juvenile myelomonocytic leukemia (JMML). Mutations responsible for NS occur in at least 11 different loci including KRAS. Here we describe a mouse model for NS induced by K-RasV14I, a recurrent KRAS mutation in NS patients. K-RasV14I–mutant mice displayed multiple NS-associated developmental defects such as growth delay, craniofacial dysmorphia, cardiac defects, and hematologic abnormalities including a severe form of MPD that resembles human JMML. Homozygous animals had perinatal lethality whose penetrance varied with genetic background. Exposure of pregnant mothers to a MEK inhibitor rescued perinatal lethality and prevented craniofacial dysmorphia and cardiac defects. However, Mek inhibition was not sufficient to correct these defects when mice were treated after weaning. Interestingly, Mek inhibition did not correct the neoplastic MPD characteristic of these mutant mice, regardless of the timing at which the mice were treated, thus suggesting that MPD is driven by additional signaling pathways. These genetically engineered K-RasV14I–mutant mice offer an experimental tool for studying the molecular mechanisms underlying the clinical manifestations of NS. Perhaps more importantly, they should be useful as a preclinical model to test new therapies aimed at preventing or ameliorating those deficits associated with this syndrome. PMID:25359213

Genetic background has a major effect on the penetrance and severity of craniofacial defects in mice heterozygous for the gene encoding the nucleolar protein Treacle.

PubMed

Dixon, Jill; Dixon, Michael James

2004-04-01

Treacher Collins syndrome (TCS) is a craniofacial disorder that results from mutations in TCOF1, which encodes the nucleolar protein Treacle. The severity of the clinical features exhibits wide variation and includes hypoplasia of the mandible and maxilla, abnormalities of the external ears and middle ear ossicles, and cleft palate. To determine the in vivo function of Treacle, we previously generated Tcof1 heterozygous mice on a mixed C57BL/6 and 129 background. These mice exhibited a lethal phenotype, which included abnormal development of the maxilla, absence of the eyes and nasal passages, and neural tube defects. Here, we show that placing the mutation onto different genetic backgrounds has a major effect on the penetrance and severity of the craniofacial and other defects. The offspring exhibit markedly variable strain-dependent phenotypes that range from extremely severe and lethal in a mixed CBA/Ca and 129 background, to apparently normal and viable in a mixed BALB/c and 129 background. In the former case, in addition to a profoundly severe craniofacial phenotype, CBA-derived heterozygous mice also exhibited delayed ossification of the long bones, rib fusions, and digit anomalies. The results of our studies indicate that factors in the different genetic backgrounds contribute extensively to the Tcof1 phenotype. Copyright 2004 Wiley-Liss, Inc.

Favipiravir can evoke lethal mutagenesis and extinction of foot-and-mouth disease virus.

PubMed

de Avila, Ana Isabel; Moreno, Elena; Perales, Celia; Domingo, Esteban

2017-04-02

Antiviral agents are increasingly considered an option for veterinary medicine. An understanding of their mechanism of activity is important to plan their administration either as monotherapy or in combination with other agents. Previous studies have shown that the broad spectrum antiviral agent favipiravir (T-705) and its derivatives T-1105 and T-1106 are efficient inhibitors of foot-and-mouth disease virus (FMDV) replication in cell culture and in vivo. However, no mechanism for their activity against FMDV has been proposed. In the present study we show that favipiravir (T-705) can act as a lethal mutagen for FMDV in cell culture. Evidence includes virus extinction associated with increase in mutation frequency in the mutant spectrum of 860 residues of the 3D (polymerase)-coding region, and a decrease of specific infectivity while the consensus nucleotide sequence of the region analyzed remained invariant. The mutational spectrum evoked by favipiravir differs from that observed with other viruses in that no predominant transition type is observed, indicating that a movement towards A,U- or G,C-rich regions of sequence space is not a prerequisite for virus extinction. We discuss prospects for the use of favipiravir to assist in the control of FMDV, and its possible broader use in veterinary medicine as an extension of its current status as antiviral agent for human influenza virus. Copyright © 2017 Elsevier B.V. All rights reserved.

Histone deacetylase inhibitor treatment induces ‘BRCAness’ and synergistic lethality with PARP inhibitor and cisplatin against human triple negative breast cancer cells

PubMed Central

Ha, Kyungsoo; Bhaskara, Srividya; Cerchietti, Leandro; Devaraj, Santhana G. T.; Shah, Bhavin; Sharma, Sunil; Chang, Jenny C.; Melnick, Ari M.; Hiebert, Scott; Bhalla, Kapil N.

2014-01-01

There is an unmet need to develop new, more effective and safe therapies for the aggressive forms of triple negative breast cancers (TNBCs). While up to 20% of women under 50 years of age with TNBC harbor germline mutations in BRCA1, and these tumors are sensitive to treatment with poly(ADP) ribose polymerase inhibitors, a majority of TNBCs lack BRCA1 mutations or loss of expression. Findings presented here demonstrate that by attenuating the levels of DNA damage response and homologous recombination proteins, pan-histone deacetylase inhibitor (HDI) treatment induces ‘BRCAness’ and sensitizes TNBC cells lacking BRCA1 to lethal effects of PARP inhibitor or cisplatin. Treatment with HDI also induced hyperacetylation of nuclear hsp90. Similar effects were observed following shRNA-mediated depletion of HDAC3, confirming its role as the deacetylase for nuclear HSP90. Furthermore, cotreatment with HDI and ABT-888 induced significantly more DNA strand breaks than either agent alone, and synergistically induced apoptosis of TNBC cells. Notably, co-treatment with HDI and ABT-888 significantly reduced in vivo tumor growth and markedly improved the survival of mice bearing TNBC cell xenografts. These findings support the rationale to interrogate the clinical activity of this novel combination against human TNBC, irrespective of its expression of mutant BRCA1. PMID:25026298

Myopathy-inducing mutation H40Y in ACTA1 hampers actin filament structure and function

DOE PAGES

Chan, Chun; Fan, Jun; Messer, Andrew E.; ...

2016-04-22

In humans, more than 200 missense mutations have been identified in the ACTA1 gene. The exact molecular mechanisms by which, these particular mutations become toxic and lead to muscle weakness and myopathies remain obscure. To address this, here, we performed a molecular dynamics simulation, and we used a broad range of biophysical assays to determine how the lethal and myopathy-related H40Y amino acid substitution in actin affects the structure, stability, and function of this protein. Interestingly, our results showed that H40Y severely disrupts the DNase I-binding-loop structure and actin filaments. In addition, we observed that normal and mutant actin monomersmore » are likely to form distinctive homopolymers, with mutant filaments being very stiff, and not supporting proper myosin binding. Lastly, these phenomena underlie the toxicity of H40Y and may be considered as important triggering factors for the contractile dysfunction, muscle weakness and disease phenotype seen in patients.« less

The Evolutionary Landscape of Localized Prostate Cancers Drives Clinical Aggression.

PubMed

Espiritu, Shadrielle Melijah G; Liu, Lydia Y; Rubanova, Yulia; Bhandari, Vinayak; Holgersen, Erle M; Szyca, Lesia M; Fox, Natalie S; Chua, Melvin L K; Yamaguchi, Takafumi N; Heisler, Lawrence E; Livingstone, Julie; Wintersinger, Jeff; Yousif, Fouad; Lalonde, Emilie; Rouette, Alexandre; Salcedo, Adriana; Houlahan, Kathleen E; Li, Constance H; Huang, Vincent; Fraser, Michael; van der Kwast, Theodorus; Morris, Quaid D; Bristow, Robert G; Boutros, Paul C

2018-05-03

The majority of newly diagnosed prostate cancers are slow growing, with a long natural life history. Yet a subset can metastasize with lethal consequences. We reconstructed the phylogenies of 293 localized prostate tumors linked to clinical outcome data. Multiple subclones were detected in 59% of patients, and specific subclonal architectures associate with adverse clinicopathological features. Early tumor development is characterized by point mutations and deletions followed by later subclonal amplifications and changes in trinucleotide mutational signatures. Specific genes are selectively mutated prior to or following subclonal diversification, including MTOR, NKX3-1, and RB1. Patients with low-risk monoclonal tumors rarely relapse after primary therapy (7%), while those with high-risk polyclonal tumors frequently do (61%). The presence of multiple subclones in an index biopsy may be necessary, but not sufficient, for relapse of localized prostate cancer, suggesting that evolution-aware biomarkers should be studied in prospective studies of low-risk tumors suitable for active surveillance. Copyright © 2018 Elsevier Inc. All rights reserved.

Mutations in KEOPS-complex genes cause nephrotic syndrome with primary microcephaly.

PubMed

Braun, Daniela A; Rao, Jia; Mollet, Geraldine; Schapiro, David; Daugeron, Marie-Claire; Tan, Weizhen; Gribouval, Olivier; Boyer, Olivia; Revy, Patrick; Jobst-Schwan, Tilman; Schmidt, Johanna Magdalena; Lawson, Jennifer A; Schanze, Denny; Ashraf, Shazia; Ullmann, Jeremy F P; Hoogstraten, Charlotte A; Boddaert, Nathalie; Collinet, Bruno; Martin, Gaëlle; Liger, Dominique; Lovric, Svjetlana; Furlano, Monica; Guerrera, I Chiara; Sanchez-Ferras, Oraly; Hu, Jennifer F; Boschat, Anne-Claire; Sanquer, Sylvia; Menten, Björn; Vergult, Sarah; De Rocker, Nina; Airik, Merlin; Hermle, Tobias; Shril, Shirlee; Widmeier, Eugen; Gee, Heon Yung; Choi, Won-Il; Sadowski, Carolin E; Pabst, Werner L; Warejko, Jillian K; Daga, Ankana; Basta, Tamara; Matejas, Verena; Scharmann, Karin; Kienast, Sandra D; Behnam, Babak; Beeson, Brendan; Begtrup, Amber; Bruce, Malcolm; Ch'ng, Gaik-Siew; Lin, Shuan-Pei; Chang, Jui-Hsing; Chen, Chao-Huei; Cho, Megan T; Gaffney, Patrick M; Gipson, Patrick E; Hsu, Chyong-Hsin; Kari, Jameela A; Ke, Yu-Yuan; Kiraly-Borri, Cathy; Lai, Wai-Ming; Lemyre, Emmanuelle; Littlejohn, Rebecca Okashah; Masri, Amira; Moghtaderi, Mastaneh; Nakamura, Kazuyuki; Ozaltin, Fatih; Praet, Marleen; Prasad, Chitra; Prytula, Agnieszka; Roeder, Elizabeth R; Rump, Patrick; Schnur, Rhonda E; Shiihara, Takashi; Sinha, Manish D; Soliman, Neveen A; Soulami, Kenza; Sweetser, David A; Tsai, Wen-Hui; Tsai, Jeng-Daw; Topaloglu, Rezan; Vester, Udo; Viskochil, David H; Vatanavicharn, Nithiwat; Waxler, Jessica L; Wierenga, Klaas J; Wolf, Matthias T F; Wong, Sik-Nin; Leidel, Sebastian A; Truglio, Gessica; Dedon, Peter C; Poduri, Annapurna; Mane, Shrikant; Lifton, Richard P; Bouchard, Maxime; Kannu, Peter; Chitayat, David; Magen, Daniella; Callewaert, Bert; van Tilbeurgh, Herman; Zenker, Martin; Antignac, Corinne; Hildebrandt, Friedhelm

2017-10-01

Galloway-Mowat syndrome (GAMOS) is an autosomal-recessive disease characterized by the combination of early-onset nephrotic syndrome (SRNS) and microcephaly with brain anomalies. Here we identified recessive mutations in OSGEP, TP53RK, TPRKB, and LAGE3, genes encoding the four subunits of the KEOPS complex, in 37 individuals from 32 families with GAMOS. CRISPR-Cas9 knockout in zebrafish and mice recapitulated the human phenotype of primary microcephaly and resulted in early lethality. Knockdown of OSGEP, TP53RK, or TPRKB inhibited cell proliferation, which human mutations did not rescue. Furthermore, knockdown of these genes impaired protein translation, caused endoplasmic reticulum stress, activated DNA-damage-response signaling, and ultimately induced apoptosis. Knockdown of OSGEP or TP53RK induced defects in the actin cytoskeleton and decreased the migration rate of human podocytes, an established intermediate phenotype of SRNS. We thus identified four new monogenic causes of GAMOS, describe a link between KEOPS function and human disease, and delineate potential pathogenic mechanisms.

Myopathy-inducing mutation H40Y in ACTA1 hampers actin filament structure and function

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

Chan, Chun; Fan, Jun; Messer, Andrew E.

In humans, more than 200 missense mutations have been identified in the ACTA1 gene. The exact molecular mechanisms by which, these particular mutations become toxic and lead to muscle weakness and myopathies remain obscure. To address this, here, we performed a molecular dynamics simulation, and we used a broad range of biophysical assays to determine how the lethal and myopathy-related H40Y amino acid substitution in actin affects the structure, stability, and function of this protein. Interestingly, our results showed that H40Y severely disrupts the DNase I-binding-loop structure and actin filaments. In addition, we observed that normal and mutant actin monomersmore » are likely to form distinctive homopolymers, with mutant filaments being very stiff, and not supporting proper myosin binding. Lastly, these phenomena underlie the toxicity of H40Y and may be considered as important triggering factors for the contractile dysfunction, muscle weakness and disease phenotype seen in patients.« less

Cardiac arrhythmia and thyroid dysfunction: a novel genetic link

PubMed Central

Purtell, Kerry; Roepke, Torsten K.; Abbott, Geoffrey W.

2010-01-01

Inherited Long QT Syndrome, a cardiac arrhythmia that predisposes to the often lethal ventricular fibrillation, is commonly linked to mutations in KCNQ1. The KCNQ1 voltage-gated K+ channel α subunit passes ventricular myocyte K+ current that helps bring a timely end to each heart-beat. KCNQ1, like many K+ channel α subunits, is regulated by KCNE β subunits, inherited mutations in which also associate with Long QT Syndrome. KCNQ1 and KCNE mutations are also associated with atrial fibrillation. It has long been known that thyroid status strongly influences cardiac function, and that thyroid dysfunction causes abnormal cardiac structure and rhythm. We recently discovered that KCNQ1 and KCNE2 form a thyroid-stimulating hormone-stimulated K+ channel in the thyroid that is required for normal thyroid hormone biosynthesis. Here, we review this novel genetic link between cardiac and thyroid physiology and pathology, and its potential influence upon future therapeutic strategies in cardiac and thyroid disease. PMID:20688187

Pathways to extinction: beyond the error threshold.

PubMed

Manrubia, Susanna C; Domingo, Esteban; Lázaro, Ester

2010-06-27

Since the introduction of the quasispecies and the error catastrophe concepts for molecular evolution by Eigen and their subsequent application to viral populations, increased mutagenesis has become a common strategy to cause the extinction of viral infectivity. Nevertheless, the high complexity of virus populations has shown that viral extinction can occur through several other pathways apart from crossing an error threshold. Increases in the mutation rate enhance the appearance of defective forms and promote the selection of mechanisms that are able to counteract the accelerated appearance of mutations. Current models of viral evolution take into account more realistic scenarios that consider compensatory and lethal mutations, a highly redundant genotype-to-phenotype map, rough fitness landscapes relating phenotype and fitness, and where phenotype is described as a set of interdependent traits. Further, viral populations cannot be understood without specifying the characteristics of the environment where they evolve and adapt. Altogether, it turns out that the pathways through which viral quasispecies go extinct are multiple and diverse.

Mutant KRAS as a critical determinant of the therapeutic response of colorectal cancer

PubMed Central

Knickelbein, Kyle; Zhang, Lin

2014-01-01

Mutations in the KRAS oncogene represent one of the most prevalent genetic alterations in colorectal cancer (CRC), the third leading cause of cancer-related death in the US. In addition to their well-characterized function in driving tumor progression, KRAS mutations have been recognized as a critical determinant of the therapeutic response of CRC. Recent studies demonstrate that KRAS-mutant tumors are intrinsically insensitive to clinically-used epidermal growth factor receptor (EGFR) targeting antibodies, including cetuximab and panitumumab. Acquired resistance to the anti-EGFR therapy was found to be associated with enrichment of KRAS-mutant tumor cells. However, the underlying molecular mechanism of mutant-KRAS-mediated therapeutic resistance has remained unclear. Despite intensive efforts, directly targeting mutant KRAS has been largely unsuccessful. This review summarizes the recent advances in understanding the biological function of KRAS mutations in determining the therapeutic response of CRC, highlighting several recently developed agents and strategies for targeting mutant KRAS, such as synthetic lethal interactions. PMID:25815366

Two New Fern Chloroplasts and Decelerated Evolution Linked to the Long Generation Time in Tree Ferns

PubMed Central

Zhong, Bojian; Fong, Richard; Collins, Lesley J.; McLenachan, Patricia A.; Penny, David

2014-01-01

We report the chloroplast genomes of a tree fern (Dicksonia squarrosa) and a “fern ally” (Tmesipteris elongata), and show that the phylogeny of early land plants is basically as expected, and the estimates of divergence time are largely unaffected after removing the fastest evolving sites. The tree fern shows the major reduction in the rate of evolution, and there has been a major slowdown in the rate of mutation in both families of tree ferns. We suggest that this is related to a generation time effect; if there is a long time period between generations, then this is probably incompatible with a high mutation rate because otherwise nearly every propagule would probably have several lethal mutations. This effect will be especially strong in organisms that have large numbers of cell divisions between generations. This shows the necessity of going beyond phylogeny and integrating its study with other properties of organisms. PMID:24787621

A homozygous mutation in the endothelin-3 gene associated with a combined Waardenburg type 2 and Hirschsprung phenotype (Shah-Waardenburg syndrome).

PubMed

Hofstra, R M; Osinga, J; Tan-Sindhunata, G; Wu, Y; Kamsteeg, E J; Stulp, R P; van Ravenswaaij-Arts, C; Majoor-Krakauer, D; Angrist, M; Chakravarti, A; Meijers, C; Buys, C H

1996-04-01

Hirschsprung disease (HSCR) or colonic aganglionosis is a congenital disorder characterized by an absence of intramural ganglia along variable lengths of the colon resulting in intestinal obstruction. The incidence of HSCR is 1 in 5,000 live births. Mutations in the RET gene, which codes for a receptor tyrosine kinase, and in EDNRB which codes for the endothelin-B receptor, have been shown to be associated with HSCR in humans. The lethal-spotted mouse which has pigment abnormalities, but also colonic aganglionosis, carries a mutation in the gene coding for endothelin 3 (Edn3), the ligand for the receptor protein encoded by EDNRB. Here, we describe a mutation of the human gene for endothelin 3 (EDN3), homozygously present in a patient with a combined Waardenburg syndrome type 2 (WS2) and HSCR phenotype (Shah-Waardenburg syndrome). The mutation, Cys159Phe, in exon 3 in the ET-3 like domain of EDN3, presumably affects the proteolytic processing of the preproendothelin to the mature peptide EDN3. The patient's parents were first cousins. A previous child in this family had been diagnosed with a similar combination of HSCR, depigmentation and deafness. Depigmentation and deafness were present in other relatives. Moreover, we present a further indication for the involvement of EDNRB in HSCR by reporting a novel mutation detected in one of 40 unselected HSCR patients.

Narrowing the wingless-2 mutation to a 227 kb candidate region on chicken chromosome 12

PubMed Central

Webb, A E; Youngworth, I A; Kaya, M; Gitter, C L; O’Hare, E A; May, B; Cheng, H H; Delany, M E

2018-01-01

ABSTRACT Wingless-2 (wg-2) is an autosomal recessive mutation in chicken that results in an embryonic lethal condition. Affected individuals exhibit a multisystem syndrome characterized by absent wings, truncated legs, and craniofacial, kidney, and feather malformations. Previously, work focused on phenotype description, establishing the autosomal recessive pattern of Mendelian inheritance and placing the mutation on an inbred genetic background to create the congenic line UCD Wingless-2.331. The research described in this paper employed the complementary tools of breeding, genetics, and genomics to map the chromosomal location of the mutation and successively narrow the size of the region for analysis of the causative element. Specifically, the wg-2 mutation was initially mapped to a 7 Mb region of chromosome 12 using an Illumina 3 K SNP array. Subsequent SNP genotyping and exon sequencing combined with analysis from improved genome assemblies narrowed the region of interest to a maximum size of 227 kb. Within this region, 3 validated and 3 predicted candidate genes are found, and these are described. The wg-2 mutation is a valuable resource to contribute to an improved understanding of the developmental pathways involved in chicken and avian limb development as well as serving as a model for human development, as the resulting syndrome shares features with human congenital disorders. PMID:29562287

The 3849 + 10 kB C-->T mutation in a 21-year-old patient with cystic fibrosis.

PubMed

Kaplan, D M; Niv, A; Aviram, M; Parvari, R; Leiberman, A; Fliss, D M

1996-12-01

Cystic fibrosis (CF) is the most common lethal inherited disease in the white population. It is characterized by exocrine gland epithelia dysfunction, which leads to pulmonary and pancreatic insufficiency. Since the cloning of the CF gene in 1989 and the identification of the most common CF mutation (delta F508), more than 400 different mutations have been described. These mutations appear to contribute to the heterogeneity of the CF phenotype and several reports have speculated on the relationship between the most common CF mutations and the patient's clinical status. We report the case of a 21-year-old woman with longstanding chronic pansinusitis, nasal polyposis, chronic cough and severe nasal crusting. During a period of five years she had been followed by her otolaryngologist and pediatric pulmonologist. Sweat tests performed at the age of 17 and 18 were within normal limits and she underwent repeated conventional sinonasal procedures, with no improvement in her clinical status. On her present admission, sweat tests showed a 70 meq/l chloride concentration. The diagnosis of CF was then confirmed by DNA analysis and the patient was found to carry the 3849 + 10 kB C-->T mutation. The early detection of this newly recognized form of CF in adults as well as in children presenting with sinonasal symptoms is critical for life expectancy and quality.

Pancreatic cancer genomes reveal aberrations in axon guidance pathway genes

PubMed Central

Biankin, Andrew V.; Waddell, Nicola; Kassahn, Karin S.; Gingras, Marie-Claude; Muthuswamy, Lakshmi B.; Johns, Amber L.; Miller, David K.; Wilson, Peter J.; Patch, Ann-Marie; Wu, Jianmin; Chang, David K.; Cowley, Mark J.; Gardiner, Brooke B.; Song, Sarah; Harliwong, Ivon; Idrisoglu, Senel; Nourse, Craig; Nourbakhsh, Ehsan; Manning, Suzanne; Wani, Shivangi; Gongora, Milena; Pajic, Marina; Scarlett, Christopher J.; Gill, Anthony J.; Pinho, Andreia V.; Rooman, Ilse; Anderson, Matthew; Holmes, Oliver; Leonard, Conrad; Taylor, Darrin; Wood, Scott; Xu, Qinying; Nones, Katia; Fink, J. Lynn; Christ, Angelika; Bruxner, Tim; Cloonan, Nicole; Kolle, Gabriel; Newell, Felicity; Pinese, Mark; Mead, R. Scott; Humphris, Jeremy L.; Kaplan, Warren; Jones, Marc D.; Colvin, Emily K.; Nagrial, Adnan M.; Humphrey, Emily S.; Chou, Angela; Chin, Venessa T.; Chantrill, Lorraine A.; Mawson, Amanda; Samra, Jaswinder S.; Kench, James G.; Lovell, Jessica A.; Daly, Roger J.; Merrett, Neil D.; Toon, Christopher; Epari, Krishna; Nguyen, Nam Q.; Barbour, Andrew; Zeps, Nikolajs; Kakkar, Nipun; Zhao, Fengmei; Wu, Yuan Qing; Wang, Min; Muzny, Donna M.; Fisher, William E.; Brunicardi, F. Charles; Hodges, Sally E.; Reid, Jeffrey G.; Drummond, Jennifer; Chang, Kyle; Han, Yi; Lewis, Lora R.; Dinh, Huyen; Buhay, Christian J.; Beck, Timothy; Timms, Lee; Sam, Michelle; Begley, Kimberly; Brown, Andrew; Pai, Deepa; Panchal, Ami; Buchner, Nicholas; De Borja, Richard; Denroche, Robert E.; Yung, Christina K.; Serra, Stefano; Onetto, Nicole; Mukhopadhyay, Debabrata; Tsao, Ming-Sound; Shaw, Patricia A.; Petersen, Gloria M.; Gallinger, Steven; Hruban, Ralph H.; Maitra, Anirban; Iacobuzio-Donahue, Christine A.; Schulick, Richard D.; Wolfgang, Christopher L.; Morgan, Richard A.; Lawlor, Rita T.; Capelli, Paola; Corbo, Vincenzo; Scardoni, Maria; Tortora, Giampaolo; Tempero, Margaret A.; Mann, Karen M.; Jenkins, Nancy A.; Perez-Mancera, Pedro A.; Adams, David J.; Largaespada, David A.; Wessels, Lodewyk F. A.; Rust, Alistair G.; Stein, Lincoln D.; Tuveson, David A.; Copeland, Neal G.; Musgrove, Elizabeth A.; Scarpa, Aldo; Eshleman, James R.; Hudson, Thomas J.; Sutherland, Robert L.; Wheeler, David A.; Pearson, John V.; McPherson, John D.; Gibbs, Richard A.; Grimmond, Sean M.

2012-01-01

Pancreatic cancer is a highly lethal malignancy with few effective therapies. We performed exome sequencing and copy number analysis to define genomic aberrations in a prospectively accrued clinical cohort (n = 142) of early (stage I and II) sporadic pancreatic ductal adenocarcinoma. Detailed analysis of 99 informative tumours identified substantial heterogeneity with 2,016 non-silent mutations and 1,628 copy-number variations. We define 16 significantly mutated genes, reaffirming known mutations (KRAS, TP53, CDKN2A, SMAD4, MLL3, TGFBR2, ARID1A and SF3B1), and uncover novel mutated genes including additional genes involved in chromatin modification (EPC1 and ARID2), DNA damage repair (ATM) and other mechanisms (ZIM2, MAP2K4, NALCN, SLC16A4 and MAGEA6). Integrative analysis with in vitro functional data and animal models provided supportive evidence for potential roles for these genetic aberrations in carcinogenesis. Pathway-based analysis of recurrently mutated genes recapitulated clustering in core signalling pathways in pancreatic ductal adenocarcinoma, and identified new mutated genes in each pathway. We also identified frequent and diverse somatic aberrations in genes described traditionally as embryonic regulators of axon guidance, particularly SLIT/ROBO signalling, which was also evident in murine Sleeping Beauty transposon-mediated somatic mutagenesis models of pancreatic cancer, providing further supportive evidence for the potential involvement of axon guidance genes in pancreatic carcinogenesis. PMID:23103869

Pancreatic cancer genomes reveal aberrations in axon guidance pathway genes.

PubMed

Biankin, Andrew V; Waddell, Nicola; Kassahn, Karin S; Gingras, Marie-Claude; Muthuswamy, Lakshmi B; Johns, Amber L; Miller, David K; Wilson, Peter J; Patch, Ann-Marie; Wu, Jianmin; Chang, David K; Cowley, Mark J; Gardiner, Brooke B; Song, Sarah; Harliwong, Ivon; Idrisoglu, Senel; Nourse, Craig; Nourbakhsh, Ehsan; Manning, Suzanne; Wani, Shivangi; Gongora, Milena; Pajic, Marina; Scarlett, Christopher J; Gill, Anthony J; Pinho, Andreia V; Rooman, Ilse; Anderson, Matthew; Holmes, Oliver; Leonard, Conrad; Taylor, Darrin; Wood, Scott; Xu, Qinying; Nones, Katia; Fink, J Lynn; Christ, Angelika; Bruxner, Tim; Cloonan, Nicole; Kolle, Gabriel; Newell, Felicity; Pinese, Mark; Mead, R Scott; Humphris, Jeremy L; Kaplan, Warren; Jones, Marc D; Colvin, Emily K; Nagrial, Adnan M; Humphrey, Emily S; Chou, Angela; Chin, Venessa T; Chantrill, Lorraine A; Mawson, Amanda; Samra, Jaswinder S; Kench, James G; Lovell, Jessica A; Daly, Roger J; Merrett, Neil D; Toon, Christopher; Epari, Krishna; Nguyen, Nam Q; Barbour, Andrew; Zeps, Nikolajs; Kakkar, Nipun; Zhao, Fengmei; Wu, Yuan Qing; Wang, Min; Muzny, Donna M; Fisher, William E; Brunicardi, F Charles; Hodges, Sally E; Reid, Jeffrey G; Drummond, Jennifer; Chang, Kyle; Han, Yi; Lewis, Lora R; Dinh, Huyen; Buhay, Christian J; Beck, Timothy; Timms, Lee; Sam, Michelle; Begley, Kimberly; Brown, Andrew; Pai, Deepa; Panchal, Ami; Buchner, Nicholas; De Borja, Richard; Denroche, Robert E; Yung, Christina K; Serra, Stefano; Onetto, Nicole; Mukhopadhyay, Debabrata; Tsao, Ming-Sound; Shaw, Patricia A; Petersen, Gloria M; Gallinger, Steven; Hruban, Ralph H; Maitra, Anirban; Iacobuzio-Donahue, Christine A; Schulick, Richard D; Wolfgang, Christopher L; Morgan, Richard A; Lawlor, Rita T; Capelli, Paola; Corbo, Vincenzo; Scardoni, Maria; Tortora, Giampaolo; Tempero, Margaret A; Mann, Karen M; Jenkins, Nancy A; Perez-Mancera, Pedro A; Adams, David J; Largaespada, David A; Wessels, Lodewyk F A; Rust, Alistair G; Stein, Lincoln D; Tuveson, David A; Copeland, Neal G; Musgrove, Elizabeth A; Scarpa, Aldo; Eshleman, James R; Hudson, Thomas J; Sutherland, Robert L; Wheeler, David A; Pearson, John V; McPherson, John D; Gibbs, Richard A; Grimmond, Sean M

2012-11-15

Pancreatic cancer is a highly lethal malignancy with few effective therapies. We performed exome sequencing and copy number analysis to define genomic aberrations in a prospectively accrued clinical cohort (n = 142) of early (stage I and II) sporadic pancreatic ductal adenocarcinoma. Detailed analysis of 99 informative tumours identified substantial heterogeneity with 2,016 non-silent mutations and 1,628 copy-number variations. We define 16 significantly mutated genes, reaffirming known mutations (KRAS, TP53, CDKN2A, SMAD4, MLL3, TGFBR2, ARID1A and SF3B1), and uncover novel mutated genes including additional genes involved in chromatin modification (EPC1 and ARID2), DNA damage repair (ATM) and other mechanisms (ZIM2, MAP2K4, NALCN, SLC16A4 and MAGEA6). Integrative analysis with in vitro functional data and animal models provided supportive evidence for potential roles for these genetic aberrations in carcinogenesis. Pathway-based analysis of recurrently mutated genes recapitulated clustering in core signalling pathways in pancreatic ductal adenocarcinoma, and identified new mutated genes in each pathway. We also identified frequent and diverse somatic aberrations in genes described traditionally as embryonic regulators of axon guidance, particularly SLIT/ROBO signalling, which was also evident in murine Sleeping Beauty transposon-mediated somatic mutagenesis models of pancreatic cancer, providing further supportive evidence for the potential involvement of axon guidance genes in pancreatic carcinogenesis.

Hereditary diffuse gastric cancer: implications of genetic testing for screening and prophylactic surgery.

PubMed

Cisco, Robin M; Ford, James M; Norton, Jeffrey A

2008-10-01

Approximately 10% of patients with gastric cancer show familial clustering, and 3% show autosomal dominance and high penetrance. Hereditary diffuse gastric cancer (HDGC) is an autosomal-dominant, inherited cancer syndrome in which affected individuals develop diffuse-type gastric cancer at a young age. Inactivating mutations in the E-cadherin gene CDH1 have been identified in 30% to 50% of patients. CDH1 mutation carriers have an approximately 70% lifetime risk of developing DGC, and affected women carry an additional 20% to 40% risk of developing lobular breast cancer. Because endoscopic surveillance is ineffective in identifying early HDGC, gene-directed prophylactic total gastrectomy currently is offered for CDH1 mutation carriers. In series of asymptomatic individuals undergoing total gastrectomy for CDH1 mutations, the removed stomachs usually contain small foci of early DGC, making surgery not prophylactic but curative. The authors of this review recommend consideration of total gastrectomy in CDH1 mutation carriers at an age 5 years younger than the youngest family member who developed gastric cancer. Individuals who choose not to undergo prophylactic gastrectomy should be followed with biannual chromoendoscopy, and women with CDH1 mutations also should undergo regular surveillance with magnetic resonance imaging studies of the breast. Because of the emergence of gene-directed gastrectomy for HDGC, today, a previously lethal disease is detected by molecular techniques, allowing curative surgery at an early stage.

The PSO4 gene is responsible for an error-prone recombinational DNA repair pathway in Saccharomyces cerevisiae.

PubMed

de Andrade, H H; Marques, E K; Schenberg, A C; Henriques, J A

1989-06-01

The induction of mitotic gene conversion and crossing-over in Saccharomyces cerevisiae diploid cells homozygous for the pso4-1 mutation was examined in comparison to the corresponding wild-type strain. The pso4-1 mutant strain was found to be completely blocked in mitotic recombination induced by photoaddition of mono- and bifunctional psoralen derivatives as well as by mono- (HN1) and bifunctional (HN2) nitrogen mustards or 254 nm UV radiation in both stationary and exponential phases of growth. Concerning the lethal effect, diploids homozygous for the pso4-1 mutation are more sensitive to all agents tested in any growth phase. However, this effect is more pronounced in the G2 phase of the cell cycle. These results imply that the ploidy effect and the resistance of budding cells are under the control of the PSO4 gene. On the other hand, the pso4-1 mutant is mutationally defective for all agents used. Therefore, the pso4-1 mutant has a generalized block in both recombination and mutation ability. This indicates that the PSO4 gene is involved in an error-prone repair pathway which relies on a recombinational mechanism, strongly suggesting an analogy between the pso4-1 mutation and the RecA or LexA mutation of Escherichia coli.

Myosin-driven rescue of contractile reserve and energetics in mouse hearts bearing familial hypertrophic cardiomyopathy-associated mutant troponin T is mutation-specific

PubMed Central

He, Huamei; Hoyer, Kirsten; Tao, Hai; Rice, Ronald; Jimenez, Jesus; Tardiff, Jil C; Ingwall, Joanne S

2012-01-01

The thin filament protein troponin T (TnT) is a regulator of sarcomere function. Whole heart energetics and contractile reserve are compromised in transgenic mice bearing missense mutations at R92 within the tropomyosin-binding domain of cTnT, despite being distal to the ATP hydrolysis domain of myosin. These mutations are associated with familial hypertrophic cardiomyopathy (FHC). Here we test the hypothesis that genetically replacing murine αα-MyHC with murine ββ-MyHC in hearts bearing the R92Q cTnT mutation, a particularly lethal FHC-associated mutation, leads to sufficiently large perturbations in sarcomere function to rescue whole heart energetics and decrease the cost of contraction. By comparing R92Q cTnT and R92L cTnT mutant hearts, we also test whether any rescue is mutation-specific. We defined the energetic state of the isolated perfused heart using 31P-NMR spectroscopy while simultaneously measuring contractile performance at four work states. We found that the cost of increasing contraction in intact mouse hearts with R92Q cTnT depends on the type of myosin present in the thick filament. We also found that the salutary effect of this manoeuvre is mutation-specific, demonstrating the major regulatory role of cTnT on sarcomere function at the whole heart level. PMID:22907055

Mutations in eukaryotic release factors 1 and 3 act as general nonsense suppressors in Drosophila.

PubMed Central

Chao, Anna T; Dierick, Herman A; Addy, Tracie M; Bejsovec, Amy

2003-01-01

In a screen for suppressors of the Drosophila wingless(PE4) nonsense allele, we isolated mutations in the two components that form eukaryotic release factor. eRF1 and eRF3 comprise the translation termination complex that recognizes stop codons and catalyzes the release of nascent polypeptide chains from ribosomes. Mutations disrupting the Drosophila eRF1 and eRF3 show a strong maternal-effect nonsense suppression due to readthrough of stop codons and are zygotically lethal during larval stages. We tested nonsense mutations in wg and in other embryonically acting genes and found that different stop codons can be suppressed but only a subset of nonsense alleles are subject to suppression. We suspect that the context of the stop codon is significant: nonsense alleles sensitive to suppression by eRF1 and eRF3 encode stop codons that are immediately followed by a cytidine. Such suppressible alleles appear to be intrinsically weak, with a low level of readthrough that is enhanced when translation termination is disrupted. Thus the eRF1 and eRF3 mutations provide a tool for identifying nonsense alleles that are leaky. Our findings have important implications for assigning null mutant phenotypes and for selecting appropriate alleles to use in suppressor screens. PMID:14573473

A comprehensive characterization of rare mitochondrial DNA variants in neuroblastoma

PubMed Central

Pignataro, Piero; Lasorsa, Vito Alessandro; Hogarty, Michael D.; Castellano, Aurora; Conte, Massimo; Tonini, Gian Paolo; Iolascon, Achille; Gasparre, Giuseppe; Capasso, Mario

2016-01-01

Background Neuroblastoma, a tumor of the developing sympathetic nervous system, is a common childhood neoplasm that is often lethal. Mitochondrial DNA (mtDNA) mutations have been found in most tumors including neuroblastoma. We extracted mtDNA data from a cohort of neuroblastoma samples that had undergone Whole Exome Sequencing (WES) and also used snap-frozen samples in which mtDNA was entirely sequenced by Sanger technology. We next undertook the challenge of determining those mutations that are relevant to, or arisen during tumor development. The bioinformatics pipeline used to extract mitochondrial variants from matched tumor/blood samples was enriched by a set of filters inclusive of heteroplasmic fraction, nucleotide variability, and in silico prediction of pathogenicity. Results Our in silico multistep workflow applied both on WES and Sanger-sequenced neuroblastoma samples, allowed us to identify a limited burden of somatic and germline mitochondrial mutations with a potential pathogenic impact. Conclusions The few singleton germline and somatic mitochondrial mutations emerged, according to our in silico analysis, do not appear to impact on the development of neuroblastoma. Our findings are consistent with the hypothesis that most mitochondrial somatic mutations can be considered as ‘passengers’ and consequently have no discernible effect in this type of cancer. PMID:27351283

Genetic and developmental study of a complex locus in the house mouse. Progress report, August 1, 1976--July 31, 1977

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

Bennett, D.

1977-06-01

We have maintained and studied certain aspects of the genetics and embryology of approximately 40 chromosome 17 mutations in the mouse, including eight newly derived t-haplotypes. Two dominant T mutations (T/sup Hp/ and T/sup Or1/) have been characterized as having a homozygous lethal phenotype different from T; they die earlier in development, at 6 to 7 days with defects of the embryonic ectoderm. The same mutations, which are both probably chromosome deletions produce mild runting in heterozygous condition, and more severe runting in compound with all t-haplotypes that have been studied. Attempts to map the position of a recessive viablemore » allele t/sup 38/ have given results that suggest that t/sup 38/ is not a point mutation, but may extend over a distance of 3 centimorgans. Data from the same set of experiments indicate that particular combinations of mutations in females may result in gametic selection, i.e., the preferential selection by the egg of one of the two classes of sperm from heterozygous males. New experiments designed to analyze the relationship between t-haplotypes and H-2 type in wild mice are in progress.« less

Clinical Utility of Circulating Tumor DNA for Molecular Assessment and Precision Medicine in Pancreatic Cancer.

PubMed

Takai, Erina; Totoki, Yasushi; Nakamura, Hiromi; Kato, Mamoru; Shibata, Tatsuhiro; Yachida, Shinichi

2016-01-01

Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal malignancies. The genomic landscape of the PDAC genome features four frequently mutated genes (KRAS, CDKN2A, TP53, and SMAD4) and dozens of candidate driver genes altered at low frequency, including potential clinical targets. Circulating cell-free DNA (cfDNA) is a promising resource to detect molecular characteristics of tumors, supporting the concept of "liquid biopsy".We determined the mutational status of KRAS in plasma cfDNA using multiplex droplet digital PCR in 259 patients with PDAC, retrospectively. Furthermore, we constructed a novel modified SureSelect-KAPA-Illumina platform and an original panel of 60 genes. We then performed targeted deep sequencing of cfDNA in 48 patients who had ≥1 % mutant allele frequencies of KRAS in plasma cfDNA.Droplet digital PCR detected KRAS mutations in plasma cfDNA in 63 of 107 (58.9 %) patients with inoperable tumors. Importantly, potentially targetable somatic mutations were identified in 14 of 48 patients (29.2 %) examined by cfDNA sequencing.Our two-step approach with plasma cfDNA, combining droplet digital PCR and targeted deep sequencing, is a feasible clinical approach. Assessment of mutations in plasma cfDNA may provide a new diagnostic tool, assisting decisions for optimal therapeutic strategies for PDAC patients.

Unexpected allelic heterogeneity and spectrum of mutations in Fowler syndrome revealed by next-generation exome sequencing.

PubMed

Lalonde, Emilie; Albrecht, Steffen; Ha, Kevin C H; Jacob, Karine; Bolduc, Nathalie; Polychronakos, Constantin; Dechelotte, Pierre; Majewski, Jacek; Jabado, Nada

2010-08-01

Protein coding genes constitute approximately 1% of the human genome but harbor 85% of the mutations with large effects on disease-related traits. Therefore, efficient strategies for selectively sequencing complete coding regions (i.e., "whole exome") have the potential to contribute our understanding of human diseases. We used a method for whole-exome sequencing coupling Agilent whole-exome capture to the Illumina DNA-sequencing platform, and investigated two unrelated fetuses from nonconsanguineous families with Fowler Syndrome (FS), a stereotyped phenotype lethal disease. We report novel germline mutations in feline leukemia virus subgroup C cellular-receptor-family member 2, FLVCR2, which has recently been shown to cause FS. Using this technology, we identified three types of genetic abnormalities: point-mutations, insertions-deletions, and intronic splice-site changes (first pathogenic report using this technology), in the fetuses who both were compound heterozygotes for the disease. Although revealing a high level of allelic heterogeneity and mutational spectrum in FS, this study further illustrates the successful application of whole-exome sequencing to uncover genetic defects in rare Mendelian disorders. Of importance, we show that we can identify genes underlying rare, monogenic and recessive diseases using a limited number of patients (n=2), in the absence of shared genetic heritage and in the presence of allelic heterogeneity.

Novel compound heterozygous DPH1 mutations in a patient with the unique clinical features of airway obstruction and external genital abnormalities.

PubMed

Nakajima, Junya; Oana, Shingo; Sakaguchi, Tomohiro; Nakashima, Mitsuko; Numabe, Hironao; Kawashima, Hisashi; Matsumoto, Naomichi; Miyake, Noriko

2018-04-01

The diphthamide biosynthesis 1 (DPH1) gene encodes one of the essential components of the enzyme catalyzing the first step of diphthamide formation on eukaryotic elongation factor 2 (EEF2). Diphthamide is the posttranslationally modified histidine residue on EEF2 that promotes protein chain elongation in the ribosome. DPH1 defects result in a failure of protein synthesis involving EEF2, leading to growth defects, embryonic lethality, and cell death. In humans, DPH1 mutations cause developmental delay with a short stature, dysmorphic features, and sparse hair, and are inherited in an autosomal recessive manner (MIM#616901). To date, only two homozygous missense mutations in DPH1 (c.17T>A, p.Met6Lys and c.701T>C, p.Leu234Pro) have been reported. We used WES to identify novel compound heterozygous mutations in DPH1 (c.289delG, p.Glu97Lysfs*8 and c.491T>C, p.Leu164Pro) in a patient from a nonconsanguineous family presenting with intellectual disability, a short stature, craniofacial abnormalities, and external genital abnormalities. The clinical phenotype of all patients with DPH1 mutations, including the current patient, revealed core features, although the external genital anomaly was newly recognized in our case.

Inactivation and mutation induction in Saccharomyces cerevisiae exposed to simulated sunlight: evaluation of action spectra.

PubMed

Schenk-Meuser, K; Pawlowsky, K; Kiefer, J

1992-07-15

The effectiveness of polychromatic light irradiation was investigated for haploid yeast cells. Inactivation and mutation induction were measured in both a RAD-wildtype strain and an excision-repair defective strain. The behaviour of vegetative "wet" cells was compared to that of dehydrated cells. The aim of the study was to assess the interaction of UVC with other wavelengths in cells of different states of humidity. The irradiation procedure was therefore carried out using a solar simulator either with full spectrum or with a UVC-blocking filter (modified sunlight) added. The results were analysed on the basis of separately determined action spectra. The summation of the efficiency of individual wavelengths was compared to the values obtained from polychromatic irradiation. It is shown that the effects caused by the whole-spectrum irradiation in wet cells can be predicted sufficiently from the calculation, while dried wildtype cells exhibit higher mutation rates. Thus it can be assumed that drying-specific damage leads to lethal and mutagenic lesions which are processed in different ways, causing a synergistic behaviour in mutation induction. Irradiation of vegetative cells with modified sunlight (UVC-) results in less inactivation and lower mutation rates than were calculated. From these results it can be concluded that this antagonistic behaviour is caused by the interaction of near-UV photoproducts.

Mismatch repair deficiency does not enhance ENU mutagenesis in the zebrafish germ line.

PubMed

Feitsma, Harma; de Bruijn, Ewart; van de Belt, Jose; Nijman, Isaac J; Cuppen, Edwin

2008-07-01

S(N)1-type alkylating agents such as N-ethyl-N-nitrosourea (ENU) are very potent mutagens. They act by transferring their alkyl group to DNA bases, which, upon mispairing during replication, can cause single base pair mutations in the next replication cycle. As DNA mismatch repair (MMR) proteins are involved in the recognition of alkylation damage, we hypothesized that ENU-induced mutation rates could be increased in a MMR-deficient background, which would be beneficial for mutagenesis approaches. We applied a standard ENU mutagenesis protocol to adult zebrafish deficient in the MMR gene msh6 and heterozygous controls to study the effect of MMR on ENU-induced DNA damage. Dose-dependent lethality was found to be similar for homozygous and heterozygous mutants, indicating that there is no difference in ENU resistance. Mutation discovery by high-throughput dideoxy resequencing of genomic targets in outcrossed progeny of the mutagenized fish did also not reveal any differences in germ line mutation frequency. These results may indicate that the maximum mutation load for zebrafish has been reached with the currently used, highly optimized ENU mutagenesis protocol. Alternatively, the MMR system in the zebrafish germ line may be saturated very rapidly, thereby having a limited effect on high-dose ENU mutagenesis.

THE COMPARATIVE MUTAGENIC EFFECT OF ETHYLENIMINE, ULTRAVIOLET AND X-RAYS (in Russian)

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

Alikhanyan, S.I.; Zhdanova, N.I.

1960-07-11

The mutagenic effects of ethylenimine were determined by the use of Actinomyces alivaceus (the product of B/sub 12/ vitamin) and compared with ultraviolet, and x ray effects. The spores were exposed to high concentrations of ethylenimine (solution of 1: 1000) for various times (30 min, 1, 2, 3, 4, and 5 hr) and to diluted concentrations (1: 3000, produced by short-time exposure to a concentrated solution; lethality at various treatments is nearly identical. The results of the comparison with ultraviolet and x rays indicate a large advantage in the use of ethylenimine. Curves of reverse mutation frequencies indicate prevailing frequenciesmore » of ethylenimine mutations over ultraviolet. This shows that regardless of genetic background changes. the advantages of ethylenimine are retained. (R.V.J.)« less

Genetic evolution of pancreatic cancer: lessons learnt from the pancreatic cancer genome sequencing project

PubMed Central

Iacobuzio-Donahue, Christine A

2012-01-01

Pancreatic cancer is a disease caused by the accumulation of genetic alterations in specific genes. Elucidation of the human genome sequence, in conjunction with technical advances in the ability to perform whole exome sequencing, have provided new insight into the mutational spectra characteristic of this lethal tumour type. Most recently, exomic sequencing has been used to clarify the clonal evolution of pancreatic cancer as well as provide time estimates of pancreatic carcinogenesis, indicating that a long window of opportunity may exist for early detection of this disease while in the curative stage. Moving forward, these mutational analyses indicate potential targets for personalised diagnostic and therapeutic intervention as well as the optimal timing for intervention based on the natural history of pancreatic carcinogenesis and progression. PMID:21749982

Uncoupling thermotolerance from the induction of heat shock proteins.

PubMed Central

Smith, B J; Yaffe, M P

1991-01-01

Exposure of cells to elevated temperatures causes a rapid increase in the synthesis of heat shock proteins (hsps) and induces thermotolerance, the increased ability of cells to survive exposure to lethal temperatures; however, the connection between hsp induction and the acquisition of thermotolerance is unclear. hsp induction in the yeast Saccharomyces cerevisiae is mediated by the activation of heat-shock transcription factor, and recently we have described a mutation, hsf1-m3, in heat-shock transcription factor that prevents the factor's activation. We now demonstrate that this mutation results in a general block in heat-shock induction but does not affect the acquisition of thermotolerance. Our results indicate that high-level induction of the major hsps is not required for cells to acquire thermotolerance. Images PMID:1763024

Bone Marrow Transplantation in Mice as a Tool to Generate Genetically Modified Animals

NASA Astrophysics Data System (ADS)

Rőszer, Tamás; Pintye, Éva; Benkő, Ilona

2008-12-01

Transgenic mice can be used either as models of known inherited human diseases or can be applied to perform phenotypic tests of genes with unknown function. In some special applications of gene modification we have to create a tissue specific mutation of a given gene. In some cases however the gene modification can be lethal in the intrauterine life, therefore we should engraft the mutated cells in the postnatal life period. After total body irradiation transplantation of bone marrow cells can be a solution to introduce mutant hematopoietic stem cells into a mature animal. Bone marrow transplantation is a useful and novel tool to study the role of hematopoietic cells in the pathogenesis of inflammation, autoimmune syndromes and many metabolic alterations coupled recently to leukocyte functions.

Intratumoral heterogeneity: Role of differentiation in a potentially lethal phenotype of testicular cancer

PubMed Central

Bilen, Mehmet Asim; Hess, Kenneth R.; Broaddus, Russell R.; Kopetz, Scott; Wei, Chongjuan; Pagliaro, Lance C.; Karam, Jose A.; Ward, John F.; Wood, Christopher G.; Rao, Priya; Tu, Zachary H.; General, Rosale; Chen, Adrienne H.; Nieto, Yago L.; Yeung, Sai‐ching J.; Lin, Sue‐Hwa; Logothetis, Christopher J.; Pisters, Louis L.

2016-01-01

BACKGROUND Intratumoral heterogeneity presents a major obstacle to the widespread implementation of precision medicine. The authors assessed the origin of intratumoral heterogeneity in nonseminomatous germ cell tumor of the testis (NSGCT) and identified distinct tumor subtypes and a potentially lethal phenotype. METHODS In this retrospective study, all consecutive patients who had been diagnosed with an NSGCT between January 2000 and December 2010 were evaluated. The histologic makeup of primary tumors and the clinical course of disease were determined for each patient. A Fine and Gray proportional hazards regression analysis was used to determine the prognostic risk factors, and the Gray test was used to detect differences in the cumulative incidence of cancer death. In a separate prospective study, next‐generation sequencing was performed on tumor samples from 9 patients to identify any actionable mutations. RESULTS Six hundred fifteen patients were included in this study. Multivariate analysis revealed that the presence of yolk sac tumor in the primary tumor (P = .0003) was associated with an unfavorable prognosis. NSGCT could be divided into 5 subgroups. Patients in the yolk sac‐seminoma subgroup had the poorest clinical outcome (P = .0015). These tumors tended to undergo somatic transformation (P < .0001). Among the 9 NSGCTs that had a yolk sac tumor phenotype, no consistent gene mutation was detected. CONCLUSIONS The current data suggest that intratumoral heterogeneity is caused in part by differentiation of pluripotent progenitor cells. Integrated or multimodal therapy may be effective at addressing intratumoral heterogeneity and treating distinct subtypes as well as a potentially lethal phenotype of NSGCT. Cancer 2016;122:1836–43. © 2016 The Authors. Cancer published by Wiley Periodicals, Inc. on behalf of American Cancer Society. This is an open access article under the terms of the Creative Commons Attribution‐NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes. PMID:27018785

Intratumoral heterogeneity: Role of differentiation in a potentially lethal phenotype of testicular cancer.

PubMed

Tu, Shi-Ming; Bilen, Mehmet Asim; Hess, Kenneth R; Broaddus, Russell R; Kopetz, Scott; Wei, Chongjuan; Pagliaro, Lance C; Karam, Jose A; Ward, John F; Wood, Christopher G; Rao, Priya; Tu, Zachary H; General, Rosale; Chen, Adrienne H; Nieto, Yago L; Yeung, Sai-Ching J; Lin, Sue-Hwa; Logothetis, Christopher J; Pisters, Louis L

2016-06-15

Intratumoral heterogeneity presents a major obstacle to the widespread implementation of precision medicine. The authors assessed the origin of intratumoral heterogeneity in nonseminomatous germ cell tumor of the testis (NSGCT) and identified distinct tumor subtypes and a potentially lethal phenotype. In this retrospective study, all consecutive patients who had been diagnosed with an NSGCT between January 2000 and December 2010 were evaluated. The histologic makeup of primary tumors and the clinical course of disease were determined for each patient. A Fine and Gray proportional hazards regression analysis was used to determine the prognostic risk factors, and the Gray test was used to detect differences in the cumulative incidence of cancer death. In a separate prospective study, next-generation sequencing was performed on tumor samples from 9 patients to identify any actionable mutations. Six hundred fifteen patients were included in this study. Multivariate analysis revealed that the presence of yolk sac tumor in the primary tumor (P = .0003) was associated with an unfavorable prognosis. NSGCT could be divided into 5 subgroups. Patients in the yolk sac-seminoma subgroup had the poorest clinical outcome (P = .0015). These tumors tended to undergo somatic transformation (P < .0001). Among the 9 NSGCTs that had a yolk sac tumor phenotype, no consistent gene mutation was detected. The current data suggest that intratumoral heterogeneity is caused in part by differentiation of pluripotent progenitor cells. Integrated or multimodal therapy may be effective at addressing intratumoral heterogeneity and treating distinct subtypes as well as a potentially lethal phenotype of NSGCT. Cancer 2016;122:1836-43. © 2016 The Authors. Cancer published by Wiley Periodicals, Inc. on behalf of American Cancer Society. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes. © 2016 American Cancer Society.

Meier–Gorlin syndrome genotype–phenotype studies: 35 individuals with pre-replication complex gene mutations and 10 without molecular diagnosis

PubMed Central

de Munnik, Sonja A; Bicknell, Louise S; Aftimos, Salim; Al-Aama, Jumana Y; van Bever, Yolande; Bober, Michael B; Clayton-Smith, Jill; Edrees, Alaa Y; Feingold, Murray; Fryer, Alan; van Hagen, Johanna M; Hennekam, Raoul C; Jansweijer, Maaike C E; Johnson, Diana; Kant, Sarina G; Opitz, John M; Ramadevi, A Radha; Reardon, Willie; Ross, Alison; Sarda, Pierre; Schrander-Stumpel, Constance T R M; Schoots, Jeroen; Temple, I Karen; Terhal, Paulien A; Toutain, Annick; Wise, Carol A; Wright, Michael; Skidmore, David L; Samuels, Mark E; Hoefsloot, Lies H; Knoers, Nine V A M; Brunner, Han G; Jackson, Andrew P; Bongers, Ernie M H F

2012-01-01

Meier–Gorlin syndrome (MGS) is an autosomal recessive disorder characterized by microtia, patellar aplasia/hypoplasia, and short stature. Recently, mutations in five genes from the pre-replication complex (ORC1, ORC4, ORC6, CDT1, and CDC6), crucial in cell-cycle progression and growth, were identified in individuals with MGS. Here, we report on genotype–phenotype studies in 45 individuals with MGS (27 females, 18 males; age 3 months–47 years). Thirty-five individuals had biallelic mutations in one of the five causative pre-replication genes. No homozygous or compound heterozygous null mutations were detected. In 10 individuals, no definitive molecular diagnosis was made. The triad of microtia, absent/hypoplastic patellae, and short stature was observed in 82% of individuals with MGS. Additional frequent clinical features were mammary hypoplasia (100%) and abnormal genitalia (42% predominantly cryptorchidism and hypoplastic labia minora/majora). One individual with ORC1 mutations only had short stature, emphasizing the highly variable clinical spectrum of MGS. Individuals with ORC1 mutations had significantly shorter stature and smaller head circumferences than individuals from other gene categories. Furthermore, compared with homozygous missense mutations, compound heterozygous mutations appeared to have a more severe effect on phenotype, causing more severe growth retardation in ORC4 and more frequently pulmonary emphysema in CDT1. A lethal phenotype was seen in four individuals with compound heterozygous ORC1 and CDT1 mutations. No other clear genotype–phenotype association was observed. Growth hormone and estrogen treatment may be of some benefit, respectively, to growth retardation and breast hypoplasia, though further studies in this patient group are needed. PMID:22333897

Mutagenic effect of accelerated heavy ions on bacterial cells

NASA Astrophysics Data System (ADS)

Boreyko, A. V.; Krasavin, E. A.

2011-11-01

The heavy ion accelerators of the Joint Institute for Nuclear Research were used to study the regularities and mechanisms of formation of different types of mutations in prokaryote cells. The induction of direct (lac-, ton B-, col B) mutations for Esherichia coli cells and reverse his- → His+ mutations of Salmonella typhimurium, Bacillus subtilis cells under the action of radiation in a wide range of linear energy transfer (LET) was studied. The regularities of formation of gene and structural (tonB trp-) mutations for Esherichia coli bacteria under the action of accelerated heavy ions were studied. It was demonstrated that the rate of gene mutations as a function of the dose under the action of Γ rays and accelerated heavy ions is described by linear-quadratic functions. For structural mutations, linear "dose-effect" dependences are typical. The quadratic character of mutagenesis dose curves is determined by the "interaction" of two independent "hitting" events in the course of SOS repair of genetic structures. The conclusion made was that gene mutations under the action of accelerated heavy ions are induced by δ electron regions of charged particle tracks. The methods of SOS chromotest, SOS lux test, and λ prophage induction were used to study the regularities of SOS response of cells under the action of radiations in a wide LET range. The following proposition was substantiated: the molecular basis for formation of gene mutations are cluster single-strand DNA breaks, and that for structural mutations, double-strand DNA breaks. It was found out that the LET dependence of the relative biological efficiency of accelerated ions is described by curves with a local maximum. It was demonstrated that the biological efficiency of ionizing radiations with different physical characteristics on cells with different genotype, estimated by the lethal action, induction of gene and deletion mutations, precision excision of transposons, is determined by the specific features of energy transfer of the radiations that affect the character of induced DNA damage, and the efficiency inducible and constitutive cell repair systems. The growth of relative biological efficiency of heavy charged particles is determined by the growth of the damage yield of the DNA participating in the formation of radiation-induced effects, and higher efficiency of inducible repair systems. It was established that the LET value ( L max) for which the maximum (according to the applied irradiation criteria) coefficients of relative biological efficiency are observed varies depending on the character of the registered radiation induced effect. It was demonstrated that for gene mutations and induction of precision excision of mobile elements the values of L max are realized in a LET range of ≈20 keV/μm. For lethal effects of irradiation and induction of deletion mutations the value of L max is ≈ 100 and 50 keV/μm, respectively. The differences in the L max for the studied radiation gene effectis are determined by the different type of DNA damage participating in the mutation process. A molecular model of the formation of gene mutations in Escherichia coli cells under the action of ionizing radiation was proposed. Basic DNA radiation damage and main repair ways were considered in the framework of this model. The basis is the idea of the decisive role of mutagenic, error-prone, branch of SOS repair in fixing premutation DNA damage into point mutations. It was demonstrated that the central mechanism in this process is the formation of an inducible multi-enzymatic complex including the DNA polymerase V (Umu C), RecA-protease, SSB proteins, subunits of DNA polymerase III, performing erroneous DNA synthesis on the damaged matrix. A mathematical model of induction of gene mutations under ultraviolet cell irradiation was developed based on the molecular model.

Paternal inheritance of classic X-linked bilateral periventricular nodular heterotopia.

PubMed

Kasper, Burkhard S; Kurzbuch, Katrin; Chang, Bernard S; Pauli, Elisabeth; Hamer, Hajo M; Winkler, Jürgen; Hehr, Ute

2013-06-01

Periventricular nodular heterotopia (PNH) is a developmental disorder of the central nervous system, characterized by heterotopic nodules of gray matter resulting from disturbed neuronal migration. The most common form of bilateral PNH is X-linked dominant inherited, caused by mutations in the Filamin A gene (FLNA) and associated with a wide variety of other clinical findings including congenital heart disease. The typical patient with FLNA-associated PNH is female and presents with difficult to treat seizures. In contrast, hemizygous FLNA loss of function mutations in males are reported to be perinatally lethal. In X-linked dominant traits like FLNA-associated PNH the causal mutation is commonly inherited from the mother. Here, we present an exceptional family with paternal transmission of classic bilateral FLNA-associated PNH from a mildly affected father with somatic and germline mosaicism for a c.5686G>A FLNA splice mutation to both daughters with strikingly variable clinical manifestation and PNH extent in cerebral MR imaging. Our observations emphasize the importance to consider in genetic counseling and risk assessment the rare genetic constellation of paternal transmission for families with X-linked dominant inherited FLNA-associated PNH. Copyright © 2013 Wiley Periodicals, Inc.

Mutations in the evolutionarily highly conserved KEOPS complex genes cause nephrotic syndrome with microcephaly

PubMed Central

Braun, Daniela A.; Rao, Jia; Mollet, Geraldine; Schapiro, David; Daugeron, Marie-Claire; Tan, Weizhen; Gribouval, Olivier; Boyer, Olivia; Revy, Patrick; Jobst-Schwan, Tilman; Schmidt, Johanna Magdalena; Lawson, Jennifer A.; Schanze, Denny; Ashraf, Shazia; Boddaert, Nathalie; Collinet, Bruno; Martin, Gaëlle; Liger, Dominique; Lovric, Svjetlana; Furlano, Monica; Guerrera, I. Chiara; Sanchez-Ferras, Oraly; Menten, Björn; Vergult, Sarah; De Rocker, Nina; Airik, Merlin; Hermle, Tobias; Shril, Shirlee; Widmeier, Eugen; Gee, Heon Yung; Choi, Won-Il; Sadowski, Carolin E.; Pabst, Werner L.; Warejko, Jillian; Daga, Ankana; LeBerre, Tamara Basta; Matejas, Verena; Behnam, Babak; Beeson, Brendan; Begtrup, Amber; Bruce, Malcolm; Ch'ng, Gaik-Siew; Lin, Shuan-Pei; Chang, Jui-Hsing; Chen, Chao-Huei; Cho, Megan T.; Gipson, Patrick E.; Hsu, Chyong-Hsin; Kari, Jameela A.; Ke, Yu-Yuan; Kiraly-Borri, Cathy; Lai, Wai-ming; Lemyre, Emmanuelle; Littlejohn, Rebecca Okasha; Masri, Amira; Moghtaderi, Mastaneh; Nakamura, Kazuyuki; Praet, Marleen; Prasad, Chitra; Prytula, Agnieszka; Roeder, Elizabeth; Rump, Patrick; Schnur, Rhonda E.; Shiihara, Takashi; Sinha, Manish; Soliman, Neveen A; Soulami, Kenza; Sweetser, David A.; Tsai, Wen-Hui; Tsai, Jeng-Daw; Vester, Udo; Viskochil, David H.; Vatanavicharn, Nithiwat; Waxler, Jessica L.; Wolf, Matthias T.F.; Wong, Sik-Nin; Poduri, Annapurna; Truglio, Gessica; Mane, Shrikant; Lifton, Richard P.; Bouchard, Maxime; Kannu, Peter; Chitayat, David; Magen, Daniella; Calleweart, Bert; van Tilbeurgh, Herman; Zenker, Martin; Antignac, Corinne; Hildebrandt, Friedhelm

2018-01-01

Galloway-Mowat syndrome (GAMOS) is a severe autosomal-recessive disease characterized by the combination of early-onset steroid-resistant nephrotic syndrome (SRNS) and microcephaly with brain anomalies. To date, mutations of WDR73 are the only known monogenic cause of GAMOS and in most affected individuals the molecular diagnosis remains elusive. We here identify recessive mutations of OSGEP, TP53RK, TPRKB, or LAGE3, encoding the 4 subunits of the KEOPS complex in 33 individuals of 30 families with GAMOS. CRISPR/Cas9 knockout in zebrafish and mice recapitulates the human phenotype of microcephaly and results in early lethality. Knockdown of OSGEP, TP53RK, or TPRKB inhibits cell proliferation, which human mutations fail to rescue, and knockdown of either gene activates DNA damage response signaling and induces apoptosis. OSGEP and TP53RK molecularly interact and co-localize with the actin-regulating ARP2/3 complex. Furthermore, knockdown of OSGEP and TP53RK induces defects of the actin cytoskeleton and reduces migration rate of human podocytes, an established intermediate phenotype of SRNS. We thus identify 4 novel monogenic causes of GAMOS, describe the first link between KEOPS function and human disease, and delineate potential pathogenic mechanisms. PMID:28805828

DNA Lesions Caused by ROS and RNOS: A Review of Interactions and Reactions Involving Guanine

NASA Astrophysics Data System (ADS)

Shukla, P. K.; Mishra, P. C.

DNA is constantly attacked by a large number of endogenous and exogenous reactive oxygen species (ROS), reactive nitrogen oxide species (RNOS), and alkylating agents which produce a wide variety of modifications of its constituents, particularly the bases. Some of these modifications (lesions) are hazardous to normal cell functioning, and are implicated in several lethal conditions including chronic inflammatory diseases, atherosclerosis, aging, mutation, cancer, and neurodegenerative disorders, such as the Alzheimer's and Parkinson's diseases.

Cu/Zn superoxide dismutase and the proton ATPase Pma1p of Saccharomyces cerevisiae

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

Baron, J. Allen; Chen, Janice S.; Culotta, Valeria C., E-mail: vculott1@jhu.edu

2015-07-03

In eukaryotes, the Cu/Zn containing superoxide dismutase (SOD1) plays a critical role in oxidative stress protection as well as in signaling. We recently demonstrated a function for Saccharomyces cerevisiae Sod1p in signaling through CK1γ casein kinases and identified the essential proton ATPase Pma1p as one likely target. The connection between Sod1p and Pma1p was explored further by testing the impact of sod1Δ mutations on cells expressing mutant alleles of Pma1p that alter activity and/or post-translational regulation of this ATPase. We report here that sod1Δ mutations are lethal when combined with the T912D allele of Pma1p in the C-terminal regulatory domain.more » This “synthetic lethality” was reversed by intragenic suppressor mutations in Pma1p, including an A906G substitution that lies within the C-terminal regulatory domain and hyper-activates Pma1p. Surprisingly the effect of sod1Δ mutations on Pma1-T912D is not mediated through the Sod1p signaling pathway involving the CK1γ casein kinases. Rather, Sod1p sustains life of cells expressing Pma1-T912D through oxidative stress protection. The synthetic lethality of sod1Δ Pma1-T912D cells is suppressed by growing cells under low oxygen conditions or by treatments with manganese-based antioxidants. We now propose a model in which Sod1p maximizes Pma1p activity in two ways: one involving signaling through CK1γ casein kinases and an independent role for Sod1p in oxidative stress protection. - Highlights: • In yeast, the anti-oxidant enzyme SOD1 promotes activity of the proton ATPase Pma1p. • Cells expressing a T912D variant of Pma1p are not viable without SOD1. • SOD1 is needed to protect Pma1-T912D expressing cells from severe oxidative damage. • SOD1 activates Pma1p through casein kinase signaling and oxidative stress protection.« less

Myosin Va Bound to Phagosomes Binds to F-Actin and Delays Microtubule-dependent Motility

PubMed Central

Al-Haddad, Ahmed; Shonn, Marion A.; Redlich, Bärbel; Blocker, Ariel; Burkhardt, Janis K.; Yu, Hanry; Hammer, John A.; Weiss, Dieter G.; Steffen, Walter; Griffiths, Gareth; Kuznetsov, Sergei A.

2001-01-01

We established a light microscopy-based assay that reconstitutes the binding of phagosomes purified from mouse macrophages to preassembled F-actin in vitro. Both endogenous myosin Va from mouse macrophages and exogenous myosin Va from chicken brain stimulated the phagosome–F-actin interaction. Myosin Va association with phagosomes correlated with their ability to bind F-actin in an ATP-regulated manner and antibodies to myosin Va specifically blocked the ATP-sensitive phagosome binding to F-actin. The uptake and retrograde transport of phagosomes from the periphery to the center of cells in bone marrow macrophages was observed in both normal mice and mice homozygous for the dilute-lethal spontaneous mutation (myosin Va null). However, in dilute-lethal macrophages the accumulation of phagosomes in the perinuclear region occurred twofold faster than in normal macrophages. Motion analysis revealed saltatory phagosome movement with temporarily reversed direction in normal macrophages, whereas almost no reversals in direction were observed in dilute-lethal macrophages. These observations demonstrate that myosin Va mediates phagosome binding to F-actin, resulting in a delay in microtubule-dependent retrograde phagosome movement toward the cell center. We propose an “antagonistic/cooperative mechanism” to explain the saltatory phagosome movement toward the cell center in normal macrophages. PMID:11553713

Inherited DNA-Repair Gene Mutations in Men with Metastatic Prostate Cancer.

PubMed

Pritchard, Colin C; Mateo, Joaquin; Walsh, Michael F; De Sarkar, Navonil; Abida, Wassim; Beltran, Himisha; Garofalo, Andrea; Gulati, Roman; Carreira, Suzanne; Eeles, Rosalind; Elemento, Olivier; Rubin, Mark A; Robinson, Dan; Lonigro, Robert; Hussain, Maha; Chinnaiyan, Arul; Vinson, Jake; Filipenko, Julie; Garraway, Levi; Taplin, Mary-Ellen; AlDubayan, Saud; Han, G Celine; Beightol, Mallory; Morrissey, Colm; Nghiem, Belinda; Cheng, Heather H; Montgomery, Bruce; Walsh, Tom; Casadei, Silvia; Berger, Michael; Zhang, Liying; Zehir, Ahmet; Vijai, Joseph; Scher, Howard I; Sawyers, Charles; Schultz, Nikolaus; Kantoff, Philip W; Solit, David; Robson, Mark; Van Allen, Eliezer M; Offit, Kenneth; de Bono, Johann; Nelson, Peter S

2016-08-04

Inherited mutations in DNA-repair genes such as BRCA2 are associated with increased risks of lethal prostate cancer. Although the prevalence of germline mutations in DNA-repair genes among men with localized prostate cancer who are unselected for family predisposition is insufficient to warrant routine testing, the frequency of such mutations in patients with metastatic prostate cancer has not been established. We recruited 692 men with documented metastatic prostate cancer who were unselected for family history of cancer or age at diagnosis. We isolated germline DNA and used multiplex sequencing assays to assess mutations in 20 DNA-repair genes associated with autosomal dominant cancer-predisposition syndromes. A total of 84 germline DNA-repair gene mutations that were presumed to be deleterious were identified in 82 men (11.8%); mutations were found in 16 genes, including BRCA2 (37 men [5.3%]), ATM (11 [1.6%]), CHEK2 (10 [1.9% of 534 men with data]), BRCA1 (6 [0.9%]), RAD51D (3 [0.4%]), and PALB2 (3 [0.4%]). Mutation frequencies did not differ according to whether a family history of prostate cancer was present or according to age at diagnosis. Overall, the frequency of germline mutations in DNA-repair genes among men with metastatic prostate cancer significantly exceeded the prevalence of 4.6% among 499 men with localized prostate cancer (P<0.001), including men with high-risk disease, and the prevalence of 2.7% in the Exome Aggregation Consortium, which includes 53,105 persons without a known cancer diagnosis (P<0.001). In our multicenter study, the incidence of germline mutations in genes mediating DNA-repair processes among men with metastatic prostate cancer was 11.8%, which was significantly higher than the incidence among men with localized prostate cancer. The frequencies of germline mutations in DNA-repair genes among men with metastatic disease did not differ significantly according to age at diagnosis or family history of prostate cancer. (Funded by Stand Up To Cancer and others.).

Identification of a pathogenic FTO mutation by next-generation sequencing in a newborn with growth retardation and developmental delay.

PubMed

Daoud, Hussein; Zhang, Dong; McMurray, Fiona; Yu, Andrea; Luco, Stephanie M; Vanstone, Jason; Jarinova, Olga; Carson, Nancy; Wickens, James; Shishodia, Shifali; Choi, Hwanho; McDonough, Michael A; Schofield, Christopher J; Harper, Mary-Ellen; Dyment, David A; Armour, Christine M

2016-03-01

A homozygous loss-of-function mutation p.(Arg316Gln) in the fat mass and obesity-associated (FTO) gene, which encodes for an iron and 2-oxoglutarate-dependent oxygenase, was previously identified in a large family in which nine affected individuals present with a lethal syndrome characterised by growth retardation and multiple malformations. To date, no other pathogenic mutation in FTO has been identified as a cause of multiple congenital malformations. We investigated a 21-month-old girl who presented distinctive facial features, failure to thrive, global developmental delay, left ventricular cardiac hypertrophy, reduced vision and bilateral hearing loss. We performed targeted next-generation sequencing of 4813 clinically relevant genes in the patient and her parents. We identified a novel FTO homozygous missense mutation (c.956C>T; p.(Ser319Phe)) in the affected individual. This mutation affects a highly conserved residue located in the same functional domain as the previously characterised mutation p.(Arg316Gln). Biochemical studies reveal that p.(Ser319Phe) FTO has reduced 2-oxoglutarate turnover and N-methyl-nucleoside demethylase activity. Our findings are consistent with previous reports that homozygous mutations in FTO can lead to rare growth retardation and developmental delay syndrome, and further support the proposal that FTO plays an important role in early development of human central nervous and cardiovascular systems. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/

De novo Uroplakin IIIa heterozygous mutations cause human renal adysplasia leading to severe kidney failure.

PubMed

Jenkins, Dagan; Bitner-Glindzicz, Maria; Malcolm, Sue; Hu, Chih-Chi A; Allison, Jennifer; Winyard, Paul J D; Gullett, Ambrose M; Thomas, David F M; Belk, Rachel A; Feather, Sally A; Sun, Tung-Tien; Woolf, Adrian S

2005-07-01

Human renal adysplasia usually occurs sporadically, and bilateral disease is the most common cause of childhood end-stage renal failure, a condition that is lethal without intervention using dialysis or transplantation. De novo heterozygous mutations in Uroplakin IIIa (UPIIIa) are reported in four of 17 children with kidney failure caused by renal adysplasia in the absence of an overt urinary tract obstruction. One girl and one boy in unrelated kindreds had a missense mutation at a CpG dinucleotide in the cytoplasmic domain of UPIIIa (Pro273Leu), both of whom had severe vesicoureteric reflux, and the girl had persistent cloaca; two other patients had de novo mutations in the 3' UTR (963 T-->G; 1003 T-->C), and they had renal adysplasia in the absence of any other anomaly. The mutations were absent in all sets of parents and in siblings, none of whom had radiologic evidence of renal adysplasia, and mutations were absent in two panels of 192 ethnically matched control chromosomes. UPIIIa was expressed in nascent urothelia in ureter and renal pelvis of human embryos, and it is suggested that perturbed urothelial differentiation may generate human kidney malformations, perhaps by altering differentiation of adjacent smooth muscle cells such that the metanephros is exposed to a functional obstruction of urine flow. With advances in renal replacement therapy, children with renal failure, who would otherwise have died, are surviving to adulthood. Therefore, although the mechanisms of action of the UPIIIa mutations have yet to be determined, these findings have important implications regarding genetic counseling of affected individuals who reach reproductive age.

The lavender plumage colour in Japanese quail is associated with a complex mutation in the region of MLPH that is related to differences in growth, feed consumption and body temperature

PubMed Central

2012-01-01

Background The lavender phenotype in quail is a dilution of both eumelanin and phaeomelanin in feathers that produces a blue-grey colour on a wild-type feather pattern background. It has been previously demonstrated by intergeneric hybridization that the lavender mutation in quail is homologous to the same phenotype in chicken, which is caused by a single base-pair change in exon 1 of MLPH. Results In this study, we have shown that a mutation of MLPH is also associated with feather colour dilution in quail, but that the mutational event is extremely different. In this species, the lavender phenotype is associated with a non-lethal complex mutation involving three consecutive overlapping chromosomal changes (two inversions and one deletion) that have consequences on the genomic organization of four genes (MLPH and the neighbouring PRLH, RAB17 and LRRFIP1). The deletion of PRLH has no effect on the level of circulating prolactin. Lavender birds have lighter body weight, lower body temperature and increased feed consumption and residual feed intake than wild-type plumage quail, indicating that this complex mutation is affecting the metabolism and the regulation of homeothermy. Conclusions An extensive overlapping chromosome rearrangement was associated with a non-pathological Mendelian trait and minor, non deleterious effects in the lavender Japanese quail which is a natural knockout for PRLH. PMID:22937744

Par-4, a Gene Required for Cytoplasmic Localization and Determination of Specific Cell Types in Caenorhabditis Elegans Embryogenesis

PubMed Central

Morton, D. G.; Roos, J. M.; Kemphues, K. J.

1992-01-01

Specification of some cell fates in the early Caenorhabditis elegans embryo is mediated by cytoplasmic localization under control of the maternal genome. Using nine newly isolated mutations, and two existing mutations, we have analyzed the role of the maternally expressed gene par-4 in cytoplasmic localization. We recovered seven new par-4 alleles in screens for maternal effect lethal mutations that result in failure to differentiate intestinal cells. Two additional par-4 mutations were identified in noncomplementation screens using strains with a high frequency of transposon mobility. All 11 mutations cause defects early in development of embryos produced by homozygous mutant mothers. Analysis with a deficiency in the region indicates that it33 is a strong loss-of-function mutation. par-4(it33) terminal stage embryos contain many cells, but show no morphogenesis, and are lacking intestinal cells. Temperature shifts with the it57ts allele suggest that the critical period for both intestinal differentiation and embryo viability begins during oogenesis, about 1.5 hr before fertilization, and ends before the four-cell stage. We propose that the primary function of the par-4 gene is to act as part of a maternally encoded system for cytoplasmic localization in the first cell cycle, with par-4 playing a particularly important role in the determination of intestine. Analysis of a par-4;par-2 double mutant suggests that par-4 and par-2 gene products interact in this system. PMID:1582558

Parasites resistant to the antimalarial atovaquone fail to transmit by mosquitoes.

PubMed

Goodman, Christopher D; Siregar, Josephine E; Mollard, Vanessa; Vega-Rodríguez, Joel; Syafruddin, Din; Matsuoka, Hiroyuki; Matsuzaki, Motomichi; Toyama, Tomoko; Sturm, Angelika; Cozijnsen, Anton; Jacobs-Lorena, Marcelo; Kita, Kiyoshi; Marzuki, Sangkot; McFadden, Geoffrey I

2016-04-15

Drug resistance compromises control of malaria. Here, we show that resistance to a commonly used antimalarial medication, atovaquone, is apparently unable to spread. Atovaquone pressure selects parasites with mutations in cytochrome b, a respiratory protein with low but essential activity in the mammalian blood phase of the parasite life cycle. Resistance mutations rescue parasites from the drug but later prove lethal in the mosquito phase, where parasites require full respiration. Unable to respire efficiently, resistant parasites fail to complete mosquito development, arresting their life cycle. Because cytochrome b is encoded by the maternally inherited parasite mitochondrion, even outcrossing with wild-type strains cannot facilitate spread of resistance. Lack of transmission suggests that resistance will be unable to spread in the field, greatly enhancing the utility of atovaquone in malaria control. Copyright © 2016, American Association for the Advancement of Science.

Cloning of the altered mRNA stability (ams) gene of Escherichia coli K-12.

PubMed Central

Claverie-Martin, F; Diaz-Torres, M R; Yancey, S D; Kushner, S R

1989-01-01

A temperature-sensitive mutation in the ams gene of Escherichia coli causes an increase in the chemical half-life of pulse-labeled RNA at the nonpermissive temperature. Using lambda clones containing DNA fragments from the 23- to 24-min region on the E. coli chromosome, we have isolated a 5.8-kilobase DNA fragment which, when present in a low-copy-number plasmid, complements the conditional lethality and increased mRNA stability associated with the ams-1 mutation. The approximate initiation site and the direction of transcription of the ams gene were determined from the size of truncated polypeptides produced by Tn1000 insertions and Bal 31 deletions. Overexpression of the ams locus by using a T7 RNA polymerase-promoter system permitted the identification of an ams-encoded polypeptide of 110 kilodaltons. Images PMID:2477358

Cloning and characterization of the Bacillus subtilis birA gene encoding a repressor of the biotin operon.

PubMed

Bower, S; Perkins, J; Yocum, R R; Serror, P; Sorokin, A; Rahaim, P; Howitt, C L; Prasad, N; Ehrlich, S D; Pero, J

1995-05-01

The Bacillus subtilis birA gene, which regulates biotin biosynthesis, has been cloned and characterized. The birA gene maps at 202 degrees on the B. subtilis chromosome and encodes a 36,200-Da protein that is 27% identical to Escherichia coli BirA protein. Three independent mutations in birA that lead to deregulation of biotin synthesis alter single amino acids in the amino-terminal end of the protein. The amino-terminal region that is affected by these three birA mutations shows sequence similarity to the helix-turn-helix DNA binding motif previously identified in E. coli BirA protein. B. subtilis BirA protein also possesses biotin-protein ligase activity, as judged by its ability to complement a conditional lethal birA mutant of E. coli.

Cloning and characterization of the Bacillus subtilis birA gene encoding a repressor of the biotin operon.

PubMed Central

Bower, S; Perkins, J; Yocum, R R; Serror, P; Sorokin, A; Rahaim, P; Howitt, C L; Prasad, N; Ehrlich, S D; Pero, J

1995-01-01

The Bacillus subtilis birA gene, which regulates biotin biosynthesis, has been cloned and characterized. The birA gene maps at 202 degrees on the B. subtilis chromosome and encodes a 36,200-Da protein that is 27% identical to Escherichia coli BirA protein. Three independent mutations in birA that lead to deregulation of biotin synthesis alter single amino acids in the amino-terminal end of the protein. The amino-terminal region that is affected by these three birA mutations shows sequence similarity to the helix-turn-helix DNA binding motif previously identified in E. coli BirA protein. B. subtilis BirA protein also possesses biotin-protein ligase activity, as judged by its ability to complement a conditional lethal birA mutant of E. coli. PMID:7730294

Flavitrack: an annotated database of flavivirus sequences

PubMed Central

Misra, Milind

2009-01-01

Motivation Properly annotated sequence data for flaviviruses, which cause diseases, such as tick-borne encephalitis (TBE), dengue fever (DF), West Nile (WN) and yellow fever (YF), can aid in the design of antiviral drugs and vaccines to prevent their spread. Flavitrack was designed to help identify conserved sequence motifs, interpret mutational and structural data and track evolution of phenotypic properties. Summary Flavitrack contains over 590 complete flavivirus genome/protein sequences and information on known mutations and literature references. Each sequence has been manually annotated according to its date and place of isolation, phenotype and lethality. Internal tools are provided to rapidly determine relationships between viruses in Flavitrack and sequences provided by the user. Availability http://carnot.utmb.edu/flavitrack Contact chschein@utmb.edu Supplementary information http://carnot.utmb.edu/flavitrack/B1S1.html PMID:17660525

Effects of Decay of Incorporated H3-Thymidine on Bacteria

PubMed Central

Person, Stanley; Leah Lewis, Hazel

1962-01-01

The killing efficiency due to the decay of incorporated H3-thymidine in three mutants of E. coli strain 15: 15T-, 15T-L-, and 15T-U- has been determined. This efficiency is comparable to that previously determined by others for P32 decay. The killing efficiency has been determined as a function of H3-thymidine specific activity, storage media and storage temperature. We have observed a latent killing effect that causes lethality under certain conditions. The kinetics of latent killing have been examined at several temperatures. Finally, mutation production induced by H3-thymidine decays was shown to occur. The results are consistent with the idea that inactivation and mutations may be caused by a process in the nuclear transmutation that is not associated with β-particle ionization damage. PMID:19431318

Mutational characterization of the P3H1/CRTAP/CypB complex in recessive osteogenesis imperfecta.

PubMed

Barbirato, C; Trancozo, M; Almeida, M G; Almeida, L S; Santos, T O; Duarte, J C G; Rebouças, M R G O; Sipolatti, V; Nunes, V R R; Paula, F

2015-12-03

Osteogenesis imperfecta (OI) is a genetic disease characterized by bone deformities and fractures. Most cases are caused by autosomal dominant mutations in the type I collagen genes COL1A1 and COL1A2; however, an increasing number of recessive mutations in other genes have been reported. The LEPRE1, CRTAP, and PPIB genes encode proteins that form the P3H1/CRTAP/CypB complex, which is responsible for posttranslational modifications of type I collagen. In general, mutations in these genes lead to severe and lethal phenotypes of recessive OI. Here, we describe sixteen genetic variations detected in LEPRE1, CRTAP, and PPIB from 25 Brazilian patients with OI. Samples were screened for mutations on single-strand conformation polymorphism gels and variants were determined by automated sequencing. Seven variants were detected in patients but were absent in control samples. LEPRE1 contained the highest number of variants, including the previously described West African allele (c.1080+1G>T) found in one patient with severe OI as well as a previously undescribed p.Trp675Leu change that is predicted to be disease causing. In CRTAP, one patient carried the c.558A>G homozygous mutation, predicted as disease causing through alteration of a splice site. Genetic variations detected in the PPIB gene are probably not pathogenic due to their localization or because of their synonymous effect. This study enhances our knowledge about the mutational pattern of the LEPRE1, CRTAP, and PPIB genes. In addition, the results strengthen the proposition that LEPRE1 should be the first gene analyzed in mutation detection studies in patients with recessive OI.

Low-fidelity Venezuelan equine encephalitis virus polymerase mutants to improve live-attenuated vaccine safety and efficacy

PubMed Central

Kautz, Tiffany F; Guerbois, Mathilde; Khanipov, Kamil; Yun, Ruimei; Warmbrod, Kelsey L; Fofanov, Yuriy; Weaver, Scott C; Forrester, Naomi L

2018-01-01

Abstract During RNA virus replication, there is the potential to incorporate mutations that affect virulence or pathogenesis. For live-attenuated vaccines, this has implications for stability, as replication may result in mutations that either restore the wild-type phenotype via reversion or compensate for the attenuating mutations by increasing virulence (pseudoreversion). Recent studies have demonstrated that altering the mutation rate of an RNA virus is an effective attenuation tool. To validate the safety of low-fidelity mutations to increase vaccine attenuation, several mutations in the RNA-dependent RNA-polymerase (RdRp) were tested in the live-attenuated Venezuelan equine encephalitis virus vaccine strain, TC-83. Next generation sequencing after passage in the presence of mutagens revealed a mutant containing three mutations in the RdRp, TC-83 3x, to have decreased replication fidelity, while a second mutant, TC-83 4x displayed no change in fidelity, but shared many phenotypic characteristics with TC-83 3x. Both mutants exhibited increased, albeit inconsistent attenuation in an infant mouse model, as well as increased immunogenicity and complete protection against lethal challenge of an adult murine model compared with the parent TC-83. During serial passaging in a highly permissive model, the mutants increased in virulence but remained less virulent than the parent TC-83. These results suggest that the incorporation of low-fidelity mutations into the RdRp of live-attenuated vaccines for RNA viruses can confer increased immunogenicity whilst showing some evidence of increased attenuation. However, while in theory such constructs may result in more effective vaccines, the instability of the vaccine phenotype decreases the likelihood of this being an effective vaccine strategy. PMID:29593882

Biallelic UFM1 and UFC1 mutations expand the essential role of ufmylation in brain development.

PubMed

Nahorski, Michael S; Maddirevula, Sateesh; Ishimura, Ryosuke; Alsahli, Saud; Brady, Angela F; Begemann, Anaïs; Mizushima, Tsunehiro; Guzmán-Vega, Francisco J; Obata, Miki; Ichimura, Yoshinobu; Alsaif, Hessa S; Anazi, Shams; Ibrahim, Niema; Abdulwahab, Firdous; Hashem, Mais; Monies, Dorota; Abouelhoda, Mohamed; Meyer, Brian F; Alfadhel, Majid; Eyaid, Wafa; Zweier, Markus; Steindl, Katharina; Rauch, Anita; Arold, Stefan T; Woods, C Geoffrey; Komatsu, Masaaki; Alkuraya, Fowzan S

2018-06-02

The post-translational modification of proteins through the addition of UFM1, also known as ufmylation, plays a critical developmental role as revealed by studies in animal models. The recent finding that biallelic mutations in UBA5 (the E1-like enzyme for ufmylation) cause severe early-onset encephalopathy with progressive microcephaly implicates ufmylation in human brain development. More recently, a homozygous UFM1 variant was proposed as a candidate aetiology of severe early-onset encephalopathy with progressive microcephaly. Here, we establish a locus for severe early-onset encephalopathy with progressive microcephaly based on two families, and map the phenotype to a novel homozygous UFM1 mutation. This mutation has a significantly diminished capacity to form thioester intermediates with UBA5 and with UFC1 (the E2-like enzyme for ufmylation), with resulting impaired ufmylation of cellular proteins. Remarkably, in four additional families where eight children have severe early-onset encephalopathy with progressive microcephaly, we identified two biallelic UFC1 mutations, which impair UFM1-UFC1 intermediate formation with resulting widespread reduction of cellular ufmylation, a pattern similar to that observed with UFM1 mutation. The striking resemblance between UFM1- and UFC1-related clinical phenotype and biochemical derangements strongly argues for an essential role for ufmylation in human brain development. The hypomorphic nature of UFM1 and UFC1 mutations and the conspicuous depletion of biallelic null mutations in the components of this pathway in human genome databases suggest that it is necessary for embryonic survival, which is consistent with the embryonic lethal nature of knockout models for the orthologous genes.

Absence of FKBP10 in Recessive Type XI Osteogenesis Imperfecta Leads to Diminished Collagen Cross-Linking and Reduced Collagen Deposition in Extracellular Matrix

PubMed Central

Barnes, Aileen M.; Cabral, Wayne A.; Weis, MaryAnn; Makareeva, Elena; Mertz, Edward L.; Leikin, Sergey; Eyre, David; Trujillo, Carlos; Marini, Joan C.

2012-01-01

Recessive osteogenesis imperfecta (OI) is caused by defects in genes whose products interact with type I collagen for modification and/or folding. We identified a Palestinian pedigree with moderate and lethal forms of recessive OI caused by mutations in FKBP10 or PPIB, which encode endoplasmic reticulum resident chaperone/isomerases FKBP65 and CyPB, respectively. In one pedigree branch, both parents carry a deletion in PPIB (c.563_566delACAG), causing lethal type IX OI in their two children. In another branch, a child with moderate type XI OI has a homozygous FKBP10 mutation (c.1271_1272delCCinsA). Proband FKBP10 transcripts are 4% of control and FKBP65 protein is absent from proband cells. Proband collagen electrophoresis reveals slight band broadening, compatible with ≈10% overmodification. Normal chain incorporation, helix folding, and collagen Tm support a minimal general collagen chaperone role for FKBP65. However, there is a dramatic decrease in collagen deposited in culture despite normal collagen secretion. Mass spectrometry reveals absence of hydroxylation of the collagen telopeptide lysine involved in cross-linking, suggesting that FKBP65 is required for lysyl hydroxylase activity or access to type I collagen telopeptide lysines, perhaps through its function as a peptidylprolyl isomerase. Proband collagen to organics ratio in matrix is approximately 30% of normal in Raman spectra. Immunofluorescence shows sparse, disorganized collagen fibrils in proband matrix. PMID:22718341

A 660-Kb Deletion with Antagonistic Effects on Fertility and Milk Production Segregates at High Frequency in Nordic Red Cattle: Additional Evidence for the Common Occurrence of Balancing Selection in Livestock

PubMed Central

Kadri, Naveen Kumar; Sahana, Goutam; Charlier, Carole; Iso-Touru, Terhi; Guldbrandtsen, Bernt; Karim, Latifa; Nielsen, Ulrik Sander; Panitz, Frank; Aamand, Gert Pedersen; Schulman, Nina; Georges, Michel; Vilkki, Johanna; Lund, Mogens Sandø; Druet, Tom

2014-01-01

In dairy cattle, the widespread use of artificial insemination has resulted in increased selection intensity, which has led to spectacular increase in productivity. However, cow fertility has concomitantly severely declined. It is generally assumed that this reduction is primarily due to the negative energy balance of high-producing cows at the peak of lactation. We herein describe the fine-mapping of a major fertility QTL in Nordic Red cattle, and identify a 660-kb deletion encompassing four genes as the causative variant. We show that the deletion is a recessive embryonically lethal mutation. This probably results from the loss of RNASEH2B, which is known to cause embryonic death in mice. Despite its dramatic effect on fertility, 13%, 23% and 32% of the animals carry the deletion in Danish, Swedish and Finnish Red Cattle, respectively. To explain this, we searched for favorable effects on other traits and found that the deletion has strong positive effects on milk yield. This study demonstrates that embryonic lethal mutations account for a non-negligible fraction of the decline in fertility of domestic cattle, and that associated positive effects on milk yield may account for part of the negative genetic correlation. Our study adds to the evidence that structural variants contribute to animal phenotypic variation, and that balancing selection might be more common in livestock species than previously appreciated. PMID:24391517

Analysis of Pax6 contiguous gene deletions in the mouse, Mus musculus, identifies regions distinct from Pax6 responsible for extreme small-eye and belly-spotting phenotypes.

PubMed

Favor, Jack; Bradley, Alan; Conte, Nathalie; Janik, Dirk; Pretsch, Walter; Reitmeir, Peter; Rosemann, Michael; Schmahl, Wolfgang; Wienberg, Johannes; Zaus, Irmgard

2009-08-01

In the mouse Pax6 function is critical in a dose-dependent manner for proper eye development. Pax6 contiguous gene deletions were shown to be homozygous lethal at an early embryonic stage. Heterozygotes express belly spotting and extreme microphthalmia. The eye phenotype is more severe than in heterozygous Pax6 intragenic null mutants, raising the possibility that deletions are functionally different from intragenic null mutations or that a region distinct from Pax6 included in the deletions affects eye phenotype. We recovered and identified the exact regions deleted in three new Pax6 deletions. All are homozygous lethal at an early embryonic stage. None express belly spotting. One expresses extreme microphthalmia and two express the milder eye phenotype similar to Pax6 intragenic null mutants. Analysis of Pax6 expression levels and the major isoforms excluded the hypothesis that the deletions expressing extreme microphthalmia are directly due to the action of Pax6 and functionally different from intragenic null mutations. A region distinct from Pax6 containing eight genes was identified for belly spotting. A second region containing one gene (Rcn1) was identified for the extreme microphthalmia phenotype. Rcn1 is a Ca(+2)-binding protein, resident in the endoplasmic reticulum, participates in the secretory pathway and expressed in the eye. Our results suggest that deletion of Rcn1 directly or indirectly contributes to the eye phenotype in Pax6 contiguous gene deletions.

Developmental and transcriptional consequences of mutations in Drosophila TAF(II)60.

PubMed

Aoyagi, N; Wassarman, D A

2001-10-01

In vitro, the TAF(II)60 component of the TFIID complex contributes to RNA polymerase II transcription initiation by serving as a coactivator that interacts with specific activator proteins and possibly as a promoter selectivity factor that interacts with the downstream promoter element. In vivo roles for TAF(II)60 in metazoan transcription are not as clear. Here we have investigated the developmental and transcriptional requirements for TAF(II)60 by analyzing four independent Drosophila melanogaster TAF(II)60 mutants. Loss-of-function mutations in Drosophila TAF(II)60 result in lethality, indicating that TAF(II)60 provides a nonredundant function in vivo. Molecular analysis of TAF(II)60 alleles revealed that essential TAF(II)60 functions are provided by two evolutionarily conserved regions located in the N-terminal half of the protein. TAF(II)60 is required at all stages of Drosophila development, in both germ cells and somatic cells. Expression of TAF(II)60 from a transgene rescued the lethality of TAF(II)60 mutants and exposed requirements for TAF(II)60 during imaginal development, spermatogenesis, and oogenesis. Phenotypes of rescued TAF(II)60 mutant flies implicate TAF(II)60 in transcriptional mechanisms that regulate cell growth and cell fate specification and suggest that TAF(II)60 is a limiting component of the machinery that regulates the transcription of dosage-sensitive genes. Finally, TAF(II)60 plays roles in developmental regulation of gene expression that are distinct from those of other TAF(II) proteins.

Genetic Studies of the Prp17 Gene of Saccharomyces Cerevisiae: A Domain Essential for Function Maps to a Nonconserved Region of the Protein

PubMed Central

Seshadri, V.; Vaidya, V. C.; Vijayraghavan, U.

1996-01-01

The PRP17 gene product is required for the second step of pre-mRNA splicing reactions. The C-terminal half of this protein bears four repeat units with homology to the β transducin repeat. Missense mutations in three temperature-sensitive prp17 mutants map to a region in the N-terminal half of the protein. We have generated, in vitro, 11 missense alleles at the β transducin repeat units and find that only one affects function in vivo. A phenotypically silent missense allele at the fourth repeat unit enhances the slow-growing phenotype conferred by an allele at the third repeat, suggesting an interaction between these domains. Although many missense mutations in highly conserved amino acids lack phenotypic effects, deletion analysis suggests an essential role for these units. Only mutations in the N-terminal nonconserved domain of PRP17 are synthetically lethal in combination with mutations in PRP16 and PRP18, two other gene products required for the second splicing reaction. A mutually allele-specific interaction between prp17 and snr7, with mutations in U5 snRNA, was observed. We therefore suggest that the functional region of Prp17p that interacts with Prp18p, Prp16p, and U5 snRNA is in the N terminal region of the protein. PMID:8722761

Lack of mutational hot spots during decitabine-mediated HIV-1 mutagenesis.

PubMed

Rawson, Jonathan M O; Landman, Sean R; Reilly, Cavan S; Bonnac, Laurent; Patterson, Steven E; Mansky, Louis M

2015-11-01

Decitabine has previously been shown to induce lethal mutagenesis of human immunodeficiency virus type 1 (HIV-1). However, the factors that determine the susceptibilities of individual sequence positions in HIV-1 to decitabine have not yet been defined. To investigate this, we performed Illumina high-throughput sequencing of multiple amplicons prepared from proviral DNA that was recovered from decitabine-treated cells infected with HIV-1. We found that decitabine induced an ≈4.1-fold increase in the total mutation frequency of HIV-1, primarily due to a striking ≈155-fold increase in the G-to-C transversion frequency. Intriguingly, decitabine also led to an ≈29-fold increase in the C-to-G transversion frequency. G-to-C frequencies varied substantially (up to ≈80-fold) depending upon sequence position, but surprisingly, mutational hot spots (defined as upper outliers within the mutation frequency distribution) were not observed. We further found that every single guanine position examined was significantly susceptible to the mutagenic effects of decitabine. Taken together, these observations demonstrate for the first time that decitabine-mediated HIV-1 mutagenesis is promiscuous and occurs in the absence of a clear bias for mutational hot spots. These data imply that decitabine-mediated G-to-C mutagenesis is a highly effective antiviral mechanism for extinguishing HIV-1 infectivity. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Mutagenesis of the lac promoter region in M13 mp10 phage DNA by 4'-hydroxymethyl-4,5',8-trimethylpsoralen

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

Piette, J.; Decuyper-Debergh, D.; Gamper, H.

Double-stranded M13 phage DNA (M13 mp10 replicative form) was photoreacted with 4'-hydroxymethyl-4,5',8-trimethylpsoralen, using light of wavelength greater than 320 nm or greater than 390 nm to generate predominantly crosslinks or monoadducts, respectively. The damaged DNAs were scored for inactivation and mutagenesis after transfection into Escherichia coli. The appearance of light-blue or colorless plaques on indicator medium showed that mutation had occurred in the lac insert of the viral DNA. A comparison of the consequences of the two phototreatments with psoralen supports the idea that crosslinks are both more lethal and more mutagenic than monoadducts. Numerous mutant clones partially or totallymore » deficient in beta-galactosidase were plaque-purified and amplified. The viral DNA of each clone was sequenced by the dideoxy chain-terminating procedure. All of the observed base-pair changes were mapped to the lac promoter region and consisted of 3 transition, 14 transversion, and 6 single base-pair frame-shift mutations. The predominant mutation was a T.A----G.C transversion.« less

Phenotypic expressions of a Gly154Arg mutation in type II collagen in two unrelated patients with spondyloepimetaphyseal dysplasia (SEMD)

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

Kaitila, I.; Marttinen, E.; Koerkkoe, J.

1996-05-03

Type II collagenopathies consist of chondrodysplasia ranging from lethal to mild in severity. A large number of mutations has been found in the COL2A1 gene. Glycine substitutions have been the most common types of mutation. Genotype-phenotype correlations in type II collagenopathies have not been established, partly because of insufficient clinical and radiographic description of the patients. We found a glycine-to-arginine substitution at position 154 in type II collagen in two unrelated isolated propositi with spondyloepimetaphyseal dysplasia and provide a comparative clinical and radiographic analysis from birth to young adulthood for this condition. The clinical phenotype was disproportionate short stature withmore » varus/valgus deformities of the lower limbs requiring corrective osteotomies, and lumbar lordosis. The skeletal radiographs showed an evolution from short tubular bones, delayed epiphyseal development, and mild vertebral involvement to severe metaphyseal dysplasia with dappling irregularities, and hip {open_quotes}dysplasia.{close_quotes} The metaphyseal abnormalities disappeared by adulthood. 27 refs., 11 figs., 1 tab.« less

Amino acid changes within the E protein hinge region that affect dengue virus type 2 infectivity and fusion

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

Butrapet, Siritorn; Childers, Thomas; Moss, Kelley J.

Fifteen mutant dengue viruses were engineered and used to identify AAs in the molecular hinge of the envelope protein that are critical to viral infection. Substitutions at Q52, A54, or E133 reduced infectivity in mammalian cells and altered the pH threshold of fusion. Mutations at F193, G266, I270, or G281 affected viral replication in mammalian and mosquito cells, but only I270W had reduced fusion activity. T280Y affected the pH threshold for fusion and reduced replication in C6/36 cells. Three different mutations at L135 were lethal in mammalian cells. Among them, L135G abrogated fusion and reduced replication in C6/36 cells, butmore » only slightly reduced the mosquito infection rate. Conversely, L135W replicated well in C6/36 cells, but had the lowest mosquito infection rate. Possible interactions between hinge residues 52 and 277, or among 53, 135, 170, 186, 265, and 276 required for hinge function were discovered by sequence analysis to identify compensatory mutations.« less

Two new fern chloroplasts and decelerated evolution linked to the long generation time in tree ferns.

PubMed

Zhong, Bojian; Fong, Richard; Collins, Lesley J; McLenachan, Patricia A; Penny, David

2014-04-30

We report the chloroplast genomes of a tree fern (Dicksonia squarrosa) and a "fern ally" (Tmesipteris elongata), and show that the phylogeny of early land plants is basically as expected, and the estimates of divergence time are largely unaffected after removing the fastest evolving sites. The tree fern shows the major reduction in the rate of evolution, and there has been a major slowdown in the rate of mutation in both families of tree ferns. We suggest that this is related to a generation time effect; if there is a long time period between generations, then this is probably incompatible with a high mutation rate because otherwise nearly every propagule would probably have several lethal mutations. This effect will be especially strong in organisms that have large numbers of cell divisions between generations. This shows the necessity of going beyond phylogeny and integrating its study with other properties of organisms. © The Author(s) 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Deciphering the mechanisms of developmental disorders: phenotype analysis of embryos from mutant mouse lines

PubMed Central

Wilson, Robert; McGuire, Christina; Mohun, Timothy

2016-01-01

The Deciphering the Mechanisms of Developmental Disorders (DMDD) consortium is a research programme set up to identify genes in the mouse, which if mutated (or knocked-out) result in embryonic lethality when homozygous, and initiate the study of why disruption of their function has such profound effects on embryo development and survival. The project uses a combination of comprehensive high resolution 3D imaging and tissue histology to identify abnormalities in embryo and placental structures of embryonic lethal lines. The image data we have collected and the phenotypes scored are freely available through the project website (http://dmdd.org.uk). In this article we describe the web interface to the images that allows the embryo data to be viewed at full resolution in different planes, discuss how to search the database for a phenotype, and our approach to organising the data for an embryo and a mutant line so it is easy to comprehend and intuitive to navigate. PMID:26519470

Impact of increased mutagenesis on adaptation to high temperature in bacteriophage Qβ.

PubMed

Arribas, María; Cabanillas, Laura; Kubota, Kirina; Lázaro, Ester

2016-10-01

RNA viruses replicate with very high error rates, which makes them more sensitive to additional increases in this parameter. This fact has inspired an antiviral strategy named lethal mutagenesis, which is based on the artificial increase of the error rate above a threshold incompatible with virus infectivity. A relevant issue concerning lethal mutagenesis is whether incomplete treatments might enhance the adaptive possibilities of viruses. We have addressed this question by subjecting an RNA virus, the bacteriophage Qβ, to different transmission regimes in the presence or the absence of sublethal concentrations of the mutagenic nucleoside analogue 5-azacytidine (AZC). Populations obtained were subsequently exposed to a non-optimal temperature and analyzed to determine their consensus sequences. Our results show that previously mutagenized populations rapidly fixed a specific set of mutations upon propagation at the new temperature, suggesting that the expansion of the mutant spectrum caused by AZC has an influence on later evolutionary behavior. Copyright © 2016 Elsevier Inc. All rights reserved.

A novel live attenuated anthrax spore vaccine based on an acapsular Bacillus anthracis Sterne strain with mutations in the htrA, lef and cya genes.

PubMed

Chitlaru, Theodor; Israeli, Ma'ayan; Rotem, Shahar; Elia, Uri; Bar-Haim, Erez; Ehrlich, Sharon; Cohen, Ofer; Shafferman, Avigdor

2017-10-20

We recently reported the development of a novel, next-generation, live attenuated anthrax spore vaccine based on disruption of the htrA (High Temperature Requirement A) gene in the Bacillus anthracis Sterne veterinary vaccine strain. This vaccine exhibited a highly significant decrease in virulence in murine, guinea pig and rabbit animal models yet preserved the protective value of the parental Sterne strain. Here, we report the evaluation of additional mutations in the lef and cya genes, encoding for the toxin components lethal factor (LF) and edema factor (EF), to further attenuate the SterneΔhtrA strain and improve its compatibility for human use. Accordingly, we constructed seven B. anthracis Sterne-derived strains exhibiting different combinations of mutations in the htrA, cya and lef genes. The various strains were indistinguishable in growth in vitro and in their ability to synthesise the protective antigen (PA, necessary for the elicitation of protection). In the sensitive murine model, we observed a gradual increase (ΔhtrA<ΔhtrAΔcya<ΔhtrAΔlef<ΔhtrAΔlefΔcya) in attenuation - up to 10 8 -fold relative to the parental Sterne vaccine strain. Most importantly, all various SterneΔhtrA derivative strains did not differ in their ability to elicit protective immunity in guinea pigs. Immunisation of guinea pigs with a single dose (10 9 spores) or double doses (>10 7 spores) of the most attenuated triple mutant strain SterneΔhtrAlef MUT Δcya induced a robust immune response, providing complete protection against a subsequent respiratory lethal challenge. Partial protection was observed in animals vaccinated with a double dose of as few as 10 5 spores. Furthermore, protective immune status was maintained in all vaccinated guinea pigs and rabbits for at least 40 and 30weeks, respectively. Copyright © 2017 Elsevier Ltd. All rights reserved.

Aberrant connexin26 hemichannels underlying keratitis-ichthyosis-deafness syndrome are potently inhibited by mefloquine

PubMed Central

Levit, Noah A.; Sellitto, Caterina; Wang, Hong-Zhan; Li, Leping; Srinivas, Miduturu; Brink, Peter R.; White, Thomas W.

2014-01-01

Keratitis-ichthyosis-deafness (KID) syndrome is an ectodermal dysplasia caused by dominant mutations of connexin26 (Cx26). Loss of Cx26 function causes non-syndromic sensorineural deafness, without consequence in the epidermis. Functional analyses have revealed that a majority of KID-causing mutations confer a novel expansion of hemichannel activity, mediated by connexin channels in a non-junctional configuration. Inappropriate Cx26 hemichannel opening is hypothesized to compromise keratinocyte integrity and epidermal homeostasis. Pharmacological modulators of Cx26 are needed to assess the pathomechanistic involvement of hemichannels in the development of hyperkeratosis in KID syndrome. We have used electrophysiological assays to evaluate small molecule analogs of quinine for suppressive effects on aberrant hemichannel currents elicited by KID mutations. Here, we show that mefloquine inhibits several mutant hemichannel forms implicated in KID syndrome when expressed in Xenopus laevis oocytes (IC50≈16µM), using an extracellular divalent cation, zinc (Zn++), as a non-specific positive control for comparison (IC50≈3µM). Furthermore, we used freshly isolated transgenic keratinocytes to show that micromolar concentrations of mefloquine attenuated increased macroscopic membrane currents in primary mouse keratinocytes expressing human Cx26-G45E, a mutation causing a lethal form of KID syndrome. PMID:25229253

Clinical utility of circulating tumor DNA for molecular assessment in pancreatic cancer.

PubMed

Takai, Erina; Totoki, Yasushi; Nakamura, Hiromi; Morizane, Chigusa; Nara, Satoshi; Hama, Natsuko; Suzuki, Masami; Furukawa, Eisaku; Kato, Mamoru; Hayashi, Hideyuki; Kohno, Takashi; Ueno, Hideki; Shimada, Kazuaki; Okusaka, Takuji; Nakagama, Hitoshi; Shibata, Tatsuhiro; Yachida, Shinichi

2015-12-16

Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal malignancies. The genomic landscape of the PDAC genome features four frequently mutated genes (KRAS, CDKN2A, TP53, and SMAD4) and dozens of candidate driver genes altered at low frequency, including potential clinical targets. Circulating cell-free DNA (cfDNA) is a promising resource to detect and monitor molecular characteristics of tumors. In the present study, we determined the mutational status of KRAS in plasma cfDNA using multiplex picoliter-droplet digital PCR in 259 patients with PDAC. We constructed a novel modified SureSelect-KAPA-Illumina platform and an original panel of 60 genes. We then performed targeted deep sequencing of cfDNA and matched germline DNA samples in 48 patients who had ≥1% mutant allele frequencies of KRAS in plasma cfDNA. Importantly, potentially targetable somatic mutations were identified in 14 of 48 patients (29.2%) examined by targeted deep sequencing of cfDNA. We also analyzed somatic copy number alterations based on the targeted sequencing data using our in-house algorithm, and potentially targetable amplifications were detected. Assessment of mutations and copy number alterations in plasma cfDNA may provide a prognostic and diagnostic tool to assist decisions regarding optimal therapeutic strategies for PDAC patients.

Mutations in FLVCR2 associated with Fowler syndrome and survival beyond infancy.

PubMed

Kvarnung, M; Taylan, F; Nilsson, D; Albåge, M; Nordenskjöld, M; Anderlid, B M; Nordgren, A; Syk Lundberg, E

2016-01-01

Proliferative vasculopathy and hydranencephaly-hydrocephaly syndrome (PVHH, OMIM 225790), also known as Fowler syndrome, is a rare autosomal recessive disorder, caused by mutations in FLVCR2. Hallmarks of the syndrome are glomerular vasculopathy in the central nervous system, severe hydrocephaly, hypokinesia and arthrogryphosis. The disorder is considered prenatally lethal. We report the first patients, a brother and a sister, with Fowler syndrome and survival beyond infancy. The patients present a phenotype of severe intellectual and neurologic disability with seizures, absence of functional movements, and no means of communication. Imaging of the brain showed calcifications, profound ventriculomegaly with only a thin edging of the cerebral cortex and hypoplastic cerebellum. Investigation with whole-exome sequencing (WES) revealed, in both patients, a homozygous pathogenic mutation in FLVCR2, c.1289C>T, compatible with a diagnosis of Fowler syndrome. The results highlight the power of combining WES with a thorough clinical examination in order to identify disease-causing mutations in patients whose clinical presentation differs from previously described cases. Specifically, the findings demonstrate that Fowler syndrome is a diagnosis to consider, not only prenatally but also in severely affected children with gross ventriculomegaly on brain imaging. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Early-progressive dilated cardiomyopathy in a family with Becker muscular dystrophy related to a novel frameshift mutation in the dystrophin gene exon 27.

PubMed

Tsuda, Takeshi; Fitzgerald, Kristi; Scavena, Mena; Gidding, Samuel; Cox, Mary O; Marks, Harold; Flanigan, Kevin M; Moore, Steven A

2015-03-01

We report a family in which two male siblings with Becker muscular dystrophy (BMD) developed severe dilated cardiomyopathy (DCM) and progressive heart failure (HF) at age 11 years; one died at age 14 years while awaiting heart transplant and the other underwent left ventricular assist device implantation at the same age. Genetic analysis of one sibling showed a novel frameshift mutation in exon 27 of Duchenne muscular dystrophy (DMD) gene (c.3779_3785delCTTTGGAinsGG), in which seven base pairs are deleted and two are inserted. Although this predicts an amino-acid substitution and premature termination (p.Thr1260Argfs*8), muscle biopsy dystrophin immunostaining instead indicates that the mutation is more likely to alter splicing. Despite relatively preserved skeletal muscular performance, both the siblings developed progressive HF secondary to early-onset DCM. In addition, their 7-year-old nephew with delayed gross motor development, mild proximal muscle weakness and markedly elevated serum creatine kinase level (>13 000 IU l(-1)) at 16 months was recently demonstrated to have the familial DMD mutation. Here, we report a novel genotype of BMD with early-onset DCM and progressive lethal HF during early adolescence.

Lack of chemically induced mutation in repair-deficient mutants of yeast.

PubMed

Prakash, L

1974-12-01

Two genes, rad6 and rad9, that confer radiation sensitivity in the yeast Saccharomyces cerevisiae also greatly reduce the frequency of chemically-induced reversions of a tester mutant cyc1-131, which is a chain initiation mutant in the structural gene determining iso-1-cytochrome c. Mutations induced by ethyl methanesulfonate (EMS), diethyl sulfate (DES), methyl methanesulfonate (MMS), dimethyl sulfate (DMS), nitroquinoline oxide (NQO), nitrosoguanidine (NTG), nitrogen mustard (HN2), beta-propiolactone, and tritiated uridine, as well as mutations induced by ultraviolet light (UV) and ionizing radiation were greatly diminished in strains homozygous for either the rad6 or rad9 gene. Nitrous acid and nitrosoimidazolidone (NIL), on the other hand, were highly mutagenic in these repair-deficient mutants, and at low doses, these mutagens acted with about the same efficiency as in the normal RAD strain. At high doses of either nitrous acid or NIL, however, reversion frequencies were significantly reduced in the two rad mutants compared to normal strains. Although both rad mutants are immutable to about the same extent, the rad9 strains tend to be less sensitive to the lethal effect of chemical mutagens than rad6 strains. It is concluded that yeast requires a functional repair system for mutation induction by chemical agents.

Genomewide Clonal Analysis of Lethal Mutations in the Drosophila melanogaster Eye: Comparison of the X Chromosome and Autosomes

PubMed Central

Call, Gerald B.; Olson, John M.; Chen, Jiong; Villarasa, Nikki; Ngo, Kathy T.; Yabroff, Allison M.; Cokus, Shawn; Pellegrini, Matteo; Bibikova, Elena; Bui, Chris; Cespedes, Albert; Chan, Cheryl; Chan, Stacy; Cheema, Amrita K.; Chhabra, Akanksha; Chitsazzadeh, Vida; Do, Minh-Tu; Fang, Q. Angela; Folick, Andrew; Goodstein, Gelsey L.; Huang, Cheng R.; Hung, Tony; Kim, Eunha; Kim, William; Kim, Yulee; Kohan, Emil; Kuoy, Edward; Kwak, Robert; Lee, Eric; Lee, JiEun; Lin, Henry; Liu, H-C. Angela; Moroz, Tatiana; Prasad, Tharani; Prashad, Sacha L.; Patananan, Alexander N.; Rangel, Alma; Rosselli, Desiree; Sidhu, Sohrab; Sitz, Daniel; Taber, Chelsea E.; Tan, Jingwen; Topp, Kasey; Tran, PhuongThao; Tran, Quynh-Minh; Unkovic, Mary; Wells, Maggie; Wickland, Jessica; Yackle, Kevin; Yavari, Amir; Zaretsky, Jesse M.; Allen, Christopher M.; Alli, Latifat; An, Ju; Anwar, Abbas; Arevalo, Sonia; Ayoub, Danny; Badal, Shawn S.; Baghdanian, Armonde; Baghdanian, Arthur H.; Baumann, Sara A.; Becerra, Vivian N.; Chan, Hei J.; Chang, Aileen E.; Cheng, Xibin A.; Chin, Mabel; Chong, Fleurette; Crisostomo, Carlyn; Datta, Sanjit; Delosreyes, Angela; Diep, Francie; Ekanayake, Preethika; Engeln, Mark; Evers, Elizabeth; Farshidi, Farzin; Fischer, Katrina; Formanes, Arlene J.; Gong, Jun; Gupta, Riju; Haas, Blake E.; Hahm, Vicky; Hsieh, Michael; Hui, James Z.; Iao, Mei L.; Jin, Sophia D.; Kim, Angela Y.; Kim, Lydia S-H.; King, Megan; Knudsen-Robbins, Chloe; Kohanchi, David; Kovshilovskaya, Bogdana; Ku, Amy; Kung, Raymond W.; Landig, Mark E. L.; Latterman, Stephanie S.; Lauw, Stephanie S.; Lee, Daniel S.; Lee, Joann S.; Lei, Kai C.; Leung, Lesley L.; Lerner, Renata; Lin, Jian-ya; Lin, Kathleen; Lim, Bryon C.; Lui, Crystal P. Y.; Liu, Tiffany Q.; Luong, Vincent; Makshanoff, Jacob; Mei, An-Chi; Meza, Miguel; Mikhaeil, Yara A.; Moarefi, Majid; Nguyen, Long H.; Pai, Shekhar S.; Pandya, Manish; Patel, Aadit R.; Picard, Paul D.; Safaee, Michael M.; Salame, Carol; Sanchez, Christian; Sanchez, Nina; Seifert, Christina C.; Shah, Abhishek; Shilgevorkyan, Oganes H.; Singh, Inderroop; Soma, Vanessa; Song, Junia J.; Srivastava, Neetika; Sta.Ana, Jennifer L.; Sun, Christie; Tan, Diane; Teruya, Alison S.; Tikia, Robyn; Tran, Trinh; Travis, Emily G.; Trinh, Jennifer D.; Vo, Diane; Walsh, Thomas; Wong, Regan S.; Wu, Katherine; Wu, Ya-Whey; Yang, Nkau X. V.; Yeranosian, Michael; Yu, James S.; Zhou, Jennifer J.; Zhu, Ran X.; Abrams, Anna; Abramson, Amanda; Amado, Latiffe; Anderson, Jenny; Bashour, Keenan; Beyer, Elsa; Bookatz, Allen; Brewer, Sarah; Buu, Natalie; Calvillo, Stephanie; Cao, Joseph; Chan, Amy; Chan, Jenny; Chang, Aileen; Chang, Daniel; Chang, Yuli; Chen, YiBing; Choi, Joo; Chou, Jeyling; Dang, Peter; Datta, Sumit; Davarifar, Ardy; Deravanesian, Artemis; Desai, Poonam; Fabrikant, Jordan; Farnad, Shahbaz; Fu, Katherine; Garcia, Eddie; Garrone, Nick; Gasparyan, Srpouhi; Gayda, Phyllis; Go, Sherrylene; Goffstein, Chad; Gonzalez, Courtney; Guirguis, Mariam; Hassid, Ryan; Hermogeno, Brenda; Hong, Julie; Hong, Aria; Hovestreydt, Lindsay; Hu, Charles; Huff, Devon; Jamshidian, Farid; Jen, James; Kahen, Katrin; Kao, Linda; Kelley, Melissa; Kho, Thomas; Kim, Yein; Kim, Sarah; Kirkpatrick, Brian; Langenbacher, Adam; Laxamana, Santino; Lee, Janet; Lee, Chris; Lee, So-Youn; Lee, ToHang S.; Lee, Toni; Lewis, Gemma; Lezcano, Sheila; Lin, Peter; Luu, Thanh; Luu, Julie; Marrs, Will; Marsh, Erin; Marshall, Jamie; Min, Sarah; Minasian, Tanya; Minye, Helena; Misra, Amit; Morimoto, Miles; Moshfegh, Yasaman; Murray, Jessica; Nguyen, Kha; Nguyen, Cynthia; Nodado, Ernesto; O'Donahue, Amanda; Onugha, Ndidi; Orjiakor, Nneka; Padhiar, Bhavin; Paul, Eric; Pavel-Dinu, Mara; Pavlenko, Alex; Paz, Edwin; Phaklides, Sarah; Pham, Lephong; Poulose, Preethi; Powell, Russell; Pusic, Aya; Ramola, Divi; Regalia, Kirsten; Ribbens, Meghann; Rifai, Bassel; Saakyan, Manyak; Saarikoski, Pamela; Segura, Miriam; Shadpour, Farnaz; Shemmassian, Aram; Singh, Ramnik; Singh, Vivek; Skinner, Emily; Solomin, Daniel; Soneji, Kosha; Spivey, Kristin; Stageberg, Erika; Stavchanskiy, Marina; Tekchandani, Leena; Thai, Leo; Thiyanaratnam, Jayantha; Tong, Maurine; Toor, Aneet; Tovar, Steve; Trangsrud, Kelly; Tsang, Wah-Yung; Uemura, Marc; Vollmer, Emily; Weiss, Emily; Wood, Damien; Wu, Joy; Wu, Sophia; Wu, Winston; Xu, Qing; Yamauchi, Yuki; Yarosh, Will; Yee, Laura; Yen, George; Banerjee, Utpal

2007-01-01

Using a large consortium of undergraduate students in an organized program at the University of California, Los Angeles (UCLA), we have undertaken a functional genomic screen in the Drosophila eye. In addition to the educational value of discovery-based learning, this article presents the first comprehensive genomewide analysis of essential genes involved in eye development. The data reveal the surprising result that the X chromosome has almost twice the frequency of essential genes involved in eye development as that found on the autosomes. PMID:17720911

Immunotherapy with mutated onchocystatin fails to enhance the efficacy of a sub-lethal oxytetracycline regimen against Onchocerca ochengi.

PubMed

Bah, Germanus S; Tanya, Vincent N; Makepeace, Benjamin L

2015-08-15

Human onchocerciasis (river blindness), caused by the filarial nematode Onchocerca volvulus, has been successfully controlled by a single drug, ivermectin, for over 25 years. Ivermectin prevents the disease symptoms of severe itching and visual impairment by killing the microfilarial stage, but does not eliminate the adult parasites, necessitating repeated annual treatments. Mass drug administration with ivermectin does not always break transmission in forest zones and is contraindicated in individuals heavily co-infected with Loa loa, while reports of reduced drug efficacy in Ghana and Cameroon may signal the development of resistance. An alternative treatment for onchocerciasis involves targeting the essential Wolbachia symbiont with tetracycline or its derivatives, which are adulticidal. However, implementation of antibiotic therapy has not occurred on a wide scale due to the prolonged treatment regimen required (several weeks). In the bovine Onchocerca ochengi system, it has been shown previously that prolonged oxytetracycline therapy increases eosinophil counts in intradermal nodules, which kill the adult worms by degranulating on their surface. Here, in an "immunochemotherapeutic" approach, we sought to enhance the efficacy of a short, sub-lethal antibiotic regimen against O. ochengi by prior immunotherapy targeting onchocystatin, an immunomodulatory protein located in the adult female worm cuticle. A key asparagine residue in onchocystatin was mutated to ablate immunomodulatory activity, which has been demonstrated previously to markedly improve the protective efficacy of this vaccine candidate when used as an immunoprophylactic. The immunochemotherapeutic regimen was compared with sub-lethal oxytetracycline therapy alone; onchocystatin immunotherapy alone; a gold-standard prolonged, intermittent oxytetracycline regimen; and no treatment (negative control) in naturally infected Cameroonian cattle. Readouts were collected over one year and comprised adult worm viability, dermal microfilarial density, anti-onchocystatin IgG in sera, and eosinophil counts in nodules. Only the gold-standard antibiotic regimen achieved significant killing of adult worms, a profound reduction in microfilarial load, and a sustained increase in local tissue eosinophilia. A small but statistically significant elevation in anti-onchocystatin IgG was observed for several weeks after immunisation in the immunotherapy-only group, but the antibody response in the immunochemotherapy group was more variable. At 12 weeks post-treatment, only a transient and non-significant increase in eosinophil counts was apparent in the immunochemotherapy group. We conclude that the addition of onchocystatin immunotherapy to a sub-lethal antibiotic regimen is insufficient to induce adulticidal activity, although with booster immunisations or the targeting of additional filarial immunomodulatory proteins, the efficacy of this strategy could be strengthened. Copyright © 2015 Elsevier B.V. All rights reserved.

Identification of ALK as the Major Familial Neuroblastoma Predisposition Gene

PubMed Central

Mossë, Yalë P; Laudenslager, Marci; Longo, Luca; Cole, Kristina A; Wood, Andrew; Attiyeh, Edward F; Laquaglia, Michael J; Sennett, Rachel; Lynch, Jill E; Perri, Patrizia; Laureys, Geneviève; Speleman, Frank; Hakonarson, Hakon; Torkamani, Ali; Schork, Nicholas J; Brodeur, Garrett M; Tonini, Gian Paolo; Rappaport, Eric; Devoto, Marcella; Maris, John M

2009-01-01

SUMMARY Survival rates for the childhood cancer neuroblastoma have not substantively improved despite dramatic escalation in chemotherapy intensity. Like most human cancers, this embryonal malignancy can be inherited, but the genetic etiology of familial and sporadically occurring neuroblastoma was largely unknown. Here we show that germline mutations in the anaplastic lymphoma kinase gene (ALK) explain the majority of hereditary neuroblastomas, and that activating mutations can also be somatically acquired. We first identified a significant linkage signal at the short arm of chromosome 2 (maximum nonparametric LOD=4.23 at rs1344063) using a whole-genome scan in neuroblastoma pedigrees. Resequencing of regional candidate genes identified three separate missense mutations in the tyrosine kinase domain of ALK (G1128A, R1192P and R1275Q) that segregated with the disease in eight separate families. Examination of 491 sporadically occurring human neuroblastoma samples showed that the ALK locus was gained in 22.8%, and highly amplified in an additional 3.3%, and that these aberrations were highly associated with death from disease (P=0.0003). Resequencing of 194 high-risk neuroblastoma samples showed somatically acquired mutations within the tyrosine kinase domain in 12.4%. Nine of the ten mutations map to critical regions of the kinase domain and were predicted to be oncogenic drivers with high probability. Mutations resulted in constitutive phosphorylation consistent with activation, and targeted knockdown of ALK mRNA resulted in profound growth inhibition of 4 of 4 cell lines harboring mutant or amplified ALK, as well as 2 of 6 wild type for ALK. Our results demonstrate that heritable mutations of ALK are the major cause of familial neuroblastoma, and that germline or acquired activation of this cell surface kinase is a tractable therapeutic target for this lethal pediatric malignancy. PMID:18724359

The heartstrings mutation in zebrafish causes heart/fin Tbx5 deficiency syndrome.

PubMed

Garrity, Deborah M; Childs, Sarah; Fishman, Mark C

2002-10-01

Holt-Oram syndrome is one of the autosomal dominant human "heart-hand" disorders, with a combination of upper limb malformations and cardiac defects. Holt-Oram syndrome is caused by mutations in the TBX5 gene, a member of a large family of T-box transcription factors that play important roles in cell-type specification and morphogenesis. In a screen for mutations affecting zebrafish cardiac function, we isolated the recessive lethal mutant heartstrings, which lacks pectoral fins and exhibits severe cardiac dysfunction, beginning with a slow heart rate and progressing to a stretched, non-functional heart. We mapped and cloned the heartstrings mutation and find it to encode the zebrafish ortholog of the TBX5 gene. The heartstrings mutation causes premature termination at amino acid 316. Homozygous mutant embryos never develop pectoral fin buds and do not express several markers of early fin differentiation. The total absence of any fin bud differentiation distinguishes heartstrings from most other mutations that affect zebrafish fin development, suggesting that Tbx5 functions very early in the pectoral fin induction pathway. Moderate reduction of Tbx5 by morpholino causes fin malformations, revealing an additional early requirement for Tbx5 in coordinating the axes of fin outgrowth. The heart of heartstrings mutant embryos appears to form and function normally through the early heart tube stage, manifesting only a slight bradycardia compared with wild-type siblings. However, the heart fails to loop and then progressively deteriorates, a process affecting the ventricle as well as the atrium. Relative to mammals, fish require lower levels of Tbx5 to produce malformed appendages and display whole-heart rather than atrial-predominant cardiac defects. However, the syndromic deficiencies of tbx5 mutation are remarkably well retained between fish and mammals.

Whole-Exome Sequencing Reveals Mutations in Genes Linked to Hemophagocytic Lymphohistiocytosis and Macrophage Activation Syndrome in Fatal Cases of H1N1 Influenza.

PubMed

Schulert, Grant S; Zhang, Mingce; Fall, Ndate; Husami, Ammar; Kissell, Diane; Hanosh, Andrew; Zhang, Kejian; Davis, Kristina; Jentzen, Jeffrey M; Napolitano, Lena; Siddiqui, Javed; Smith, Lauren B; Harms, Paul W; Grom, Alexei A; Cron, Randy Q

2016-04-01

Severe H1N1 influenza can be lethal in otherwise healthy individuals and can have features of reactive hemophagocytic lymphohistiocytosis (HLH). HLH is associated with mutations in lymphocyte cytolytic pathway genes, which have not been previously explored in H1N1 influenza. Sixteen cases of fatal influenza A(H1N1) infection, 81% with histopathologic hemophagocytosis, were identified and analyzed for clinical and laboratory features of HLH, using modified HLH-2004 and macrophage activation syndrome (MAS) criteria. Fourteen specimens were subject to whole-exome sequencing. Sequence alignment and variant filtering detected HLH gene mutations and potential disease-causing variants. Cytolytic function of the PRF1 p.A91V mutation was tested in lentiviral-transduced NK-92 natural killer (NK) cells. Despite several lacking variables, cases of influenza A(H1N1) infection met 44% and 81% of modified HLH-2004 and MAS criteria, respectively. Five subjects (36%) carried one of 3 heterozygous LYST mutations, 2 of whom also possessed the p.A91V PRF1 mutation, which was shown to decrease NK cell cytolytic function. Several patients also carried rare variants in other genes previously observed in MAS. This cohort of fatal influenza A(H1N1) infections confirms the presence of hemophagocytosis and HLH pathology. Moreover, the high percentage of HLH gene mutations suggests they are risk factors for mortality among individuals with influenza A(H1N1) infection. © The Author 2015. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

The Somatic Genomic Landscape of Glioblastoma

PubMed Central

Brennan, Cameron W.; Verhaak, Roel G.W.; McKenna, Aaron; Campos, Benito; Noushmehr, Houtan; Salama, Sofie R.; Zheng, Siyuan; Chakravarty, Debyani; Sanborn, J. Zachary; Berman, Samuel H.; Beroukhim, Rameen; Bernard, Brady; Wu, Chang-Jiun; Genovese, Giannicola; Shmulevich, Ilya; Barnholtz-Sloan, Jill; Zou, Lihua; Vegesna, Rahulsimham; Shukla, Sachet A.; Ciriello, Giovanni; Yung, WK; Zhang, Wei; Sougnez, Carrie; Mikkelsen, Tom; Aldape, Kenneth; Bigner, Darell D.; Van Meir, Erwin G.; Prados, Michael; Sloan, Andrew; Black, Keith L.; Eschbacher, Jennifer; Finocchiaro, Gaetano; Friedman, William; Andrews, David W.; Guha, Abhijit; Iacocca, Mary; O’Neill, Brian P.; Foltz, Greg; Myers, Jerome; Weisenberger, Daniel J.; Penny, Robert; Kucherlapati, Raju; Perou, Charles M.; Hayes, D. Neil; Gibbs, Richard; Marra, Marco; Mills, Gordon B.; Lander, Eric; Spellman, Paul; Wilson, Richard; Sander, Chris; Weinstein, John; Meyerson, Matthew; Gabriel, Stacey; Laird, Peter W.; Haussler, David; Getz, Gad; Chin, Lynda

2013-01-01

We describe the landscape of somatic genomic alterations based on multi-dimensional and comprehensive characterization of more than 500 glioblastoma tumors (GBMs). We identify several novel mutated genes as well as complex rearrangements of signature receptors including EGFR and PDGFRA. TERT promoter mutations are shown to correlate with elevated mRNA expression, supporting a role in telomerase reactivation. Correlative analyses confirm that the survival advantage of the proneural subtype is conferred by the G-CIMP phenotype, and MGMT DNA methylation may be a predictive biomarker for treatment response only in classical subtype GBM. Integrative analysis of genomic and proteomic profiles challenges the notion of therapeutic inhibition of a pathway as an alternative to inhibition of the target itself. These data will facilitate the discovery of therapeutic and diagnostic target candidates, the validation of research and clinical observations and the generation of unanticipated hypotheses that can advance our molecular understanding of this lethal cancer. PMID:24120142

EXOSC8 mutations alter mRNA metabolism and cause hypomyelination with spinal muscular atrophy and cerebellar hypoplasia

PubMed Central

Boczonadi, Veronika; Müller, Juliane S.; Pyle, Angela; Munkley, Jennifer; Dor, Talya; Quartararo, Jade; Ferrero, Ileana; Karcagi, Veronika; Giunta, Michele; Polvikoski, Tuomo; Birchall, Daniel; Princzinger, Agota; Cinnamon, Yuval; Lützkendorf, Susanne; Piko, Henriett; Reza, Mojgan; Florez, Laura; Santibanez-Koref, Mauro; Griffin, Helen; Schuelke, Markus; Elpeleg, Orly; Kalaydjieva, Luba; Lochmüller, Hanns; Elliott, David J.; Chinnery, Patrick F.; Edvardson, Shimon; Horvath, Rita

2014-01-01

The exosome is a multi-protein complex, required for the degradation of AU-rich element (ARE) containing messenger RNAs (mRNAs). EXOSC8 is an essential protein of the exosome core, as its depletion causes a severe growth defect in yeast. Here we show that homozygous missense mutations in EXOSC8 cause progressive and lethal neurological disease in 22 infants from three independent pedigrees. Affected individuals have cerebellar and corpus callosum hypoplasia, abnormal myelination of the central nervous system or spinal motor neuron disease. Experimental downregulation of EXOSC8 in human oligodendroglia cells and in zebrafish induce a specific increase in ARE mRNAs encoding myelin proteins, showing that the imbalanced supply of myelin proteins causes the disruption of myelin, and explaining the clinical presentation. These findings show the central role of the exosomal pathway in neurodegenerative disease. PMID:24989451

Molecular determinants of Ebola virus virulence in mice.

PubMed

Ebihara, Hideki; Takada, Ayato; Kobasa, Darwyn; Jones, Steven; Neumann, Gabriele; Theriault, Steven; Bray, Mike; Feldmann, Heinz; Kawaoka, Yoshihiro

2006-07-01

Zaire ebolavirus (ZEBOV) causes severe hemorrhagic fever in humans and nonhuman primates, with fatality rates in humans of up to 90%. The molecular basis for the extreme virulence of ZEBOV remains elusive. While adult mice resist ZEBOV infection, the Mayinga strain of the virus has been adapted to cause lethal infection in these animals. To understand the pathogenesis underlying the extreme virulence of Ebola virus (EBOV), here we identified the mutations responsible for the acquisition of the high virulence of the adapted Mayinga strain in mice, by using reverse genetics. We found that mutations in viral protein 24 and in the nucleoprotein were primarily responsible for the acquisition of high virulence. Moreover, the role of these proteins in virulence correlated with their ability to evade type I interferon-stimulated antiviral responses. These findings suggest a critical role for overcoming the interferon-induced antiviral state in the pathogenicity of EBOV and offer new insights into the pathogenesis of EBOV infection.

The maintenance of single-locus polymorphism. IV. Models with mutation from existing alleles.

PubMed

Spencer, H G; Marks, R W

1992-01-01

The ability of viability selection to maintain allelic polymorphism is investigated using a constructionist approach. In extensions to the models we have previously proposed, a population is bombarded with a series of mutations whose fitnesses in conjunction with other alleles are functions of the corresponding fitnesses with a particular allele, the parent allele, already in the population. Allele frequencies are iterated simultaneously, thus allowing alleles to be driven to extinction by selection. Such models allow very high levels of polymorphism to evolve: up to 38 alleles in one case. Alleles that are lethal as homozygotes can evolve to surprisingly high frequencies. The joint evolution of allele frequencies and viabilities highlights the necessity to consider more than the current morphology of a population. Comparisons are made with the neutral theory of evolution and it is suggested that failure to reject neutrality using the Ewens-Watterson test cannot be regarded as evidence for the neutral theory.

The somatic genomic landscape of glioblastoma.

PubMed

Brennan, Cameron W; Verhaak, Roel G W; McKenna, Aaron; Campos, Benito; Noushmehr, Houtan; Salama, Sofie R; Zheng, Siyuan; Chakravarty, Debyani; Sanborn, J Zachary; Berman, Samuel H; Beroukhim, Rameen; Bernard, Brady; Wu, Chang-Jiun; Genovese, Giannicola; Shmulevich, Ilya; Barnholtz-Sloan, Jill; Zou, Lihua; Vegesna, Rahulsimham; Shukla, Sachet A; Ciriello, Giovanni; Yung, W K; Zhang, Wei; Sougnez, Carrie; Mikkelsen, Tom; Aldape, Kenneth; Bigner, Darell D; Van Meir, Erwin G; Prados, Michael; Sloan, Andrew; Black, Keith L; Eschbacher, Jennifer; Finocchiaro, Gaetano; Friedman, William; Andrews, David W; Guha, Abhijit; Iacocca, Mary; O'Neill, Brian P; Foltz, Greg; Myers, Jerome; Weisenberger, Daniel J; Penny, Robert; Kucherlapati, Raju; Perou, Charles M; Hayes, D Neil; Gibbs, Richard; Marra, Marco; Mills, Gordon B; Lander, Eric; Spellman, Paul; Wilson, Richard; Sander, Chris; Weinstein, John; Meyerson, Matthew; Gabriel, Stacey; Laird, Peter W; Haussler, David; Getz, Gad; Chin, Lynda

2013-10-10

We describe the landscape of somatic genomic alterations based on multidimensional and comprehensive characterization of more than 500 glioblastoma tumors (GBMs). We identify several novel mutated genes as well as complex rearrangements of signature receptors, including EGFR and PDGFRA. TERT promoter mutations are shown to correlate with elevated mRNA expression, supporting a role in telomerase reactivation. Correlative analyses confirm that the survival advantage of the proneural subtype is conferred by the G-CIMP phenotype, and MGMT DNA methylation may be a predictive biomarker for treatment response only in classical subtype GBM. Integrative analysis of genomic and proteomic profiles challenges the notion of therapeutic inhibition of a pathway as an alternative to inhibition of the target itself. These data will facilitate the discovery of therapeutic and diagnostic target candidates, the validation of research and clinical observations and the generation of unanticipated hypotheses that can advance our molecular understanding of this lethal cancer. Copyright © 2013 Elsevier Inc. All rights reserved.

Kinase-dead ATM protein causes genomic instability and early embryonic lethality in mice.

PubMed

Yamamoto, Kenta; Wang, Yunyue; Jiang, Wenxia; Liu, Xiangyu; Dubois, Richard L; Lin, Chyuan-Sheng; Ludwig, Thomas; Bakkenist, Christopher J; Zha, Shan

2012-08-06

Ataxia telangiectasia (A-T) mutated (ATM) kinase orchestrates deoxyribonucleic acid (DNA) damage responses by phosphorylating numerous substrates implicated in DNA repair and cell cycle checkpoint activation. A-T patients and mouse models that express no ATM protein undergo normal embryonic development but exhibit pleiotropic DNA repair defects. In this paper, we report that mice carrying homozygous kinase-dead mutations in Atm (Atm(KD/KD)) died during early embryonic development. Atm(KD/-) cells exhibited proliferation defects and genomic instability, especially chromatid breaks, at levels higher than Atm(-/-) cells. Despite this increased genomic instability, Atm(KD/-) lymphocytes progressed through variable, diversity, and joining recombination and immunoglobulin class switch recombination, two events requiring nonhomologous end joining, at levels comparable to Atm(-/-) lymphocytes. Together, these results reveal an essential function of ATM during embryogenesis and an important function of catalytically inactive ATM protein in DNA repair.

Investigations for transmitted genetic effects of hycanthone in mice. [9H-Thioxanthen-9-one-, 1-((2-(diethylamino) ethyl) amino)-4-(hydroxymethyl)

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

Russell, W.L.; Generoso, W.M.

1976-01-01

Repeated as well as single doses of hycanthone were administered to two different strains of mice for tests on transmitted deficiencies and gene mutations. No dominant lethal effects were found in male mice following intraperitoneal injection of 50 times the therapeutic dose and no mutations were observed in more than 16,000 offspring from injected males. When females were injected intraperitoneally, there was a reduction in litter size and an increase in frequency of x-chromosome loss. However, when injections were given intramuscularly or subcutaneously, there was no effect on litter size and the effect on x-chromosome loss was greatly reduced. Resultsmore » of these studies suggest that there is no genetic basis for changing the WHO conclusion that there are no reasons sufficient to justify a recommendation that the use of hycanthone for the treatment of schistosomiasis should cease. (HLW)« less

Favipiravir elicits antiviral mutagenesis during virus replication in vivo.

PubMed

Arias, Armando; Thorne, Lucy; Goodfellow, Ian

2014-10-21

Lethal mutagenesis has emerged as a novel potential therapeutic approach to treat viral infections. Several studies have demonstrated that increases in the high mutation rates inherent to RNA viruses lead to viral extinction in cell culture, but evidence during infections in vivo is limited. In this study, we show that the broad-range antiviral nucleoside favipiravir reduces viral load in vivo by exerting antiviral mutagenesis in a mouse model for norovirus infection. Increased mutation frequencies were observed in samples from treated mice and were accompanied with lower or in some cases undetectable levels of infectious virus in faeces and tissues. Viral RNA isolated from treated animals showed reduced infectivity, a feature of populations approaching extinction during antiviral mutagenesis. These results suggest that favipiravir can induce norovirus mutagenesis in vivo, which in some cases leads to virus extinction, providing a proof-of-principle for the use of favipiravir derivatives or mutagenic nucleosides in the clinical treatment of noroviruses.

Lack of a peroxiredoxin suppresses the lethality of cells devoid of electron donors by channelling electrons to oxidized ribonucleotide reductase.

PubMed

Boronat, Susanna; Domènech, Alba; Carmona, Mercè; García-Santamarina, Sarela; Bañó, M Carmen; Ayté, José; Hidalgo, Elena

2017-06-01

The thioredoxin and glutaredoxin pathways are responsible of recycling several enzymes which undergo intramolecular disulfide bond formation as part of their catalytic cycles such as the peroxide scavengers peroxiredoxins or the enzyme ribonucleotide reductase (RNR). RNR, the rate-limiting enzyme of deoxyribonucleotide synthesis, is an essential enzyme relying on these electron flow cascades for recycling. RNR is tightly regulated in a cell cycle-dependent manner at different levels, but little is known about the participation of electron donors in such regulation. Here, we show that cytosolic thioredoxins Trx1 and Trx3 are the primary electron donors for RNR in fission yeast. Unexpectedly, trx1 transcript and Trx1 protein levels are up-regulated in a G1-to-S phase-dependent manner, indicating that the supply of electron donors is also cell cycle-regulated. Indeed, genetic depletion of thioredoxins triggers a DNA replication checkpoint ruled by Rad3 and Cds1, with the final goal of up-regulating transcription of S phase genes and constitutive RNR synthesis. Regarding the thioredoxin and glutaredoxin cascades, one combination of gene deletions is synthetic lethal in fission yeast: cells lacking both thioredoxin reductase and cytosolic dithiol glutaredoxin. We have isolated a suppressor of this lethal phenotype: a mutation at the Tpx1-coding gene, leading to a frame shift and a loss-of-function of Tpx1, the main client of electron donors. We propose that in a mutant strain compromised in reducing equivalents, the absence of an abundant and competitive substrate such as the peroxiredoxin Tpx1 has been selected as a lethality suppressor to favor RNR function at the expense of the non-essential peroxide scavenging function, to allow DNA synthesis and cell growth.

Redox crisis underlies conditional light–dark lethality in cyanobacterial mutants that lack the circadian regulator, RpaA

PubMed Central

Diamond, Spencer; Rubin, Benjamin E.; Shultzaberger, Ryan K.; Chen, You; Barber, Chase D.; Golden, Susan S.

2017-01-01

Cyanobacteria evolved a robust circadian clock, which has a profound influence on fitness and metabolism under daily light–dark (LD) cycles. In the model cyanobacterium Synechococcus elongatus PCC 7942, a functional clock is not required for diurnal growth, but mutants defective for the response regulator that mediates transcriptional rhythms in the wild-type, regulator of phycobilisome association A (RpaA), cannot be cultured under LD conditions. We found that rpaA-null mutants are inviable after several hours in the dark and compared the metabolomes of wild-type and rpaA-null strains to identify the source of lethality. Here, we show that the wild-type metabolome is very stable throughout the night, and this stability is lost in the absence of RpaA. Additionally, an rpaA mutant accumulates excessive reactive oxygen species (ROS) during the day and is unable to clear it during the night. The rpaA-null metabolome indicates that these cells are reductant-starved in the dark, likely because enzymes of the primary nighttime NADPH-producing pathway are direct targets of RpaA. Because NADPH is required for processes that detoxify ROS, conditional LD lethality likely results from inability of the mutant to activate reductant-requiring pathways that detoxify ROS when photosynthesis is not active. We identified second-site mutations and growth conditions that suppress LD lethality in the mutant background that support these conclusions. These results provide a mechanistic explanation as to why rpaA-null mutants die in the dark, further connect the clock to metabolism under diurnal growth, and indicate that RpaA likely has important unidentified functions during the day. PMID:28074036

Short-Rib Polydactyly and Jeune Syndromes Are Caused by Mutations in WDR60

PubMed Central

McInerney-Leo, Aideen M.; Schmidts, Miriam; Cortés, Claudio R.; Leo, Paul J.; Gener, Blanca; Courtney, Andrew D.; Gardiner, Brooke; Harris, Jessica A.; Lu, Yeping; Marshall, Mhairi; Scambler, Peter J.; Beales, Philip L.; Brown, Matthew A.; Zankl, Andreas; Mitchison, Hannah M.; Duncan, Emma L.; Wicking, Carol

2013-01-01

Short-rib polydactyly syndromes (SRPS I–V) are a group of lethal congenital disorders characterized by shortening of the ribs and long bones, polydactyly, and a range of extraskeletal phenotypes. A number of other disorders in this grouping, including Jeune and Ellis-van Creveld syndromes, have an overlapping but generally milder phenotype. Collectively, these short-rib dysplasias (with or without polydactyly) share a common underlying defect in primary cilium function and form a subset of the ciliopathy disease spectrum. By using whole-exome capture and massive parallel sequencing of DNA from an affected Australian individual with SRPS type III, we detected two novel heterozygous mutations in WDR60, a relatively uncharacterized gene. These mutations segregated appropriately in the unaffected parents and another affected family member, confirming compound heterozygosity, and both were predicted to have a damaging effect on the protein. Analysis of an additional 54 skeletal ciliopathy exomes identified compound heterozygous mutations in WDR60 in a Spanish individual with Jeune syndrome of relatively mild presentation. Of note, these two families share one novel WDR60 missense mutation, although haplotype analysis suggested no shared ancestry. We further show that WDR60 localizes at the base of the primary cilium in wild-type human chondrocytes, and analysis of fibroblasts from affected individuals revealed a defect in ciliogenesis and aberrant accumulation of the GLI2 transcription factor at the centrosome or basal body in the absence of an obvious axoneme. These findings show that WDR60 mutations can cause skeletal ciliopathies and suggest a role for WDR60 in ciliogenesis. PMID:23910462

Dync1h1 Mutation Causes Proprioceptive Sensory Neuron Loss and Impaired Retrograde Axonal Transport of Dorsal Root Ganglion Neurons.

PubMed

Zhao, Jing; Wang, Yi; Xu, Huan; Fu, Yuan; Qian, Ting; Bo, Deng; Lu, Yan-Xin; Xiong, Yi; Wan, Jun; Zhang, Xiang; Dong, Qiang; Chen, Xiang-Jun

2016-07-01

Sprawling (Swl) is a radiation-induced mutation which has been identified to have a nine base pair deletion in dynein heavy chain 1 (DYNC1H1: encoded by a single gene Dync1h1). This study is to investigate the phenotype and the underlying mechanism of the Dync1h1 mutant. To display the phenotype of Swl mutant mice, we examined the embryos of homozygous (Swl/Swl) and heterozygous (Swl/+) mice and their postnatal dorsal root ganglion (DRG) of surviving Swl/+ mice. The Swl/+ mice could survive for a normal life span, while Swl/Swl could only survive till embryonic (E) 8.5 days. Excessive apoptosis of Swl/+ DRG neurons was revealed during E11.5-E15.5 days, and the peak rate was at E13.5 days. In vitro study of mutated DRG neurons showed impaired retrograde transport of dynein-driven nerve growth factor (NGF). Mitochondria, another dynein-driven cargo, demonstrated much slower retrograde transport velocity in Swl/+ neurons than in wild-type (WT) neurons. Nevertheless, the Swl, Loa, and Cra mutations did not affect homodimerization of DYNC1H1. The Swl/Swl mutation of Dync1h1 gene led to embryonic mal-development and lethality, whereas the Swl/+ DRG neurons demonstrated deficient retrograde transport in dynein-driven cargos and excessive apoptosis during mid- to late-developmental stages. The underlying mechanism of the mutation may not be due to impaired homodimerization of DYNC1H1. © 2016 John Wiley & Sons Ltd.

PPIB mutations cause severe osteogenesis imperfecta.

PubMed

van Dijk, Fleur S; Nesbitt, Isabel M; Zwikstra, Eline H; Nikkels, Peter G J; Piersma, Sander R; Fratantoni, Silvina A; Jimenez, Connie R; Huizer, Margriet; Morsman, Alice C; Cobben, Jan M; van Roij, Mirjam H H; Elting, Mariet W; Verbeke, Jonathan I M L; Wijnaendts, Liliane C D; Shaw, Nick J; Högler, Wolfgang; McKeown, Carole; Sistermans, Erik A; Dalton, Ann; Meijers-Heijboer, Hanne; Pals, Gerard

2009-10-01

Deficiency of cartilage-associated protein (CRTAP) or prolyl 3-hydroxylase 1(P3H1) has been reported in autosomal-recessive lethal or severe osteogenesis imperfecta (OI). CRTAP, P3H1, and cyclophilin B (CyPB) form an intracellular collagen-modifying complex that 3-hydroxylates proline at position 986 (P986) in the alpha1 chains of collagen type I. This 3-prolyl hydroxylation is decreased in patients with CRTAP and P3H1 deficiency. It was suspected that mutations in the PPIB gene encoding CyPB would also cause OI with decreased collagen 3-prolyl hydroxylation. To our knowledge we present the first two families with recessive OI caused by PPIB gene mutations. The clinical phenotype is compatible with OI Sillence type II-B/III as seen with COL1A1/2, CRTAP, and LEPRE1 mutations. The percentage of 3-hydroxylated P986 residues in patients with PPIB mutations is decreased in comparison to normal, but it is higher than in patients with CRTAP and LEPRE1 mutations. This result and the fact that CyPB is demonstrable independent of CRTAP and P3H1, along with reported decreased 3-prolyl hydroxylation due to deficiency of CRTAP lacking the catalytic hydroxylation domain and the known function of CyPB as a cis-trans isomerase, suggest that recessive OI is caused by a dysfunctional P3H1/CRTAP/CyPB complex rather than by the lack of 3-prolyl hydroxylation of a single proline residue in the alpha1 chains of collagen type I.

A novel germline PIGA mutation in Ferro-Cerebro-Cutaneous syndrome: a neurodegenerative X-linked epileptic encephalopathy with systemic iron-overload.

PubMed

Swoboda, Kathryn J; Margraf, Rebecca L; Carey, John C; Zhou, Holly; Newcomb, Tara M; Coonrod, Emily; Durtschi, Jacob; Mallempati, Kalyan; Kumanovics, Attila; Katz, Ben E; Voelkerding, Karl V; Opitz, John M

2014-01-01

Three related males presented with a newly recognized x-linked syndrome associated with neurodegeneration, cutaneous abnormalities, and systemic iron overload. Linkage studies demonstrated that they shared a haplotype on Xp21.3-Xp22.2 and exome sequencing was used to identify candidate variants. Of the segregating variants, only a PIGA mutation segregated with disease in the family. The c.328_330delCCT PIGA variant predicts, p.Leu110del (or c.1030_1032delCTT, p.Leu344del depending on the reference sequence). The unaffected great-grandfather shared his X allele with the proband but he did not have the PIGA mutation, indicating that the mutation arose de novo in his daughter. A single family with a germline PIGA mutation has been reported; affected males had a phenotype characterized by multiple congenital anomalies and severe neurologic impairment resulting in infantile lethality. In contrast, affected boys in the family described here were born without anomalies and were neurologically normal prior to onset of seizures after 6 months of age, with two surviving to the second decade. PIGA encodes an enzyme in the GPI anchor biosynthesis pathway. An affected individual in the family studied here was deficient in GPI anchor proteins on granulocytes but not erythrocytes. In conclusion, the PIGA mutation in this family likely causes a reduction in GPI anchor protein cell surface expression in various cell types, resulting in the observed pleiotropic phenotype involving central nervous system, skin, and iron metabolism. © 2013 Wiley Periodicals, Inc.

A novel ICK mutation causes ciliary disruption and lethal endocrine-cerebro-osteodysplasia syndrome.

PubMed

Oud, Machteld M; Bonnard, Carine; Mans, Dorus A; Altunoglu, Umut; Tohari, Sumanty; Ng, Alvin Yu Jin; Eskin, Ascia; Lee, Hane; Rupar, C Anthony; de Wagenaar, Nathalie P; Wu, Ka Man; Lahiry, Piya; Pazour, Gregory J; Nelson, Stanley F; Hegele, Robert A; Roepman, Ronald; Kayserili, Hülya; Venkatesh, Byrappa; Siu, Victoria M; Reversade, Bruno; Arts, Heleen H

2016-01-01

Endocrine-cerebro-osteodysplasia (ECO) syndrome [MIM:612651] caused by a recessive mutation (p.R272Q) in Intestinal cell kinase (ICK) shows significant clinical overlap with ciliary disorders. Similarities are strongest between ECO syndrome, the Majewski and Mohr-Majewski short-rib thoracic dysplasia (SRTD) with polydactyly syndromes, and hydrolethalus syndrome. In this study, we present a novel homozygous ICK mutation in a fetus with ECO syndrome and compare the effect of this mutation with the previously reported ICK variant on ciliogenesis and cilium morphology. Through homozygosity mapping and whole-exome sequencing, we identified a second variant (c.358G > T; p.G120C) in ICK in a Turkish fetus presenting with ECO syndrome. In vitro studies of wild-type and mutant mRFP-ICK (p.G120C and p.R272Q) revealed that, in contrast to the wild-type protein that localizes along the ciliary axoneme and/or is present in the ciliary base, mutant proteins rather enrich in the ciliary tip. In addition, immunocytochemistry revealed a decreased number of cilia in ICK p.R272Q-affected cells. Through identification of a novel ICK mutation, we confirm that disruption of ICK causes ECO syndrome, which clinically overlaps with the spectrum of ciliopathies. Expression of ICK-mutated proteins result in an abnormal ciliary localization compared to wild-type protein. Primary fibroblasts derived from an individual with ECO syndrome display ciliogenesis defects. In aggregate, our findings are consistent with recent reports that show that ICK regulates ciliary biology in vitro and in mice, confirming that ECO syndrome is a severe ciliopathy.

Saccharomyces cerevisiae mutants with enhanced induced mutation and altered mitotic gene conversion.

PubMed

Ivanov, E L; Kovaltzova, S V; Korolev, V G

1989-08-01

We have developed a method to isolate yeast (Saccharomyces cerevisiae) mutants with enhanced induced mutagenesis based on nitrous acid-induced reversion of the ade2-42 allele. Six mutants have been isolated and designated him (high induced mutagenesis), and 4 of them were studied in more detail. The him mutants displayed enhanced reversion of the ade2-42 allele, either spontaneous or induced by nitrous acid, UV light, and the base analog 6-N-hydroxylaminopurine, but not by gamma-irradiation. It is worth noting that the him mutants turned out not to be sensitive to the lethal effects of the mutagens used. The enhancement in mutation induced by nitrous acid, UV light, and 6-N-hydroxylaminopurine has been confirmed in a forward-mutation assay (induction of mutations in the ADE1, ADE2 genes). The latter agent revealed the most apparent differences between the him mutants and the wild-type strain and was, therefore, chosen for the genetic analysis of mutants, him mutations analyzed behaved as a single Mendelian trait; complementation tests indicated 3 complementation groups (HIM1, HIM2, and HIM3), each containing 1 mutant allele. Uracil-DNA glycosylase activity was determined in crude cell extracts, and no significant differences between the wild-type and him strains were detected. Spontaneous mitotic gene conversion at the ADE2 locus is altered in him1 strains, either increased or decreased, depending on the particular heteroallelic combination. Genetic evidence strongly suggests him mutations to be involved in a process of mismatch correction of molecular heteroduplexes.

A synthetic lethal screen identifies ATR-inhibition as a novel therapeutic approach for POLD1-deficient cancers

PubMed Central

Hocke, Sandra; Guo, Yang; Job, Albert; Orth, Michael; Ziesch, Andreas; Lauber, Kirsten; De Toni, Enrico N; Gress, Thomas M.; Herbst, Andreas; Göke, Burkhard; Gallmeier, Eike

2016-01-01

The phosphoinositide 3-kinase-related kinase ATR represents a central checkpoint regulator and mediator of DNA-repair. Its inhibition selectively eliminates certain subsets of cancer cells in various tumor types, but the underlying genetic determinants remain enigmatic. Here, we applied a synthetic lethal screen directed against 288 DNA-repair genes using the well-defined ATR knock-in model of DLD1 colorectal cancer cells to identify potential DNA-repair defects mediating these effects. We identified a set of DNA-repair proteins, whose knockdown selectively killed ATR-deficient cancer cells. From this set, we further investigated the profound synthetic lethal interaction between ATR and POLD1. ATR-dependent POLD1 knockdown-induced cell killing was reproducible pharmacologically in POLD1-depleted DLD1 cells and a panel of other colorectal cancer cell lines by using chemical inhibitors of ATR or its major effector kinase CHK1. Mechanistically, POLD1 depletion in ATR-deficient cells caused caspase-dependent apoptosis without preceding cell cycle arrest and increased DNA-damage along with impaired DNA-repair. Our data could have clinical implications regarding tumor genotype-based cancer therapy, as inactivating POLD1 mutations have recently been identified in small subsets of colorectal and endometrial cancers. POLD1 deficiency might thus represent a predictive marker for treatment response towards ATR- or CHK1-inhibitors that are currently tested in clinical trials. PMID:26755646

Development and characterization of a guinea pig model for Marburg virus.

PubMed

Wong, Gary; Cao, Wen-Guang; He, Shi-Hua; Zhang, Zi-Rui; Zhu, Wen-Jun; Moffat, Estella; Ebihara, Hideki; Embury-Hyatt, Carissa; Qiu, Xiang-Guo

2018-01-18

The Angolan strain of Marburg virus (MARV/Ang) can cause lethal disease in humans with a case fatality rate of up to 90%, but infection of immunocompetent rodents do not result in any observable symptoms. Our previous work includes the development and characterization of a MARV/Ang variant that can cause lethal disease in mice (MARV/Ang-MA), with the aim of using this tool to screen for promising prophylactic and therapeutic candidates. An intermediate animal model is needed to confirm any findings from mice studies before testing in the gold-standard non-human primate (NHP) model. In this study, we serially passaged the clinical isolate of MARV/Ang in the livers and spleens of guinea pigs until a variant emerged that causes 100% lethality in guinea pigs (MARV/Ang-GA). Animals infected with MARV/Ang-GA showed signs of filovirus infection including lymphocytopenia, thrombocytopenia, and high viremia leading to spread to major organs, including the liver, spleen, lungs, and kidneys. The MARV/Ang-GA guinea pigs died between 7-9 days after infection, and the LD 50 was calculated to be 1.1×10 -1 TCID 50 (median tissue culture infective dose). Mutations in MARV/Ang-GA were identified and compared to sequences of known rodent-adapted MARV/Ang variants, which may benefit future studies characterizing important host adaptation sites in the MARV/Ang viral genome.

Dominant lethal mutations in Drosophila melanogaster natural populations flown on board ISS.

NASA Astrophysics Data System (ADS)

Larina, Olga; Bekker, Anna

The resistance to mutagenic impacts represents an important issue of manned space missions. However the reasons of its individual variability as well as the factors which could induce mutations in space flight are not fully understood. Drosophila studies accomplished by several research teams at real space flights, revealed pronounced increase of mutations in somatic and reproductive cells, nonetheless, quite an opposite spaceflight effects also occurred, i.e., mei-41 laboratory strain showed postflight mutation rates lower than that in ground control. In order to monitor the influence of space flight on the mutational process, 4 series of space experiment with D. melanogaster wild type populations were performed at International Space Station (ISS). The appliance “Drosophila-2” used for breeding of drosophila in spaceflight conditions, enabled to conduct synchronous studies with two samples of fly populations. First instar drosophila larvae were placed into the experimental appliance 12 hours before the start of transport spacecraft. The duration of experiments was 7.9 through 19.7 days. In 19.7-day experiment, two generations of the flies were raised during the space flight, and then delivered to the earth. The frequency of dominant lethal mutations (DLM) was evaluated as the percentage of embryonic death in the progeny of experimental drosophila samples. DLM tests in VV-09 and Chas-09 natural populations, performed after the exposure to 10.9-day flight, showed the increase of DLM rate in Chas-09 (0.077 in flight series vs. 0.43 in earth-based control) while post-flight DLM value in VV-09 did not diverge from on-earth sample (0.025 and 0.027 correspondingly). The same results for VV-09 were obtained after the 14.7-day and 7.9-day flights with the only exception: 7.9-day flight experiment employed DLM measurements in two VV-09 spaceflight samples, differing by the age of the flies, and the above DLM rates were detected in “younger” VV-09 sample only. DLM in the “elder” sample which returned to the earth at the late pupae stage (0.049) was 2 times higher than in both “young” flight and ground control series. To elucidate the factors underlying these discrepancies, DLM evaluation after the subsequent, 19.6-day flight experiment, was performed in three fractions of second in-flight VV-09 generation, each of them comprised imagoes with definite hatching date (postflight days 2, 3, and 5). The results revealed a gradual decrease of the proportion of embryonic death in the progeny of the second in-flight generation from 0.113 to 0.032 (which is close to baseline values). The ionizing radiation at low Earth orbits alone could not produce considerable impact on mutational frequency. By the return to the earth the flies of the first fractions had attained the pre-imaginal ontogenetic stages which display decreased tolerance to unfavourable environmental conditions, which could probably affect the mutation rate. The results obtained show that native D. melanogaster populations display different susceptibility to mutagenic impacts of space flight. Mutation rate depends on the stage of ontogenetic development and thus could present the source of discrepancies in the results of space experiments.

Thermodynamic stability, unfolding kinetics, and aggregation of the N-terminal actin-binding domains of utrophin and dystrophin.

PubMed

Singh, Surinder M; Molas, Justine F; Kongari, Narsimulu; Bandi, Swati; Armstrong, Geoffrey S; Winder, Steve J; Mallela, Krishna M G

2012-05-01

Muscular dystrophy (MD) is the most common genetic lethal disorder in children. Mutations in dystrophin trigger the most common form of MD, Duchenne, and its allelic variant Becker MD. Utrophin is the closest homologue and has been shown to compensate for the loss of dystrophin in human disease animal models. However, the structural and functional similarities and differences between utrophin and dystrophin are less understood. Both proteins interact with actin through their N-terminal actin-binding domain (N-ABD). In this study, we examined the thermodynamic stability and aggregation of utrophin N-ABD and compared with that of dystrophin. Our results show that utrophin N-ABD has spectroscopic properties similar to dystrophin N-ABD. However, utrophin N-ABD has decreased denaturant and thermal stability, unfolds faster, and is correspondingly more susceptible to proteolysis, which might account for its decreased in vivo half-life compared to dystrophin. In addition, utrophin N-ABD aggregates to a lesser extent compared with dystrophin N-ABD, contrary to the general behavior of proteins in which decreased stability enhances protein aggregation. Despite these differences in stability and aggregation, both proteins exhibit deleterious effects of mutations. When utrophin N-ABD mutations analogous in position to the dystrophin disease-causing mutations were generated, they behaved similarly to dystrophin mutants in terms of decreased stability and the formation of cross-β aggregates, indicating a possible role for utrophin mutations in disease mechanisms. Copyright © 2012 Wiley Periodicals, Inc.

Thermodynamic stability, unfolding kinetics, and aggregation of the N-terminal actin binding domains of utrophin and dystrophin†

PubMed Central

Singh, Surinder M.; Molas, Justine F.; Kongari, Narsimulu; Bandi, Swati; Armstrong, Geoffrey S.; Winder, Steve J.; Mallela, Krishna M.G.

2012-01-01

Muscular dystrophy (MD) is the most common genetic lethal disorder in children. Mutations in dystrophin trigger the most common form of MD, Duchenne and its allelic variant Becker MD. Utrophin is the closest homologue and has been shown to compensate for the loss of dystrophin in human disease animal models. However, the structural and functional similarities and differences between utrophin and dystrophin are less understood. Both proteins interact with actin through their N-terminal actin-binding domain (N-ABD). In this study, we examined the thermodynamic stability and aggregation of utrophin N-ABD and compared with that of dystrophin. Our results show that utrophin N-ABD has spectroscopic properties similar to dystrophin N-ABD. However, utrophin N-ABD has decreased denaturant and thermal stability, unfolds faster, and is correspondingly more susceptible to proteolysis, which might account for its decreased in-vivo half-life compared to dystrophin. In addition, utrophin N-ABD aggregates to a lesser extent compared with dystrophin N-ABD, contrary to the general behavior of proteins in which decreased stability enhances protein aggregation. Despite these differences in stability and aggregation, both proteins exhibit deleterious effects of mutations. When utrophin N-ABD mutations analogous in position to the dystrophin disease-causing mutations were generated, they behaved similarly to dystrophin mutants in terms of decreased stability and the formation of cross-β aggregates, indicating a possible role for utrophin mutations in disease mechanisms. PMID:22275054

FLT3-ITD cooperates with inv(16) to promote progression to acute myeloid leukemia

PubMed Central

Kim, Hyung-Gyoon; Kojima, Kyoko; Swindle, C. Scott; Cotta, Claudiu V.; Huo, Yongliang; Reddy, Vishnu

2008-01-01

The inversion of chromosome 16 in the inv(16)(p13q22) is one of the most frequent cytogenetic abnormalities observed in acute myeloid leukemia (AML). The inv(16) fuses the core binding factor (CBF) beta subunit with the coiled-coil rod domain of smooth muscle myosin heavy chain (SMMHC). Expression of CBFβ-SMMHC in mice does not promote AML in the absence of secondary mutations. Patient samples with the inv(16) also possess mutually exclusive activating mutations in either N-RAS, K-RAS, or the receptor tyrosine kinases, c-KIT and FLT3, in almost 70% of cases. To test whether an activating mutation of FLT3 (FLT3-ITD) would cooperate with CBFβ-SMMHC to promote AML, we coexpressed both mutations in hematopoietic progenitor cells used to reconstitute lethally irradiated mice. Analysis of transplanted animals showed strong selection for CBFβ-SMMHC/FLT3-ITD–expressing cells in bone marrow and peripheral blood. Compared with animals transplanted with only CBFβ-SMMHC–expressing cells, FLT3-ITD further restricted early myeloid differentiation and promoted peripheralization of primitive myeloblasts as early as 2.5 weeks after transplantation. FLT3-ITD also accelerated disease progression in all CBFβ-SMMHC/FLT3-ITD–reconstituted animals, which died of a highly aggressive and transplantable AML within 3 to 5 months. These results indicate that FLT3-activating mutations can cooperate with CBFβ-SMMHC in an animal model of inv(16)-associated AML. PMID:17967943

PSO4: a novel gene involved in error-prone repair in Saccharomyces cerevisiae.

PubMed

Henriques, J A; Vicente, E J; Leandro da Silva, K V; Schenberg, A C

1989-09-01

The haploid xs9 mutant, originally selected for on the basis of a slight sensitivity to the lethal effect of X-rays, was found to be extremely sensitive to inactivation by 8-methoxypsoralen (8MOP) photoaddition, especially when cells are treated in the G2 phase of the cell cycle. As the xs9 mutation showed no allelism with any of the 3 known pso mutations, it was now given the name of pso4-1. Regarding inactivation, the pso4-1 mutant is also sensitive to mono- (HN1) or bi-functional (HN2) nitrogen mustards, it is slightly sensitive to 254 nm UV radiation (UV), and shows nearly normal sensitivity to 3-carbethoxypsoralen (3-CPs) photoaddition or methyl methanesulfonate (MMS). Regarding mutagenesis, the pso4-1 mutation completely blocks reverse and forward mutations induced by either 8MOP or 3CPs photoaddition, or by gamma-rays. In the cases of UV, HN1, HN2 or MMS treatments, while reversion induction is still completely abolished, forward mutagenesis is only partially inhibited for UV, HN1, or MMS, and it is unaffected for HN2. Besides severely inhibiting induced mutagenesis, the pso4-1 mutation was found to be semi-dominant, to block sporulation, to abolish the diploid resistance effect, and to block induced mitotic recombination, which indicates that the PSO4 gene is involved in a recombinational pathway of error-prone repair, comparable to the E. coli SOS repair pathway.

A targeted deletion/insertion in the mouse Pcsk1 locus is associated with homozygous embryo preimplantation lethality, mutant allele preferential transmission and heterozygous female susceptibility to dietary fat.

PubMed

Mbikay, Majambu; Croissandeau, Gilles; Sirois, Francine; Anini, Younes; Mayne, Janice; Seidah, Nabil G; Chrétien, Michel

2007-06-15

Proprotein convertase 1 (PC1) is a neuroendocrine proteinase involved in the proteolytic activation of precursors to hormones and neuropeptides. To determine the physiological importance of PC1, we produced a mutant mouse from embryonic stem cells in which its locus (Pcsk1) had been inactivated by homologous recombination. The inactivating mutation consisted of a 32.7-kb internal deletion and a 1.8 kb insertion of the bacterial neomycin resistance gene (neo) under the mouse phosphoglycerate kinase 1 protein (PGKneo). Intercross of Pcsk1(+/-) mice produced no Pcsk1(-/-) offspring or blastocysts; in addition, more than 80% of the offspring were Pcsk1(+/-). These observations suggested that the mutation caused preimplantation lethality of homozygous embryos and preferential transmission of the mutant allele. Interestingly, RT-PCR analysis on RNA from endocrine tissues from Pcsk1(+/-) mice revealed the presence of aberrant transcripts specifying the N-terminal half of the PC1 propeptide fused to neo gene product. Mass spectrometric profiles of proopiomelanocortin-derived peptides in the anterior pituitary were similar between Pcsk1(+/-) and Pcsk1(+/+) mice, but significantly different between male and female mice of the same genotype. Relative to their wild-type counterparts, female mutant mice exhibited stunted growth under a low fat diet, and catch-up growth under a high-fat diet. The complex phenotype exhibited by this Pcsk1 mutant mouse model may be due to PC1 deficiency aggravated by expression of aberrant gene products from the mutant allele.

Bacterial Signaling Nucleotides Inhibit Yeast Cell Growth by Impacting Mitochondrial and Other Specifically Eukaryotic Functions

PubMed Central

Vergnano, Marta; Wan, Chris

2017-01-01

ABSTRACT We have engineered Saccharomyces cerevisiae to inducibly synthesize the prokaryotic signaling nucleotides cyclic di-GMP (cdiGMP), cdiAMP, and ppGpp in order to characterize the range of effects these nucleotides exert on eukaryotic cell function during bacterial pathogenesis. Synthetic genetic array (SGA) and transcriptome analyses indicated that, while these compounds elicit some common reactions in yeast, there are also complex and distinctive responses to each of the three nucleotides. All three are capable of inhibiting eukaryotic cell growth, with the guanine nucleotides exhibiting stronger effects than cdiAMP. Mutations compromising mitochondrial function and chromatin remodeling show negative epistatic interactions with all three nucleotides. In contrast, certain mutations that cause defects in chromatin modification and ribosomal protein function show positive epistasis, alleviating growth inhibition by at least two of the three nucleotides. Uniquely, cdiGMP is lethal both to cells growing by respiration on acetate and to obligately fermentative petite mutants. cdiGMP is also synthetically lethal with the ribonucleotide reductase (RNR) inhibitor hydroxyurea. Heterologous expression of the human ppGpp hydrolase Mesh1p prevented the accumulation of ppGpp in the engineered yeast and restored cell growth. Extensive in vivo interactions between bacterial signaling molecules and eukaryotic gene function occur, resulting in outcomes ranging from growth inhibition to death. cdiGMP functions through a mechanism that must be compensated by unhindered RNR activity or by functionally competent mitochondria. Mesh1p may be required for abrogating the damaging effects of ppGpp in human cells subjected to bacterial infection. PMID:28743817

Tautomerism provides a molecular explanation for the mutagenic properties of the anti-HIV nucleoside 5-aza-5,6-dihydro-2'-deoxycytidine.

PubMed

Li, Deyu; Fedeles, Bogdan I; Singh, Vipender; Peng, Chunte Sam; Silvestre, Katherine J; Simi, Allison K; Simpson, Jeffrey H; Tokmakoff, Andrei; Essigmann, John M

2014-08-12

Viral lethal mutagenesis is a strategy whereby the innate immune system or mutagenic pool nucleotides increase the error rate of viral replication above the error catastrophe limit. Lethal mutagenesis has been proposed as a mechanism for several antiviral compounds, including the drug candidate 5-aza-5,6-dihydro-2'-deoxycytidine (KP1212), which causes A-to-G and G-to-A mutations in the HIV genome, both in tissue culture and in HIV positive patients undergoing KP1212 monotherapy. This work explored the molecular mechanism(s) underlying the mutagenicity of KP1212, and specifically whether tautomerism, a previously proposed hypothesis, could explain the biological consequences of this nucleoside analog. Establishing tautomerism of nucleic acid bases under physiological conditions has been challenging because of the lack of sensitive methods. This study investigated tautomerism using an array of spectroscopic, theoretical, and chemical biology approaches. Variable temperature NMR and 2D infrared spectroscopic methods demonstrated that KP1212 existed as a broad ensemble of interconverting tautomers, among which enolic forms dominated. The mutagenic properties of KP1212 were determined empirically by in vitro and in vivo replication of a single-stranded vector containing a single KP1212. It was found that KP1212 paired with both A (10%) and G (90%), which is in accord with clinical observations. Moreover, this mutation frequency is sufficient for pushing a viral population over its error catastrophe limit, as observed before in cell culture studies. Finally, a model is proposed that correlates the mutagenicity of KP1212 with its tautomeric distribution in solution.

Neonatal carnitine palmitoyltransferase II deficiency associated with Dandy-Walker syndrome and sudden death.

PubMed

Yahyaoui, Raquel; Espinosa, María Gracia; Gómez, Celia; Dayaldasani, Anita; Rueda, Inmaculada; Roldán, Ana; Ugarte, Magdalena; Lastra, Gonzalo; Pérez, Vidal

2011-11-01

Neonatal onset of carnitine palmitoyltransferase II (CPT II) deficiency is an autosomal recessive, often lethal disorder of the mitochondrial beta-oxidation of long-chain fatty acids. It is a rare multiorgan disease which includes hypoketotic hypoglycemia, severe hepatomuscular symptoms, cardiac abnormalities, seizures and lethargy, as well as dysmorphic features. Until now, only 22 affected families have been described in the literature. An increasing number of mutations are being identified in the CPT2 gene, with a distinct genotype-phenotype correlation in most cases. Herein we report a new case of neonatal CPT II deficiency associated with Dandy-Walker syndrome and sudden death at 13 days of life. CPT II deficiency was suggested by acylcarnitine analysis of dried-blood on filter paper in the expanded newborn screening. Genetic analysis of the CPT2 gene identified the presence of a previously described mutation in homozygosity (c.534_558del25bpinsT). All lethal neonatal CPT II deficiency patients previously described presented severe symptoms during the first week of life, although this was not the case in our patient, who remained stable and without apparent vital risk during the first 11 days of life. The introduction of tandem mass spectrometry to newborn screening has substantially improved our ability to detect metabolic diseases in the newborn period. This case illustrates the value of expanded newborn screening in a neonate with an unusual clinical presentation, combining hydrocephalus and sudden death, that might not commonly lead to the suspicion of an inborn error of metabolism. Copyright © 2011 Elsevier Inc. All rights reserved.

ATP7A Gene Addition to the Choroid Plexus Results in Long-term Rescue of the Lethal Copper Transport Defect in a Menkes Disease Mouse Model

PubMed Central

Donsante, Anthony; Yi, Ling; Zerfas, Patricia M; Brinster, Lauren R; Sullivan, Patricia; Goldstein, David S; Prohaska, Joseph; Centeno, Jose A; Rushing, Elisabeth; Kaler, Stephen G

2011-01-01

Menkes disease is a lethal infantile neurodegenerative disorder of copper metabolism caused by mutations in a P-type ATPase, ATP7A. Currently available treatment (daily subcutaneous copper injections) is not entirely effective in the majority of affected individuals. The mottled-brindled (mo-br) mouse recapitulates the Menkes phenotype, including abnormal copper transport to the brain owing to mutation in the murine homolog, Atp7a, and dies by 14 days of age. We documented that mo-br mice on C57BL/6 background were not rescued by peripheral copper administration, and used this model to evaluate brain-directed therapies. Neonatal mo-br mice received lateral ventricle injections of either adeno-associated virus serotype 5 (AAV5) harboring a reduced-size human ATP7A (rsATP7A) complementary DNA (cDNA), copper chloride, or both. AAV5-rsATP7A showed selective transduction of choroid plexus epithelia and AAV5-rsATP7A plus copper combination treatment rescued mo-br mice; 86% survived to weaning (21 days), median survival increased to 43 days, 37% lived beyond 100 days, and 22% survived to the study end point (300 days). This synergistic treatment effect correlated with increased brain copper levels, enhanced activity of dopamine-β-hydroxylase, a copper-dependent enzyme, and correction of brain pathology. Our findings provide the first definitive evidence that gene therapy may have clinical utility in the treatment of Menkes disease. PMID:21878905

Parallel universes of Black Six biology.

PubMed

Kraev, Alexander

2014-07-19

Creation of lethal and synthetic lethal mutations in an experimental organism is a cornerstone of genetic dissection of gene function, and is related to the concept of an essential gene. Common inbred mouse strains carry background mutations, which can act as genetic modifiers, interfering with the assignment of gene essentiality. The inbred strain C57BL/6J, commonly known as "Black Six", stands out, as it carries a spontaneous homozygous deletion in the nicotinamide nucleotide transhydrogenase (Nnt) gene [GenBank: AH009385.2], resulting in impairment of steroidogenic mitochondria of the adrenal gland, and a multitude of indirect modifier effects, coming from alteration of glucocorticoid-regulated processes. Over time, the popular strain has been used, by means of gene targeting technology, to assign "essential" and "redundant" qualifiers to numerous genes, thus creating an internally consistent "parallel universe" of knowledge. It is unrealistic to suggest phasing-out of this strain, given the scope of shared resources built around it, however, continuing on the road of "strain-unawareness" will result in profound waste of effort, particularly where translational research is concerned. The review analyzes the historical roots of this phenomenon and proposes that building of "parallel universes" should be urgently made visible to a critical reader by obligatory use of unambiguous and persistent tags in publications and databases, such as hypertext links, pointing to a vendor's strain description web page, or to a digital object identifier (d.o.i.) of the original publication, so that any research done exclusively in C57BL/6J, could be easily identified. This article was reviewed by Dr. Neil Smalheiser and Dr. Miguel Andrade-Navarro.

Structure–function analysis and genetic interactions of the SmG, SmE, and SmF subunits of the yeast Sm protein ring

PubMed Central

Schwer, Beate; Kruchten, Joshua; Shuman, Stewart

2016-01-01

A seven-subunit Sm protein ring forms a core scaffold of the U1, U2, U4, and U5 snRNPs that direct pre-mRNA splicing. Using human snRNP structures to guide mutagenesis in Saccharomyces cerevisiae, we gained new insights into structure–function relationships of the SmG, SmE, and SmF subunits. An alanine scan of 19 conserved amino acids of these three proteins, comprising the Sm RNA binding sites or inter-subunit interfaces, revealed that, with the exception of Arg74 in SmF, none are essential for yeast growth. Yet, for SmG, SmE, and SmF, as for many components of the yeast spliceosome, the effects of perturbing protein–RNA and protein–protein interactions are masked by built-in functional redundancies of the splicing machine. For example, tests for genetic interactions with non-Sm splicing factors showed that many benign mutations of SmG, SmE, and SmF (and of SmB and SmD3) were synthetically lethal with null alleles of U2 snRNP subunits Lea1 and Msl1. Tests of pairwise combinations of SmG, SmE, SmF, SmB, and SmD3 alleles highlighted the inherent redundancies within the Sm ring, whereby simultaneous mutations of the RNA binding sites of any two of the Sm subunits are lethal. Our results suggest that six intact RNA binding sites in the Sm ring suffice for function but five sites may not. PMID:27417296

Murine lethal milk mutation causes maternal accumulation of zinc in intestine and kidney and reduced zinc transport to milk

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

Dohyeel Lee; Cousins, R.J.

1991-03-15

The lethal milk (Lm) mutation is autosomal recessive in C57BL/6J mice and causes Zn deficiency in pups nursed by Lm dams. The genetic defect may cause a shift in the tissue Zn distribution in Lm dams since their milk has a 34-45% lower Zn concentration than milk of normal (N) dams. To examine tissue Zn distribution and Zn transport to milk and pups, 1 {mu}Ci of {sup 65}Zn was administered ip to lactating N and Lm dams. They also received 800 {mu}g Zn/ml in their drinking water to preclude short term, terminal zinc deficiency in the neonates nursed by Lmmore » dams. {sup 65}Zn content of milk and tissues of dams and tissues of pups was measured. Transport of {sup 65}Zn to milk of Lm dams was about 50% compared to milk of N dams. The percentage of the {sup 65}Zn dose recovered in the intestine, liver, and kidney of N pups nursed by LM dams was markedly lower than those of N pups nursed by N dams. In contrast, the percentage of {sup 65}Zn in the intestine and kidney of Lm dams was about twice that of N dams. The elevated intestinal {sup 65}Zn was paralleled by and elevated metallothionein concentration, but the increased {sup 65}Zn in the kidney was not. The Lm gene defect might limit Zn transport to milk by shifting the Zn distribution in lactating dams to the intestine, kidney, and perhaps other tissues.« less

An anthrax toxin variant with an improved activity in tumor targeting

PubMed Central

Wein, Alexander N.; Peters, Diane E.; Valivullah, Zaheer; Hoover, Benjamin J.; Tatineni, Aparna; Ma, Qian; Fattah, Rasem; Bugge, Thomas H.; Leppla, Stephen H.; Liu, Shihui

2015-01-01

Anthrax lethal toxin (LT) is an A-B type toxin secreted by Bacillus anthracis, consisting of the cellular binding moiety, protective antigen (PA), and the catalytic moiety, lethal factor (LF). To target cells, PA binds to cell-surface receptors and is then proteolytically processed forming a LF-binding competent PA oligomer where each LF binding site is comprised of three subsites on two adjacent PA monomers. We previously generated PA-U2-R200A, a urokinase-activated PA variant with LF-binding subsite II residue Arg200 mutated to Ala, and PA-L1-I210A, a matrix metalloproteinase-activated PA variant with subsite III residue Ile210 mutated to Ala. PA-U2-R200A and PA-L1-I210A displayed reduced cytotoxicity when used singly. However, when combined, they formed LF-binding competent heterogeneous oligomers by intermolecular complementation, and achieved high specificity in tumor targeting. Nevertheless, each of these proteins, in particular PA-L1-I210A, retained residual LF-binding ability. In this work, we screened a library containing all possible amino acid substitutions for LF-binding site to find variants with activity strictly dependent upon intermolecular complementation. PA-I207R was identified as an excellent replacement for the original clockwise-side variant, PA-I210A. Consequently, the new combination of PA-L1-I207R and PA-U2-R200A showed potent anti-tumor activity and low toxicity, exceeding the performance of the original combination, and warranting further investigation. PMID:26584669

Loss of Anticodon Wobble Uridine Modifications Affects tRNALys Function and Protein Levels in Saccharomyces cerevisiae

PubMed Central

Klassen, Roland; Grunewald, Pia; Thüring, Kathrin L.; Eichler, Christian; Helm, Mark; Schaffrath, Raffael

2015-01-01

In eukaryotes, wobble uridines in the anticodons of tRNALys UUU, tRNAGlu UUC and tRNAGln UUG are modified to 5-methoxy-carbonyl-methyl-2-thio-uridine (mcm5s2U). While mutations in subunits of the Elongator complex (Elp1-Elp6), which disable mcm5 side chain formation, or removal of components of the thiolation pathway (Ncs2/Ncs6, Urm1, Uba4) are individually tolerated, the combination of both modification defects has been reported to have lethal effects on Saccharomyces cerevisiae. Contrary to such absolute requirement of mcm5s2U for viability, we demonstrate here that in the S. cerevisiae S288C-derived background, both pathways can be simultaneously inactivated, resulting in combined loss of tRNA anticodon modifications (mcm5U and s2U) without a lethal effect. However, an elp3 disruption strain displays synthetic sick interaction and synergistic temperature sensitivity when combined with either uba4 or urm1 mutations, suggesting major translational defects in the absence of mcm5s2U modifications. Consistent with this notion, we find cellular protein levels drastically decreased in an elp3uba4 double mutant and show that this effect as well as growth phenotypes can be partially rescued by excess of tRNALys UUU. These results may indicate a global translational or protein homeostasis defect in cells simultaneously lacking mcm5 and s2 wobble uridine modification that could account for growth impairment and mainly originates from tRNALys UUU hypomodification and malfunction. PMID:25747122

Loss of anticodon wobble uridine modifications affects tRNA(Lys) function and protein levels in Saccharomyces cerevisiae.

PubMed

Klassen, Roland; Grunewald, Pia; Thüring, Kathrin L; Eichler, Christian; Helm, Mark; Schaffrath, Raffael

2015-01-01

In eukaryotes, wobble uridines in the anticodons of tRNA(Lys)UUU, tRNA(Glu)UUC and tRNA(Gln)UUG are modified to 5-methoxy-carbonyl-methyl-2-thio-uridine (mcm5s2U). While mutations in subunits of the Elongator complex (Elp1-Elp6), which disable mcm5 side chain formation, or removal of components of the thiolation pathway (Ncs2/Ncs6, Urm1, Uba4) are individually tolerated, the combination of both modification defects has been reported to have lethal effects on Saccharomyces cerevisiae. Contrary to such absolute requirement of mcm5s2U for viability, we demonstrate here that in the S. cerevisiae S288C-derived background, both pathways can be simultaneously inactivated, resulting in combined loss of tRNA anticodon modifications (mcm5U and s2U) without a lethal effect. However, an elp3 disruption strain displays synthetic sick interaction and synergistic temperature sensitivity when combined with either uba4 or urm1 mutations, suggesting major translational defects in the absence of mcm5s2U modifications. Consistent with this notion, we find cellular protein levels drastically decreased in an elp3uba4 double mutant and show that this effect as well as growth phenotypes can be partially rescued by excess of tRNA(Lys)UUU. These results may indicate a global translational or protein homeostasis defect in cells simultaneously lacking mcm5 and s2 wobble uridine modification that could account for growth impairment and mainly originates from tRNA(Lys)UUU hypomodification and malfunction.

Loss of imprinting at the Dlk1-Gtl2 locus caused by insertional mutagenesis in the Gtl2 5' region

PubMed Central

Steshina, Ekaterina Y; Carr, Michael S; Glick, Elena A; Yevtodiyenko, Aleksey; Appelbe, Oliver K; Schmidt, Jennifer V

2006-01-01

Background The Dlk1 and Gtl2 genes define a region of mouse chromosome 12 that is subject to genomic imprinting, the parental allele-specific expression of a gene. Although imprinted genes play important roles in growth and development, the mechanisms by which imprinting is established and maintained are poorly understood. Differentially methylated regions (DMRs), which carry methylation on only one parental allele, are involved in imprinting control at many loci. The Dlk1-Gtl2 region contains three known DMRs, the Dlk1 DMR in the 3' region of Dlk1, the intergenic DMR 15 kb upstream of Gtl2, and the Gtl2 DMR at the Gtl2 promoter. Three mouse models are analyzed here that provide new information about the regulation of Dlk1-Gtl2 imprinting. Results A previously existing insertional mutation (Gtl2lacZ), and a targeted deletion in which the Gtl2 upstream region was replaced by a Neo cassette (Gtl2Δ5'Neo), display partial lethality and dwarfism upon paternal inheritance. Molecular characterization shows that both mutations cause loss of imprinting and changes in expression of the Dlk1, Gtl2 and Meg8/Rian genes. Dlk1 levels are decreased upon paternal inheritance of either mutation, suggesting Dlk1 may be causative for the lethality and dwarfism. Loss of imprinting on the paternal chromosome in both Gtl2lacZ and Gtl2Δ5'Neo mice is accompanied by the loss of paternal-specific Gtl2 DMR methylation, while maternal loss of imprinting suggests a previously unknown regulatory role for the maternal Gtl2 DMR. Unexpectedly, when the Neo gene is excised, Gtl2Δ5' animals are of normal size, imprinting is unchanged and the Gtl2 DMR is properly methylated. The exogenous DNA sequences integrated upstream of Gtl2 are therefore responsible for the growth and imprinting effects. Conclusion These data provide further evidence for the coregulation of the imprinted Dlk1 and Gtl2 genes, and support a role for Dlk1 as an important neonatal growth factor. The ability of the Gtl2lacZ and Gtl2Δ5'Neo mutations to cause long-range changes in imprinting and gene expression suggest that regional imprinting regulatory elements may lie in proximity to the integration site. PMID:17014736

Mutation-Linked Defective Inter-Domain Interactions within Ryanodine Receptor Cause Aberrant Ca2+ Release Leading to Catecholaminergic Polymorphic Ventricular Tachycardia

PubMed Central

Suetomi, Takeshi; Yano, Masafumi; Uchinoumi, Hitoshi; Fukuda, Masakazu; Hino, Akihiro; Ono, Makoto; Xu, Xiaojuan; Tateishi, Hiroki; Okuda, Shinichi; Doi, Masahiro; Kobayashi, Shigeki; Ikeda, Yasuhiho; Yamamoto, Takeshi; Ikemoto, Noriaki; Matsuzaki, Masunori

2011-01-01

Background The molecular mechanism by which catecholaminergic polymorphic ventricular tachycardia (CPVT) is induced by single amino acid mutations within the cardiac ryanodine receptor (RyR2) remains elusive. Here, we investigated mutation-induced conformational defects of RyR2 using a knock-in (KI) mouse model expressing the human CPVT-associated RyR2 mutant (S2246L; Serine to Leucine mutation at the residue 2246). Methods and Results All KI mice we examined produced VT after exercise on a treadmill. cAMP-dependent increase in the frequency of Ca2+ sparks was more pronounced in saponin-permeabilized KI cardiomyocytes than in WT cardiomyocytes. Site-directed fluorescent labeling and quartz microbalance assays of the specific binding of DP2246 (a peptide corresponding to the 2232–2266 region: the 2246 domain) showed that DP2246 binds with the K201-binding sequence of RyR2 (1741– 2270). Introduction of S2246L mutation into the DP2246 increased the affinity of peptide binding. Fluorescence quench assays of inter-domain interactions within RyR2 showed that tight interaction of the 2246 domain/K201-binding domain is coupled with domain unzipping of the N-terminal (1-600)/central (2000–2500) domain pair in an allosteric manner. Dantrolene corrected the mutation-caused domain unzipping of the domain switch, and stopped the exercise-induced ventricular tachycardia. Conclusions The CPVT-linked mutation of RyR2, S2246L, causes an abnormally tight local sub-domain/sub-domain interaction within the central domain involving the mutation site, which induces defective interaction between the N-terminal and central domains. This results in an erroneous activation of Ca2+ channel in a diastolic state reflecting on the increased Ca2+ spark frequency, which then leads to lethal arrhythmia. PMID:21768539

Novel insertion mutation in a non-Jewish Caucasian type 1 Gaucher disease patient

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

Choy, F.Y.M.; Humphries, M.L.; Ferreira, P.

1997-01-20

Gaucher disease is the most prevalent lysosomal storage disorder. It is autosomal recessive, resulting in lysosomal glucocerebrosidase deficiency. Three clinical forms of Gaucher disease have been described: type 1 (nonneuronopathic), type 2 (acute neuronopathic), and type 3 (subacute neuronopathic). We performed PCR-thermal cycle sequence analysis of glucocerebrosidase genomic DNA and identified a novel mutation in a non-Jewish type 1 Gaucher disease patient. It is a C insertion in exon 3 at cDNA nucleotide position 122 and genomic nucleotide position 1626. This mutation causes a frameshift and, subsequently, four of the five codons immediately downstream of the insertion were changed whilemore » the sixth was converted to a stop codon, resulting in premature termination of protein translation. The 122CC insertion abolishes a Cac81 restriction endonuclease cleavage site, allowing a convenient and reliable method for detection using RFLP analysis of PCR-amplified glucocerebrosidase genomic DNA. The mutation in the other Gaucher allele was found to be an A{r_arrow}G substitution at glucocerebrosidase cDNA nucleotide position 1226 that so far has only been reported among type 1 Gaucher disease patients. Since mutation 122CC causes a frameshift and early termination of protein translation, it most likely results in a meaningless transcript and subsequently no residual glucocerebrosidase enzyme activity. We speculate that mutation 122CC may result in a worse prognosis than mutations associated with partial activity. When present in the homozygous form, it could be a lethal allele similar to what has been postulated for the other known insertion mutation, 84GG. Our patient, who is a compound heterozygote 122CC/1226G, has moderately severe type 1 Gaucher disease. Her clinical response to Ceredase{reg_sign} therapy that began 31 months ago has been favorable, though incomplete. 30 refs., 3 figs., 2 tabs.« less

Rare Autosomal Recessive Cardiac Valvular Form of Ehlers-Danlos Syndrome Results from Mutations in the COL1A2 Gene That Activate the Nonsense-Mediated RNA Decay Pathway

PubMed Central

Schwarze, Ulrike; Hata, Ryu-Ichiro; McKusick, Victor A.; Shinkai, Hiroshi; Hoyme, H. Eugene; Pyeritz, Reed E.; Byers, Peter H.

2004-01-01

Splice site mutations in the COL1A2 gene of type I collagen can give rise to forms of Ehlers-Danlos syndrome (EDS) because of partial or complete skipping of exon 6, as well as to mild, moderate, or lethal forms of osteogenesis imperfecta as a consequence of skipping of other exons. We identified three unrelated individuals with a rare recessively inherited form of EDS (characterized by joint hypermobility, skin hyperextensibility, and cardiac valvular defects); in two of them, COL1A2 messenger RNA (mRNA) instability results from compound heterozygosity for splice site mutations in the COL1A2 gene, and, in the third, it results from homozygosity for a nonsense codon. The splice site mutations led to use of cryptic splice donor sites, creation of a downstream premature termination codon, and extremely unstable mRNA. In the wild-type allele, the two introns (IVS11 and IVS24) in which these mutations occurred were usually spliced slowly in relation to their respective immediate upstream introns. In the mutant alleles, the upstream intron was removed, so that exon skipping could not occur. In the context of the mutation in IVS24, computer-generated folding of a short stretch of mRNA surrounding the mutation site demonstrated realignment of the relationships between the donor and acceptor sites that could facilitate use of a cryptic donor site. These findings suggest that the order of intron removal is an important variable in prediction of mutation outcome at splice sites and that folding of the nascent mRNA could be one element that contributes to determination of order of splicing. The complete absence of proα2(I) chains has the surprising effect of producing cardiac valvular disease without bone involvement. PMID:15077201

Rare autosomal recessive cardiac valvular form of Ehlers-Danlos syndrome results from mutations in the COL1A2 gene that activate the nonsense-mediated RNA decay pathway.

PubMed

Schwarze, Ulrike; Hata, Ryu-Ichiro; McKusick, Victor A; Shinkai, Hiroshi; Hoyme, H Eugene; Pyeritz, Reed E; Byers, Peter H

2004-05-01

Splice site mutations in the COL1A2 gene of type I collagen can give rise to forms of Ehlers-Danlos syndrome (EDS) because of partial or complete skipping of exon 6, as well as to mild, moderate, or lethal forms of osteogenesis imperfecta as a consequence of skipping of other exons. We identified three unrelated individuals with a rare recessively inherited form of EDS (characterized by joint hypermobility, skin hyperextensibility, and cardiac valvular defects); in two of them, COL1A2 messenger RNA (mRNA) instability results from compound heterozygosity for splice site mutations in the COL1A2 gene, and, in the third, it results from homozygosity for a nonsense codon. The splice site mutations led to use of cryptic splice donor sites, creation of a downstream premature termination codon, and extremely unstable mRNA. In the wild-type allele, the two introns (IVS11 and IVS24) in which these mutations occurred were usually spliced slowly in relation to their respective immediate upstream introns. In the mutant alleles, the upstream intron was removed, so that exon skipping could not occur. In the context of the mutation in IVS24, computer-generated folding of a short stretch of mRNA surrounding the mutation site demonstrated realignment of the relationships between the donor and acceptor sites that could facilitate use of a cryptic donor site. These findings suggest that the order of intron removal is an important variable in prediction of mutation outcome at splice sites and that folding of the nascent mRNA could be one element that contributes to determination of order of splicing. The complete absence of pro alpha 2(I) chains has the surprising effect of producing cardiac valvular disease without bone involvement.

Mutation-linked defective interdomain interactions within ryanodine receptor cause aberrant Ca²⁺release leading to catecholaminergic polymorphic ventricular tachycardia.

PubMed

Suetomi, Takeshi; Yano, Masafumi; Uchinoumi, Hitoshi; Fukuda, Masakazu; Hino, Akihiro; Ono, Makoto; Xu, Xiaojuan; Tateishi, Hiroki; Okuda, Shinichi; Doi, Masahiro; Kobayashi, Shigeki; Ikeda, Yasuhiro; Yamamoto, Takeshi; Ikemoto, Noriaki; Matsuzaki, Masunori

2011-08-09

The molecular mechanism by which catecholaminergic polymorphic ventricular tachycardia is induced by single amino acid mutations within the cardiac ryanodine receptor (RyR2) remains elusive. In the present study, we investigated mutation-induced conformational defects of RyR2 using a knockin mouse model expressing the human catecholaminergic polymorphic ventricular tachycardia-associated RyR2 mutant (S2246L; serine to leucine mutation at the residue 2246). All knockin mice we examined produced ventricular tachycardia after exercise on a treadmill. cAMP-dependent increase in the frequency of Ca²⁺ sparks was more pronounced in saponin-permeabilized knockin cardiomyocytes than in wild-type cardiomyocytes. Site-directed fluorescent labeling and quartz microbalance assays of the specific binding of DP2246 (a peptide corresponding to the 2232 to 2266 region: the 2246 domain) showed that DP2246 binds with the K201-binding sequence of RyR2 (1741 to 2270). Introduction of S2246L mutation into the DP2246 increased the affinity of peptide binding. Fluorescence quench assays of interdomain interactions within RyR2 showed that tight interaction of the 2246 domain/K201-binding domain is coupled with domain unzipping of the N-terminal (1 to 600)/central (2000 to 2500) domain pair in an allosteric manner. Dantrolene corrected the mutation-caused domain unzipping of the domain switch and stopped the exercise-induced ventricular tachycardia. The catecholaminergic polymorphic ventricular tachycardia-linked mutation of RyR2, S2246L, causes an abnormally tight local subdomain-subdomain interaction within the central domain involving the mutation site, which induces defective interaction between the N-terminal and central domains. This results in an erroneous activation of Ca²⁺ channel in a diastolic state reflecting on the increased Ca²⁺ spark frequency, which then leads to lethal arrhythmia.

Activation of multiple signaling pathways causes developmental defects in mice with a Noonan syndrome–associated Sos1 mutation

PubMed Central

Chen, Peng-Chieh; Wakimoto, Hiroko; Conner, David; Araki, Toshiyuki; Yuan, Tao; Roberts, Amy; Seidman, Christine E.; Bronson, Roderick; Neel, Benjamin G.; Seidman, Jonathan G.; Kucherlapati, Raju

2010-01-01

Noonan syndrome (NS) is an autosomal dominant genetic disorder characterized by short stature, unique facial features, and congenital heart disease. About 10%–15% of individuals with NS have mutations in son of sevenless 1 (SOS1), which encodes a RAS and RAC guanine nucleotide exchange factor (GEF). To understand the role of SOS1 in the pathogenesis of NS, we generated mice with the NS-associated Sos1E846K gain-of-function mutation. Both heterozygous and homozygous mutant mice showed many NS-associated phenotypes, including growth delay, distinctive facial dysmorphia, hematologic abnormalities, and cardiac defects. We found that the Ras/MAPK pathway as well as Rac and Stat3 were activated in the mutant hearts. These data provide in vivo molecular and cellular evidence that Sos1 is a GEF for Rac under physiological conditions and suggest that Rac and Stat3 activation might contribute to NS phenotypes. Furthermore, prenatal administration of a MEK inhibitor ameliorated the embryonic lethality, cardiac defects, and NS features of the homozygous mutant mice, demonstrating that this signaling pathway might represent a promising therapeutic target for NS. PMID:21041952

A Brg1 mutation that uncouples ATPase activity from chromatin remodeling reveals an essential role for SWI/SNF-related complexes in β-globin expression and erythroid development

PubMed Central

Bultman, Scott J.; Gebuhr, Thomas C.; Magnuson, Terry

2005-01-01

The Brg1 catalytic subunit of SWI/SNF-related complexes has been implicated in many developmental and physiological processes, but null homozygotes die as blastocysts prior to implantation. To circumvent this early embryonic lethality, we performed an ENU mutagenesis screen and generated a Brg1 hypomorph mutation in the ATPase domain. The mutant Brg1 protein is stable, assembles into SWI/SNF-related complexes, and exhibits normal ATPase activity but is unable to establish DNase I hypersensitivity sites characteristic of open chromatin. Mutant embryos develop normally until midgestation but then exhibit a distinct block in the development of the erythroid lineage, leading to anemia and death. The mutant Brg1 protein is recruited to the β-globin locus, but chromatin remodeling and transcription are perturbed. Histone acetylation and DNA methylation are also affected. To our knowledge, Brg1 is the first chromatin-modifying factor shown to be required for β-globin regulation and erythropoiesis in vivo. Not only does this mutation establish a role for Brg1 during organogenesis, it also demonstrates that ATPase activity can be uncoupled from chromatin remodeling. PMID:16287714

SMK-1/PPH-4.1–mediated silencing of the CHK-1 response to DNA damage in early C. elegans embryos

PubMed Central

Kim, Seung-Hwan; Holway, Antonia H.; Wolff, Suzanne; Dillin, Andrew; Michael, W. Matthew

2007-01-01

During early embryogenesis in Caenorhabditis elegans, the ATL-1–CHK-1 (ataxia telangiectasia mutated and Rad3 related–Chk1) checkpoint controls the timing of cell division in the future germ line, or P lineage, of the animal. Activation of the CHK-1 pathway by its canonical stimulus DNA damage is actively suppressed in early embryos so that P lineage cell divisions may occur on schedule. We recently found that the rad-2 mutation alleviates this checkpoint silent DNA damage response and, by doing so, causes damage-dependent delays in early embryonic cell cycle progression and subsequent lethality. In this study, we report that mutations in the smk-1 gene cause the rad-2 phenotype. SMK-1 is a regulatory subunit of the PPH-4.1 (protein phosphatase 4) protein phosphatase, and we show that SMK-1 recruits PPH-4.1 to replicating chromatin, where it silences the CHK-1 response to DNA damage. These results identify the SMK-1–PPH-4.1 complex as a critical regulator of the CHK-1 pathway in a developmentally relevant context. PMID:17908915

Human COL7A1-corrected induced pluripotent stem cells for the treatment of recessive dystrophic epidermolysis bullosa

PubMed Central

Sebastiano, Vittorio; Zhen, Hanson Hui; Haddad, Bahareh; Bashkirova, Elizaveta; Melo, Sandra P.; Wang, Pei; Leung, Thomas L.; Siprashvili, Zurab; Tichy, Andrea; Li, Jiang; Ameen, Mohammed; Hawkins, John; Lee, Susie; Li, Lingjie; Schwertschkow, Aaron; Bauer, Gerhard; Lisowski, Leszek; Kay, Mark A.; Kim, Seung K.; Lane, Alfred T.; Wernig, Marius; Oro, Anthony E.

2015-01-01

Patients with recessive dystrophic epidermolysis bullosa (RDEB) lack functional type VII collagen owing to mutations in the gene COL7A1 and suffer severe blistering and chronic wounds that ultimately lead to infection and development of lethal squamous cell carcinoma. The discovery of induced pluripotent stem cells (iPSCs) and the ability to edit the genome bring the possibility to provide definitive genetic therapy through corrected autologous tissues. We generated patient-derived COL7A1-corrected epithelial keratinocyte sheets for autologous grafting. We demonstrate the utility of sequential reprogramming and adenovirus-associated viral genome editing to generate corrected iPSC banks. iPSC-derived keratinocytes were produced with minimal heterogeneity, and these cells secreted wild-type type VII collagen, resulting in stratified epidermis in vitro in organotypic cultures and in vivo in mice. Sequencing of corrected cell lines before tissue formation revealed heterogeneity of cancer-predisposing mutations, allowing us to select COL7A1-corrected banks with minimal mutational burden for downstream epidermis production. Our results provide a clinical platform to use iPSCs in the treatment of debilitating genodermatoses, such as RDEB. PMID:25429056

Genetic control of cuticle formation during embryonic development of Drosophila melanogaster.

PubMed Central

Ostrowski, Stephen; Dierick, Herman A; Bejsovec, Amy

2002-01-01

The embryonic cuticle of Drosophila melanogaster is deposited by the epidermal epithelium during stage 16 of development. This tough, waterproof layer is essential for maintaining the structural integrity of the larval body. We have characterized mutations in a set of genes required for proper deposition and/or morphogenesis of the cuticle. Zygotic disruption of any one of these genes results in embryonic lethality. Mutant embryos are hyperactive within the eggshell, resulting in a high proportion reversed within the eggshell (the "retroactive" phenotype), and all show poor cuticle integrity when embryos are mechanically devitellinized. This last property results in embryonic cuticle preparations that appear grossly inflated compared to wild-type cuticles (the "blimp" phenotype). We find that one of these genes, krotzkopf verkehrt (kkv), encodes the Drosophila chitin synthase enzyme and that a closely linked gene, knickkopf (knk), encodes a novel protein that shows genetic interaction with the Drosophila E-cadherin, shotgun. We also demonstrate that two other known mutants, grainy head (grh) and retroactive (rtv), show the blimp phenotype when devitellinized, and we describe a new mutation, called zeppelin (zep), that shows the blimp phenotype but does not produce defects in the head cuticle as the other mutations do. PMID:12019232

Drosophila Vps16A is required for trafficking to lysosomes and biogenesis of pigment granules.

PubMed

Pulipparacharuvil, Suprabha; Akbar, Mohammed Ali; Ray, Sanchali; Sevrioukov, Evgueny A; Haberman, Adam S; Rohrer, Jack; Krämer, Helmut

2005-08-15

Mutations that disrupt trafficking to lysosomes and lysosome-related organelles cause multiple diseases, including Hermansky-Pudlak syndrome. The Drosophila eye is a model system for analyzing such mutations. The eye-color genes carnation and deep orange encode two subunits of the Vps-C protein complex required for endosomal trafficking and pigment-granule biogenesis. Here we demonstrate that dVps16A (CG8454) encodes another Vps-C subunit. Biochemical experiments revealed a specific interaction between the dVps16A C-terminus and the Sec1/Munc18 homolog Carnation but not its closest homolog, dVps33B. Instead, dVps33B interacted with a related protein, dVps16B (CG18112). Deep orange bound both Vps16 homologs. Like a deep orange null mutation, eye-specific RNAi-induced knockdown of dVps16A inhibited lysosomal delivery of internalized ligands and interfered with biogenesis of pigment granules. Ubiquitous knockdown of dVps16A was lethal. Together, these findings demonstrate that Drosophila Vps16A is essential for lysosomal trafficking. Furthermore, metazoans have two types of Vps-C complexes with non-redundant functions.

Mechanisms and significance of fungicide resistance†

PubMed Central

Deising, Holger B.; Reimann, Sven; Pascholati, Sérgio F.

2008-01-01

In this review article, we show that occurrence of fungicide resistance is one of the most important issues in modern agriculture. Fungicide resistance may be due to mutations of genes encoding fungicide targets (qualitative fungicide resistance) or to different mechanisms that are induced by sub-lethal fungicide stress. These mechanisms result in different and varying levels of resistance (quantitative fungicide resistance). We discuss whether or not extensive use of fungicides in agricultural environments is related to the occurrence of fungicide resistance in clinical environments. Furthermore, we provide recommendations of how development of fungicide resistant pathogen populations may be prevented or delayed. PMID:24031218

Current Perspectives on HIV-1 Antiretroviral Drug Resistance

PubMed Central

Iyidogan, Pinar; Anderson, Karen S.

2014-01-01

Current advancements in antiretroviral therapy (ART) have turned HIV-1 infection into a chronic and manageable disease. However, treatment is only effective until HIV-1 develops resistance against the administered drugs. The most recent antiretroviral drugs have become superior at delaying the evolution of acquired drug resistance. In this review, the viral fitness and its correlation to HIV-1 mutation rates and drug resistance are discussed while emphasizing the concept of lethal mutagenesis as an alternative therapy. The development of resistance to the different classes of approved drugs and the importance of monitoring antiretroviral drug resistance are also summarized briefly. PMID:25341668

Report of a factor VIII inhibitor in a patient with autoimmune lymphoproliferative syndrome.

PubMed

Fang, B S; Sneller, M C; Straus, S E; Frenkel, L; Dale, J K; Rick, M E

2000-07-01

The occurrence of factor VIII inhibitors in non-hemophilic patients is a rare event with a potentially lethal outcome. Despite its infrequent occurrence, the association of this inhibitor with multiple autoimmune diseases is well recognized. We report the case of a patient with the recently described autoimmune lymphoproliferative syndrome (ALPS) who developed an inhibitor to factor VIII. ALPS is a disease characterized by defective lymphocyte apoptosis due to inherited mutations in genes that regulate apoptosis, with the resulting enlargement of lymphoid organs and a variety of autoimmune manifestations. Published 2000 Wiley-Liss, Inc.

Fox transcription factors: from development to disease.

PubMed

Golson, Maria L; Kaestner, Klaus H

2016-12-15

Forkhead box (Fox) transcription factors are evolutionarily conserved in organisms ranging from yeast to humans. They regulate diverse biological processes both during development and throughout adult life. Mutations in many Fox genes are associated with human disease and, as such, various animal models have been generated to study the function of these transcription factors in mechanistic detail. In many cases, the absence of even a single Fox transcription factor is lethal. In this Primer, we provide an overview of the Fox family, highlighting several key Fox transcription factor families that are important for mammalian development. © 2016. Published by The Company of Biologists Ltd.

Update on the evaluation and treatment of osteogenesis imperfecta.

PubMed

Harrington, Jennifer; Sochett, Etienne; Howard, Andrew

2014-12-01

Osteogenesis imperfecta (OI) is a heritable bone fragility disorder that presents with a wide clinical phenotype spectrum: from perinatal lethality and severe deformities to very mild forms without fractures. Most cases of OI are due to autosomal dominant mutations of the type I collagen genes. A multidisciplinary approach with rehabilitation, orthopedic surgery, and consideration of medical therapy with bisphosphonates underpins current management. Greater understanding of the pathogenesis of OI may lead to novel, therapeutic approaches to help improve clinical symptoms of children with OI in the future. Copyright © 2014 Elsevier Inc. All rights reserved.

Unusual Father-to-Daughter Transmission of Incontinentia Pigmenti Due to Mosaicism in IP Males.

PubMed

Fusco, Francesca; Conte, Matilde Immacolata; Diociaiuti, Andrea; Bigoni, Stefania; Branda, Maria Francesca; Ferlini, Alessandra; El Hachem, Maya; Ursini, Matilde Valeria

2017-09-01

Incontinentia pigmenti (IP; Online Mendelian Inheritance in Man catalog #308300) is an X-linked dominant ectodermal disorder caused by mutations of the inhibitor of κ polypeptide gene enchancer in B cells, kinase γ ( IKBKG )/ nuclear factor κB, essential modulator ( NEMO ) gene. Hemizygous IKBKG/NEMO loss-of-function (LoF) mutations are lethal in males, thus patients are female, and the disease is always transmitted from an IP-affected mother to her daughter. We present 2 families with father-to-daughter transmission of IP and provide for the first time molecular evidence that the combination of somatic and germ-line mosaicism for IKBKG/NEMO loss of function mutations in IP males resulted in the transmission of the disease to a female child. We searched for the IKBKG/NEMO mutant allele in blood, urine, skin, and sperm DNA and found that the 2 fathers were somatic and germ-line mosaics for the p.Gln132×mutation or the exon 4-10 deletion of IKBKG/NEMO , respectively. The highest level of IKBKG/NEMO mutant cells was detected in the sperm, which might explain the recurrence of the disease. We therefore recommend careful clinical evaluation in IP male cases and the genetic investigation in sperm DNA to ensure correct genetic counseling and prevent the risk of paternal transmission of IP. Copyright © 2017 by the American Academy of Pediatrics.

Larger aggregates of mutant seipin in Celia's Encephalopathy, a new protein misfolding neurodegenerative disease.

PubMed

Ruiz-Riquelme, Alejandro; Sánchez-Iglesias, Sofía; Rábano, Alberto; Guillén-Navarro, Encarna; Domingo-Jiménez, Rosario; Ramos, Adriana; Rosa, Isaac; Senra, Ana; Nilsson, Peter; García, Ángel; Araújo-Vilar, David; Requena, Jesús R

2015-11-01

Celia's Encephalopathy (MIM #615924) is a recently discovered fatal neurodegenerative syndrome associated with a new BSCL2 mutation (c.985C>T) that results in an aberrant isoform of seipin (Celia seipin). This mutation is lethal in both homozygosity and compounded heterozygosity with a lipodystrophic BSCL2 mutation, resulting in a progressive encephalopathy with fatal outcomes at ages 6-8. Strikingly, heterozygous carriers are asymptomatic, conflicting with the gain of toxic function attributed to this mutation. Here we report new key insights about the molecular pathogenic mechanism of this new syndrome. Intranuclear inclusions containing mutant seipin were found in brain tissue from a homozygous patient suggesting a pathogenic mechanism similar to other neurodegenerative diseases featuring brain accumulation of aggregated, misfolded proteins. Sucrose gradient distribution showed that mutant seipin forms much larger aggregates as compared with wild type (wt) seipin, indicating an impaired oligomerization. On the other hand, the interaction between wt and Celia seipin confirmed by coimmunoprecipitation (CoIP) assays, together with the identification of mixed oligomers in sucrose gradient fractionation experiments can explain the lack of symptoms in heterozygous carriers. We propose that the increased aggregation and subsequent impaired oligomerization of Celia seipin leads to cell death. In heterozygous carriers, wt seipin might prevent the damage caused by mutant seipin through its sequestration into harmless mixed oligomers. Copyright © 2015 Elsevier Inc. All rights reserved.

Involvement of the pagR gene of pXO2 in anthrax pathogenesis

PubMed Central

Liang, Xudong; Zhang, Enmin; Zhang, Huijuan; Wei, Jianchun; Li, Wei; Zhu, Jin; Wang, Bingxiang; Dong, Shulin

2016-01-01

Anthrax is a disease caused by Bacillus anthracis. Specifically, the anthrax toxins and capsules encoded by the pXO1 and pXO2 plasmids, respectively, are the major virulence factors. We previously reported that the pXO1 plasmid was retained in the attenuated strain of B. anthracis vaccine strains even after subculturing at high temperatures. In the present study, we reinvestigate the attenuation mechanism of Pasteur II. Sequencing of pXO1 and pXO2 from Pasteur II strain revealed mutations in these plasmids as compared to the reference sequences. Two deletions on these plasmids, one each on pXO1 and pXO2, were confirmed to be unique to the Pasteur II strain as compared to the wild-type strains. Gene replacement with homologous recombination revealed that the mutation in the promoter region of the pagR gene on pXO2, but not the mutation on pXO1, contributes to lethal levels of toxin production. This result was further confirmed by RT-PCR, western blot, and animal toxicity assays. Taken together, our results signify that the attenuation of the Pasteur II vaccine strain is caused by a mutation in the pagR gene on its pXO2 plasmid. Moreover, these data suggest that pXO2 plasmid encoded proteins are involved in the virulence of B. anthracis. PMID:27363681

Drosophila Lyra mutations are gain-of-function mutations of senseless

NASA Technical Reports Server (NTRS)

Nolo, R.; Abbott, L. A.; Bellen, H. J.

2001-01-01

The Lyra mutation was first described by Jerry Coyne in 1935. Lyra causes recessive pupal lethality and adult heterozygous Lyra mutants exhibit a dominant loss of the anterior and posterior wing margins. Unlike many mutations that cause loss of wing tissue (e.g., scalloped, Beadex, cut, and apterous-Xasta), Lyra wing discs do not exhibit increased necrotic or apoptotic cell death, nor do they show altered BrdU incorporation. However, during wing disc eversion, loss of the anterior and posterior wing margins is apparent. We have previously shown that senseless, a gene that is necessary and sufficient for peripheral nervous system (PNS) development, is allelic to Lyra. Here we show by several genetic criteria that Lyra alleles are neomorphic alleles of senseless that cause ectopic expression of SENSELESS in the wing pouch. Similarly, overexpression of SENSELESS in the wing disc causes loss of wing margin tissue, thereby mimicking the Lyra phenotype. Lyra mutants display aberrant expression of DELTA, VESTIGIAL, WINGLESS, and CUT. As in Lyra mutants, overexpression of SENSELESS in some areas of the wing pouch also leads to loss of WINGLESS and CUT. In summary, our data indicate that overexpression of SENSELESS causes a severe reduction in NOTCH signaling that in turn may lead to decreased transcription of several key genes required for wing development, leading to a failure in cell proliferation and loss of wing margin tissue.

Functional Studies of Tyrosine Hydroxylase Missense Variants Reveal Distinct Patterns of Molecular Defects in Dopa-Responsive Dystonia

PubMed Central

Fossbakk, Agnete; Kleppe, Rune; Knappskog, Per M; Martinez, Aurora; Haavik, Jan

2014-01-01

Congenital tyrosine hydroxylase deficiency (THD) is found in autosomal-recessive Dopa-responsive dystonia and related neurological syndromes. The clinical manifestations of THD are variable, ranging from early-onset lethal disease to mild Parkinson disease-like symptoms appearing in adolescence. Until 2014, approximately 70 THD patients with a total of 40 different disease-related missense mutations, five nonsense mutations, and three mutations in the promoter region of the tyrosine hydroxylase (TH) gene have been reported. We collected clinical and biochemical data in the literature for all variants, and also generated mutant forms of TH variants previously not studied (N = 23). We compared the in vitro solubility, thermal stability, and kinetic properties of the TH variants to determine the cause(s) of their impaired enzyme activity, and found great heterogeneity in all these properties among the mutated forms. Some TH variants had specific kinetic anomalies and phenylalanine hydroxylase, and Dopa oxidase activities were measured for variants that showed signs of altered substrate binding. p.Arg233His, p.Gly247Ser, and p.Phe375Leu had shifted substrate specificity from tyrosine to phenylalanine and Dopa, whereas p.Cys359Phe had an impaired activity toward these substrates. The new data about pathogenic mechanisms presented are expected to contribute to develop individualized therapy for THD patients. PMID:24753243

ESCRT-III-Associated Protein ALIX Mediates High-Affinity Phosphate Transporter Trafficking to Maintain Phosphate Homeostasis in Arabidopsis

PubMed Central

Cardona-López, Ximena; Cuyas, Laura; Marín, Elena; Irigoyen, María Luisa; Gil, Erica; Puga, María Isabel; Bligny, Richard; Nussaume, Laurent; Geldner, Niko; Paz-Ares, Javier

2015-01-01

Prior to the release of their cargoes into the vacuolar lumen, sorting endosomes mature into multivesicular bodies (MVBs) through the action of ENDOSOMAL COMPLEX REQUIRED FOR TRANSPORT (ESCRT) protein complexes. MVB-mediated sorting of high-affinity phosphate transporters (PHT1) to the vacuole limits their plasma membrane levels under phosphate-sufficient conditions, a process that allows plants to maintain phosphate homeostasis. Here, we describe ALIX, a cytosolic protein that associates with MVB by interacting with ESCRT-III subunit SNF7 and mediates PHT1;1 trafficking to the vacuole in Arabidopsis thaliana. We show that the partial loss-of-function mutant alix-1 displays reduced vacuolar degradation of PHT1;1. ALIX derivatives containing the alix-1 mutation showed reduced interaction with SNF7, providing a simple molecular explanation for impaired cargo trafficking in alix-1 mutants. In fact, the alix-1 mutation also hampered vacuolar sorting of the brassinosteroid receptor BRI1. We also show that alix-1 displays altered vacuole morphogenesis, implying a new role for ALIX proteins in vacuolar biogenesis, likely acting as part of ESCRT-III complexes. In line with a presumed broad target spectrum, the alix-1 mutation is pleiotropic, leading to reduced plant growth and late flowering, with stronger alix mutations being lethal, indicating that ALIX participates in diverse processes in plants essential for their life. PMID:26342016

Female receptivity phenotype of icebox mutants caused by a mutation in the L1-type cell adhesion molecule neuroglian.

PubMed

Carhan, A; Allen, F; Armstrong, J D; Hortsch, M; Goodwin, S F; O'Dell, K M C

2005-11-01

Relatively little is known about the genes and brain structures that enable virgin female Drosophila to make the decision to mate or not. Classical genetic approaches have identified several mutant females that have a reluctance-to-mate phenotype, but most of these have additional behavioral defects. However, the icebox (ibx) mutation was previously reported to lower the sexual receptivity of females, without apparently affecting any other aspect of female behavior. We have shown that the ibx mutation maps to the 7F region of the Drosophila X chromosome to form a complex complementation group with both lethal and viable alleles of neuroglian (nrg). The L1-type cell adhesion molecule encoded by nrg consists of six immunoglobulin-like domains, five fibronectin-like domains, one transmembrane domain and one alternatively spliced intracellular domain. The ibx strain has a missense mutation causing a glycine-to-arginine change at amino acid 92 in the first immunoglobulin domain of nrg. Defects in the central brain of ibx mutants are similar to those observed in another nrg mutant, central brain deranged(1) (ceb(1)). However, both ceb(1) homozygous and ceb(1)/ibx heterozygous females are receptive. The expression of a transgene containing the non-neural isoform of nrg rescues both the receptivity and the brain structure phenotypes of ibx females.

Zebrafish cdc6 hypomorphic mutation causes Meier-Gorlin syndrome-like phenotype.

PubMed

Yao, Likun; Chen, Jing; Wu, Xiaotong; Jia, Shunji; Meng, Anming

2017-11-01

Cell Division Cycle 6 (Cdc6) is a component of pre-replicative complex (preRC) forming on DNA replication origins in eukaryotes. Recessive mutations in ORC1, ORC4, ORC6, CDT1 or CDC6 of the preRC in human cause Meier-Gorlin syndrome (MGS) that is characterized by impaired post-natal growth, short stature and microcephaly. However, vertebrate models of MGS have not been reported. Through N-ethyl-N-nitrosourea mutagenesis and Cas9 knockout, we generate several cdc6 mutant lines in zebrafish. Loss-of-function mutations of cdc6, as manifested by cdc6tsu4305 and cdc6tsu7cd mutants, lead to embryonic lethality due to cell cycle arrest at the S phase and extensive apoptosis. Embryos homozygous for a cdc6 hypomorphic mutation, cdc6tsu21cd, develop normally during embryogenesis. Later on, compared with their wild-type (WT) siblings, cdc6tsu21cd mutant fish show growth retardation, and their body weight and length in adulthood are greatly reduced, which resemble human MGS. Surprisingly, cdc6tsu21cd mutant fish become males with a short life and fail to mate with WT females, suggesting defective reproduction. Overexpression of Cdc6 mutant forms, which mimic human CDC6(T323R) mutation found in a MGS patient, in zebrafish cdc6tsu4305 mutant embryos partially represses cell death phenotype, suggesting that the human CDC6(T323R) mutation is a hypomorph. cdc6tsu21cd mutant fish will be useful to detect more tissue defects and develop medical treatment strategies for MGS patients. © The Author 2017. Published by Oxford University Press.

Short-rib polydactyly and Jeune syndromes are caused by mutations in WDR60.

PubMed

McInerney-Leo, Aideen M; Schmidts, Miriam; Cortés, Claudio R; Leo, Paul J; Gener, Blanca; Courtney, Andrew D; Gardiner, Brooke; Harris, Jessica A; Lu, Yeping; Marshall, Mhairi; Scambler, Peter J; Beales, Philip L; Brown, Matthew A; Zankl, Andreas; Mitchison, Hannah M; Duncan, Emma L; Wicking, Carol

2013-09-05

Short-rib polydactyly syndromes (SRPS I-V) are a group of lethal congenital disorders characterized by shortening of the ribs and long bones, polydactyly, and a range of extraskeletal phenotypes. A number of other disorders in this grouping, including Jeune and Ellis-van Creveld syndromes, have an overlapping but generally milder phenotype. Collectively, these short-rib dysplasias (with or without polydactyly) share a common underlying defect in primary cilium function and form a subset of the ciliopathy disease spectrum. By using whole-exome capture and massive parallel sequencing of DNA from an affected Australian individual with SRPS type III, we detected two novel heterozygous mutations in WDR60, a relatively uncharacterized gene. These mutations segregated appropriately in the unaffected parents and another affected family member, confirming compound heterozygosity, and both were predicted to have a damaging effect on the protein. Analysis of an additional 54 skeletal ciliopathy exomes identified compound heterozygous mutations in WDR60 in a Spanish individual with Jeune syndrome of relatively mild presentation. Of note, these two families share one novel WDR60 missense mutation, although haplotype analysis suggested no shared ancestry. We further show that WDR60 localizes at the base of the primary cilium in wild-type human chondrocytes, and analysis of fibroblasts from affected individuals revealed a defect in ciliogenesis and aberrant accumulation of the GLI2 transcription factor at the centrosome or basal body in the absence of an obvious axoneme. These findings show that WDR60 mutations can cause skeletal ciliopathies and suggest a role for WDR60 in ciliogenesis. Copyright © 2013 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

Mitochondrial Hsp60 Chaperonopathy Causes an Autosomal-Recessive Neurodegenerative Disorder Linked to Brain Hypomyelination and Leukodystrophy

PubMed Central

Magen, Daniella; Georgopoulos, Costa; Bross, Peter; Ang, Debbie; Segev, Yardena; Goldsher, Dorit; Nemirovski, Alexandra; Shahar, Eli; Ravid, Sarit; Luder, Anthony; Heno, Bayan; Gershoni-Baruch, Ruth; Skorecki, Karl; Mandel, Hanna

2008-01-01

Hypomyelinating leukodystrophies (HMLs) are disorders involving aberrant myelin formation. The prototype of primary HMLs is the X-linked Pelizaeus-Merzbacher disease (PMD) caused by mutations in PLP1. Recently, homozygous mutations in GJA12 encoding connexin 47 were found in patients with autosomal-recessive Pelizaeus-Merzbacher-like disease (PMLD). However, many patients of both genders with PMLD carry neither PLP1 nor GJA12 mutations. We report a consanguineous Israeli Bedouin kindred with clinical and radiological findings compatible with PMLD, in which linkage to PLP1 and GJA12 was excluded. Using homozygosity mapping and mutation analysis, we have identified a homozygous missense mutation (D29G) not previously described in HSPD1, encoding the mitochondrial heat-shock protein 60 (Hsp60) in all affected individuals. The D29G mutation completely segregates with the disease-associated phenotype. The pathogenic effect of D29G on Hsp60-chaperonin activity was verified by an in vivo E. coli complementation assay, which demonstrated compromised ability of the D29G-Hsp60 mutant protein to support E. coli survival, especially at high temperatures. The disorder, which we have termed MitCHAP-60 disease, can be distinguished from spastic paraplegia 13 (SPG13), another Hsp60-associated autosomal-dominant neurodegenerative disorder, by its autosomal-recessive inheritance pattern, as well as by its early-onset, profound cerebral involvement and lethality. Our findings suggest that Hsp60 defects can cause neurodegenerative pathologies of varying severity, not previously suspected on the basis of the SPG13 phenotype. These findings should help to clarify the important role of Hsp60 in myelinogenesis and neurodegeneration. PMID:18571143

CHK1-targeted therapy to deplete DNA replication-stressed, p53-deficient, hyperdiploid colorectal cancer stem cells

PubMed Central

Russo, Giorgio; Corradi, Francesca; Siteni, Silvia; Musella, Martina; Vitale, Sara; De Angelis, Maria Laura; Pallocca, Matteo; Amoreo, Carla Azzurra; Sperati, Francesca; Di Franco, Simone; Barresi, Sabina; Policicchio, Eleonora; De Luca, Gabriele; De Nicola, Francesca; Mottolese, Marcella; Zeuner, Ann; Fanciulli, Maurizio; Stassi, Giorgio; Maugeri-Saccà, Marcello; Baiocchi, Marta; Tartaglia, Marco

2018-01-01

Objective Cancer stem cells (CSCs) are responsible for tumour formation and spreading, and their targeting is required for tumour eradication. There are limited therapeutic options for advanced colorectal cancer (CRC), particularly for tumours carrying RAS-activating mutations. The aim of this study was to identify novel CSC-targeting strategies. Design To discover potential therapeutics to be clinically investigated as single agent, we performed a screening with a panel of FDA-approved or investigational drugs on primary CRC cells enriched for CSCs (CRC-SCs) isolated from 27 patients. Candidate predictive biomarkers of efficacy were identified by integrating genomic, reverse-phase protein microarray (RPPA) and cytogenetic analyses, and validated by immunostainings. DNA replication stress (RS) was increased by employing DNA replication-perturbing or polyploidising agents. Results The drug-library screening led to the identification of LY2606368 as a potent anti-CSC agent acting in vitro and in vivo in tumour cells from a considerable number of patients (∼36%). By inhibiting checkpoint kinase (CHK)1, LY2606368 affected DNA replication in most CRC-SCs, including RAS-mutated ones, forcing them into premature, lethal mitoses. Parallel genomic, RPPA and cytogenetic analyses indicated that CRC-SCs sensitive to LY2606368 displayed signs of ongoing RS response, including the phosphorylation of RPA32 and ataxia telangiectasia mutated serine/threonine kinase (ATM). This was associated with mutation(s) in TP53 and hyperdiploidy, and made these CRC-SCs exquisitely dependent on CHK1 function. Accordingly, experimental increase of RS sensitised resistant CRC-SCs to LY2606368. Conclusions LY2606368 selectively eliminates replication-stressed, p53-deficient and hyperdiploid CRC-SCs independently of RAS mutational status. These results provide a strong rationale for biomarker-driven clinical trials with LY2606368 in patients with CRC. PMID:28389531

Mitochondrial encephalomyopathy and retinoblastoma explained by compound heterozygosity of SUCLA2 point mutation and 13q14 deletion

PubMed Central

Matilainen, Sanna; Isohanni, Pirjo; Euro, Liliya; Lönnqvist, Tuula; Pihko, Helena; Kivelä, Tero; Knuutila, Sakari; Suomalainen, Anu

2015-01-01

Mutations in SUCLA2, encoding the ß-subunit of succinyl-CoA synthetase of Krebs cycle, are one cause of mitochondrial DNA depletion syndrome. Patients have been reported to have severe progressive childhood-onset encephalomyopathy, and methylmalonic aciduria, often leading to death in childhood. We studied two families, with children manifesting with slowly progressive mitochondrial encephalomyopathy, hearing impairment and transient methylmalonic aciduria, without mtDNA depletion. The other family also showed dominant inheritance of bilateral retinoblastoma, which coexisted with mitochondrial encephalomyopathy in one patient. We found a variant in SUCLA2 leading to Asp333Gly change, homozygous in one patient and compound heterozygous in one. The latter patient also carried a deletion of 13q14 of the other allele, discovered with molecular karyotyping. The deletion spanned both SUCLA2 and RB1 gene regions, leading to manifestation of both mitochondrial disease and retinoblastoma. We made a homology model for human succinyl-CoA synthetase and used it for structure–function analysis of all reported pathogenic mutations in SUCLA2. On the basis of our model, all previously described mutations were predicted to result in decreased amounts of incorrectly assembled protein or disruption of ADP phosphorylation, explaining the severe early lethal manifestations. However, the Asp333Gly change was predicted to reduce the activity of the otherwise functional enzyme. On the basis of our findings, SUCLA2 mutations should be analyzed in patients with slowly progressive encephalomyopathy, even in the absence of methylmalonic aciduria or mitochondrial DNA depletion. In addition, an encephalomyopathy in a patient with retinoblastoma suggests mutations affecting SUCLA2. PMID:24986829

Thioredoxin Reductase 2 (TXNRD2) mutation associated with familial glucocorticoid deficiency (FGD).

PubMed

Prasad, Rathi; Chan, Li F; Hughes, Claire R; Kaski, Juan P; Kowalczyk, Julia C; Savage, Martin O; Peters, Catherine J; Nathwani, Nisha; Clark, Adrian J L; Storr, Helen L; Metherell, Louise A

2014-08-01

Classic ACTH resistance, due to disruption of ACTH signaling, accounts for the majority of cases of familial glucocorticoid deficiency (FGD). Recently FGD cases caused by mutations in the mitochondrial antioxidant, nicotinamide nucleotide transhydrogenase, have highlighted the importance of redox regulation in steroidogenesis. We hypothesized that other components of mitochondrial antioxidant systems would be good candidates in the etiology of FGD. Whole-exome sequencing was performed on three related patients, and segregation of putative causal variants confirmed by Sanger sequencing of all family members. A TXNRD2-knockdown H295R cell line was created to investigate redox homeostasis. The study was conducted on patients from three pediatric centers in the United Kingdom. Seven individuals from a consanguineous Kashmiri kindred, six of whom presented with FGD between 0.1 and 10.8 years, participated in the study. There were no interventions. Identification and functional interrogation of a novel homozygous mutation segregating with the disease trait were measured. A stop gain mutation, p.Y447X in TXNRD2, encoding the mitochondrial selenoprotein thioredoxin reductase 2 (TXNRD2) was identified and segregated with disease in this extended kindred. RT-PCR and Western blotting revealed complete absence of TXNRD2 in patients homozygous for the mutation. TXNRD2 deficiency leads to impaired redox homeostasis in a human adrenocortical cell line. In contrast to the Txnrd2-knockout mouse model, in which embryonic lethality as a consequence of hematopoietic and cardiac defects is described, absence of TXNRD2 in humans leads to glucocorticoid deficiency. This is the first report of a homozygous mutation in any component of the thioredoxin antioxidant system leading to inherited disease in humans.

Zebrafish cdc6 hypomorphic mutation causes Meier-Gorlin syndrome-like phenotype

PubMed Central

Yao, Likun; Chen, Jing; Wu, Xiaotong; Jia, Shunji; Meng, Anming

2017-01-01

Abstract Cell Division Cycle 6 (Cdc6) is a component of pre-replicative complex (preRC) forming on DNA replication origins in eukaryotes. Recessive mutations in ORC1, ORC4, ORC6, CDT1 or CDC6 of the preRC in human cause Meier-Gorlin syndrome (MGS) that is characterized by impaired post-natal growth, short stature and microcephaly. However, vertebrate models of MGS have not been reported. Through N-ethyl-N-nitrosourea mutagenesis and Cas9 knockout, we generate several cdc6 mutant lines in zebrafish. Loss-of-function mutations of cdc6, as manifested by cdc6tsu4305 and cdc6tsu7cd mutants, lead to embryonic lethality due to cell cycle arrest at the S phase and extensive apoptosis. Embryos homozygous for a cdc6 hypomorphic mutation, cdc6tsu21cd, develop normally during embryogenesis. Later on, compared with their wild-type (WT) siblings, cdc6tsu21cd mutant fish show growth retardation, and their body weight and length in adulthood are greatly reduced, which resemble human MGS. Surprisingly, cdc6tsu21cd mutant fish become males with a short life and fail to mate with WT females, suggesting defective reproduction. Overexpression of Cdc6 mutant forms, which mimic human CDC6(T323R) mutation found in a MGS patient, in zebrafish cdc6tsu4305 mutant embryos partially represses cell death phenotype, suggesting that the human CDC6(T323R) mutation is a hypomorph. cdc6tsu21cd mutant fish will be useful to detect more tissue defects and develop medical treatment strategies for MGS patients. PMID:28985365

DNA Glycosylases Involved in Base Excision Repair May Be Associated with Cancer Risk in BRCA1 and BRCA2 Mutation Carriers

PubMed Central

Osorio, Ana; Milne, Roger L.; Kuchenbaecker, Karoline; Vaclová, Tereza; Pita, Guillermo; Alonso, Rosario; Peterlongo, Paolo; Blanco, Ignacio; de la Hoya, Miguel; Duran, Mercedes; Díez, Orland; Ramón y Cajal, Teresa; Konstantopoulou, Irene; Martínez-Bouzas, Cristina; Andrés Conejero, Raquel; Soucy, Penny; McGuffog, Lesley; Barrowdale, Daniel; Lee, Andrew; SWE-BRCA; Arver, Brita; Rantala, Johanna; Loman, Niklas; Ehrencrona, Hans; Olopade, Olufunmilayo I.; Beattie, Mary S.; Domchek, Susan M.; Nathanson, Katherine; Rebbeck, Timothy R.; Arun, Banu K.; Karlan, Beth Y.; Walsh, Christine; Lester, Jenny; John, Esther M.; Whittemore, Alice S.; Daly, Mary B.; Southey, Melissa; Hopper, John; Terry, Mary B.; Buys, Saundra S.; Janavicius, Ramunas; Dorfling, Cecilia M.; van Rensburg, Elizabeth J.; Steele, Linda; Neuhausen, Susan L.; Ding, Yuan Chun; Hansen, Thomas v. O.; Jønson, Lars; Ejlertsen, Bent; Gerdes, Anne-Marie; Infante, Mar; Herráez, Belén; Moreno, Leticia Thais; Weitzel, Jeffrey N.; Herzog, Josef; Weeman, Kisa; Manoukian, Siranoush; Peissel, Bernard; Zaffaroni, Daniela; Scuvera, Giulietta; Bonanni, Bernardo; Mariette, Frederique; Volorio, Sara; Viel, Alessandra; Varesco, Liliana; Papi, Laura; Ottini, Laura; Tibiletti, Maria Grazia; Radice, Paolo; Yannoukakos, Drakoulis; Garber, Judy; Ellis, Steve; Frost, Debra; Platte, Radka; Fineberg, Elena; Evans, Gareth; Lalloo, Fiona; Izatt, Louise; Eeles, Ros; Adlard, Julian; Davidson, Rosemarie; Cole, Trevor; Eccles, Diana; Cook, Jackie; Hodgson, Shirley; Brewer, Carole; Tischkowitz, Marc; Douglas, Fiona; Porteous, Mary; Side, Lucy; Walker, Lisa; Morrison, Patrick; Donaldson, Alan; Kennedy, John; Foo, Claire; Godwin, Andrew K.; Schmutzler, Rita Katharina; Wappenschmidt, Barbara; Rhiem, Kerstin; Engel, Christoph; Meindl, Alfons; Ditsch, Nina; Arnold, Norbert; Plendl, Hans Jörg; Niederacher, Dieter; Sutter, Christian; Wang-Gohrke, Shan; Steinemann, Doris; Preisler-Adams, Sabine; Kast, Karin; Varon-Mateeva, Raymonda; Gehrig, Andrea; Stoppa-Lyonnet, Dominique; Sinilnikova, Olga M.; Mazoyer, Sylvie; Damiola, Francesca; Poppe, Bruce; Claes, Kathleen; Piedmonte, Marion; Tucker, Kathy; Backes, Floor; Rodríguez, Gustavo; Brewster, Wendy; Wakeley, Katie; Rutherford, Thomas; Caldés, Trinidad; Nevanlinna, Heli; Aittomäki, Kristiina; Rookus, Matti A.; van Os, Theo A. M.; van der Kolk, Lizet; de Lange, J. L.; Meijers-Heijboer, Hanne E. J.; van der Hout, A. H.; van Asperen, Christi J.; Gómez Garcia, Encarna B.; Hoogerbrugge, Nicoline; Collée, J. Margriet; van Deurzen, Carolien H. M.; van der Luijt, Rob B.; Devilee, Peter; HEBON; Olah, Edith; Lázaro, Conxi; Teulé, Alex; Menéndez, Mireia; Jakubowska, Anna; Cybulski, Cezary; Gronwald, Jacek; Lubinski, Jan; Durda, Katarzyna; Jaworska-Bieniek, Katarzyna; Johannsson, Oskar Th.; Maugard, Christine; Montagna, Marco; Tognazzo, Silvia; Teixeira, Manuel R.; Healey, Sue; Investigators, kConFab; Olswold, Curtis; Guidugli, Lucia; Lindor, Noralane; Slager, Susan; Szabo, Csilla I.; Vijai, Joseph; Robson, Mark; Kauff, Noah; Zhang, Liying; Rau-Murthy, Rohini; Fink-Retter, Anneliese; Singer, Christian F.; Rappaport, Christine; Geschwantler Kaulich, Daphne; Pfeiler, Georg; Tea, Muy-Kheng; Berger, Andreas; Phelan, Catherine M.; Greene, Mark H.; Mai, Phuong L.; Lejbkowicz, Flavio; Andrulis, Irene; Mulligan, Anna Marie; Glendon, Gord; Toland, Amanda Ewart; Bojesen, Anders; Pedersen, Inge Sokilde; Sunde, Lone; Thomassen, Mads; Kruse, Torben A.; Jensen, Uffe Birk; Friedman, Eitan; Laitman, Yael; Shimon, Shani Paluch; Simard, Jacques; Easton, Douglas F.; Offit, Kenneth; Couch, Fergus J.; Chenevix-Trench, Georgia; Antoniou, Antonis C.; Benitez, Javier

2014-01-01

Single Nucleotide Polymorphisms (SNPs) in genes involved in the DNA Base Excision Repair (BER) pathway could be associated with cancer risk in carriers of mutations in the high-penetrance susceptibility genes BRCA1 and BRCA2, given the relation of synthetic lethality that exists between one of the components of the BER pathway, PARP1 (poly ADP ribose polymerase), and both BRCA1 and BRCA2. In the present study, we have performed a comprehensive analysis of 18 genes involved in BER using a tagging SNP approach in a large series of BRCA1 and BRCA2 mutation carriers. 144 SNPs were analyzed in a two stage study involving 23,463 carriers from the CIMBA consortium (the Consortium of Investigators of Modifiers of BRCA1 and BRCA2). Eleven SNPs showed evidence of association with breast and/or ovarian cancer at p<0.05 in the combined analysis. Four of the five genes for which strongest evidence of association was observed were DNA glycosylases. The strongest evidence was for rs1466785 in the NEIL2 (endonuclease VIII-like 2) gene (HR: 1.09, 95% CI (1.03–1.16), p = 2.7×10−3) for association with breast cancer risk in BRCA2 mutation carriers, and rs2304277 in the OGG1 (8-guanine DNA glycosylase) gene, with ovarian cancer risk in BRCA1 mutation carriers (HR: 1.12 95%CI: 1.03–1.21, p = 4.8×10−3). DNA glycosylases involved in the first steps of the BER pathway may be associated with cancer risk in BRCA1/2 mutation carriers and should be more comprehensively studied. PMID:24698998

Polε Instability Drives Replication Stress, Abnormal Development, and Tumorigenesis.

PubMed

Bellelli, Roberto; Borel, Valerie; Logan, Clare; Svendsen, Jennifer; Cox, Danielle E; Nye, Emma; Metcalfe, Kay; O'Connell, Susan M; Stamp, Gordon; Flynn, Helen R; Snijders, Ambrosius P; Lassailly, François; Jackson, Andrew; Boulton, Simon J

2018-05-17

DNA polymerase ε (POLE) is a four-subunit complex and the major leading strand polymerase in eukaryotes. Budding yeast orthologs of POLE3 and POLE4 promote Polε processivity in vitro but are dispensable for viability in vivo. Here, we report that POLE4 deficiency in mice destabilizes the entire Polε complex, leading to embryonic lethality in inbred strains and extensive developmental abnormalities, leukopenia, and tumor predisposition in outbred strains. Comparable phenotypes of growth retardation and immunodeficiency are also observed in human patients harboring destabilizing mutations in POLE1. In both Pole4 -/- mouse and POLE1 mutant human cells, Polε hypomorphy is associated with replication stress and p53 activation, which we attribute to inefficient replication origin firing. Strikingly, removing p53 is sufficient to rescue embryonic lethality and all developmental abnormalities in Pole4 null mice. However, Pole4 -/- p53 +/- mice exhibit accelerated tumorigenesis, revealing an important role for controlled CMG and origin activation in normal development and tumor prevention. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.