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Sample records for genes encoding nicotinamide

  1. Saccharomyces cerevisiae YOR071C encodes the high affinity nicotinamide riboside transporter Nrt1.

    PubMed

    Belenky, Peter A; Moga, Tiberiu G; Brenner, Charles

    2008-03-28

    NAD(+) is an essential coenzyme for hydride transfer enzymes and a substrate of sirtuins and other NAD(+)-consuming enzymes. Nicotinamide riboside is a recently discovered eukaryotic NAD(+) precursor converted to NAD(+) via the nicotinamide riboside kinase pathway and by nucleosidase activity and nicotinamide salvage. Nicotinamide riboside supplementation of yeast extends replicative life span on high glucose medium. The molecular basis for nicotinamide riboside uptake was unknown in any eukaryote. Here, we show that deletion of a single gene, YOR071C, abrogates nicotinamide riboside uptake without altering nicotinic acid or nicotinamide import. The gene, which is negatively regulated by Sum1, Hst1, and Rfm1, fully restores nicotinamide riboside import and utilization when resupplied to mutant yeast cells. The encoded polypeptide, Nrt1, is a predicted deca-spanning membrane protein related to the thiamine transporter, which functions as a pH-dependent facilitator with a K(m) for nicotinamide riboside of 22 microm. Nrt1-related molecules are conserved in particular fungi, suggesting a similar basis for nicotinamide riboside uptake.

  2. Maternal nicotinamide supplementation causes global DNA hypomethylation, uracil hypo-incorporation and gene expression changes in fetal rats.

    PubMed

    Tian, Yan-Jie; Luo, Ning; Chen, Na-Na; Lun, Yong-Zhi; Gu, Xin-Yi; Li, Zhi; Ma, Qiang; Zhou, Shi-Sheng

    2014-05-01

    Recent evidence shows that excess nicotinamide can cause epigenetic changes in developing rats. The aim of the present study was to investigate the effects of maternal nicotinamide supplementation on the fetus. Female rats were randomised into four groups fed a standard chow diet (control group) or diets supplemented with 1 g/kg of nicotinamide (low-dose group), 4 g/kg of nicotinamide (high-dose group) or 4 g/kg of nicotinamide plus 2 g/kg of betaine (betaine group) for 14-16 d before mating and throughout the study. Fetal tissue samples were collected on the 20th day of pregnancy. Compared with the control group, the high-dose group had a higher fetal death rate, and the average fetal body weight was higher in the low-dose group but lower in the high-dose group. Nicotinamide supplementation led to a decrease in placental and fetal hepatic genomic DNA methylation and genomic uracil contents (a factor modifying DNA for diversity) in the placenta and fetal liver and brain, which could be completely or partially prevented by betaine. Moreover, nicotinamide supplementation induced tissue-specific alterations in the mRNA expression of the genes encoding nicotinamide N-methyltransferase, DNA methyltransferase 1, catalase and tumour protein p53 in the placenta and fetal liver. High-dose nicotinamide supplementation increased fetal hepatic α-fetoprotein mRNA level, which was prevented by betaine supplementation. It is concluded that maternal nicotinamide supplementation can induce changes in fetal epigenetic modification and DNA base composition. The present study raises the concern that maternal nicotinamide supplementation may play a role in the development of epigenetic-related diseases in the offspring.

  3. Nucleotide sequence of yeast GDH1 encoding nicotinamide adenine dinucleotide phosphate-dependent glutamate dehydrogenase.

    PubMed

    Moye, W S; Amuro, N; Rao, J K; Zalkin, H

    1985-07-15

    The yeast GDH1 gene encodes NADP-dependent glutamate dehydrogenase. This gene was isolated by complementation of an Escherichia coli glutamate auxotroph. NADP-dependent glutamate dehydrogenase was overproduced 6-10-fold in Saccharomyces cerevisiae bearing GDH1 on a multicopy plasmid. The nucleotide sequence of the 1362-base pair coding region and 5' and 3' flanking sequences were determined. Transcription start sites were located by S1 nuclease mapping. Regulation of GDH1 was not maintained when the gene was present on a multicopy plasmid. Protein secondary structure predictions identified a region with potential to form the dinucleotide-binding domain. The amino acid sequences of the yeast and Neurospora crassa enzymes are 63% conserved. Unlike the N. crassa gene, yeast GDH1 has no introns.

  4. Gene encoding plant asparagine synthetase

    DOEpatents

    Coruzzi, Gloria M.; Tsai, Fong-Ying

    1993-10-26

    The identification and cloning of the gene(s) for plant asparagine synthetase (AS), an important enzyme involved in the formation of asparagine, a major nitrogen transport compound of higher plants is described. Expression vectors constructed with the AS coding sequence may be utilized to produce plant AS; to engineer herbicide resistant plants, salt/drought tolerant plants or pathogen resistant plants; as a dominant selectable marker; or to select for novel herbicides or compounds useful as agents that synchronize plant cells in culture. The promoter for plant AS, which directs high levels of gene expression and is induced in an organ specific manner and by darkness, is also described. The AS promoter may be used to direct the expression of heterologous coding sequences in appropriate hosts.

  5. Gene encoding acetyl-coenzyme A carboxylase

    DOEpatents

    Roessler, P.G.; Ohlrogge, J.B.

    1996-09-24

    A DNA encoding an acetyl-coenzyme A carboxylase (ACCase) from a photosynthetic organism and functional derivatives are disclosed which are resistant to inhibition from certain herbicides. This gene can be placed in organisms to increase their fatty acid content or to render them resistant to certain herbicides. 5 figs.

  6. Gene encoding acetyl-coenzyme A carboxylase

    DOEpatents

    Roessler, Paul G.; Ohlrogge, John B.

    1996-01-01

    A DNA encoding an acetyl-coenzyme A carboxylase (ACCase) from a photosynthetic organism and functional derivatives thereof which are resistant to inhibition from certain herbicides. This gene can be placed in organisms to increase their fatty acid content or to render them resistant to certain herbicides.

  7. Physiological Study on Association between Nicotinamide N-Methyltransferase Gene Polymorphisms and Hyperlipidemia

    PubMed Central

    Zhu, Xiao-Juan; Lin, Ya-Jun; Chen, Wei; Wang, Ya-Hui; Qiu, Li-Qiang; Cai, Can-Xin; Xiong, Qun; Chen, Fei; Chen, Li-Hui; Zhou, Qiong

    2016-01-01

    Nicotinamide N-methyltransferase (NNMT) catalyzes the methylation of nicotinamide. Our previous works indicate that NNMT is involved in the body mass index and energy metabolism, and recently the association between a SNP (rs694539) of NNMT and a variety of cardiovascular diseases was reported. At present, more than 200 NNMT single nucleotide polymorphisms (SNPs) have been identified in the databases of the human genome projects; however, the association between rs694539 variation and hyperlipidemia has not been reported yet, and whether there are any SNPs in NNMT significantly associated with hyperlipidemia is still unclear. In this paper, we selected 19 SNPs in NNMT as the tagSNPs using Haploview software (Haploview 4.2) first and then performed a case-control study to observe the association between these tagSNPs and hyperlipidemia and finally applied physiological approaches to explore the possible mechanisms through which the NNMT polymorphism induces hyperlipidemia. The results show that a SNP (rs1941404) in NNMT is significantly associated with hyperlipidemia, and the influence of rs1941404 variation on the resting energy expenditure may be the possible mechanism for rs1941404 variation to induce hyperlipidemia. PMID:27999813

  8. The First Gene-encoded Amphibian Neurotoxin*

    PubMed Central

    You, Dewen; Hong, Jing; Rong, Mingqiang; Yu, Haining; Liang, Songping; Ma, Yufang; Yang, Hailong; Wu, Jing; Lin, Donghai; Lai, Ren

    2009-01-01

    Many gene-encoded neurotoxins with various functions have been discovered in fish, reptiles, and mammals. A novel 60-residue neurotoxin peptide (anntoxin) that inhibited tetrodotoxin-sensitive (TTX-S) voltage-gated sodium channel (VGSC) was purified and characterized from the skin secretions of the tree frog Hyla annectans (Jerdon). This is the first gene-encoded neurotoxin found in amphibians. The IC50 of anntoxin for the TTX-S channel was about 3.4 μm. Anntoxin shares sequence homology with Kunitz-type toxins but contains only two of three highly conserved cysteine bridges, which are typically found in these small, basic neurotoxin modules, i.e. snake dendrotoxins. Anntoxin showed an inhibitory ability against trypsin with an inhibitory constant (Ki) of 0.025 μm. Anntoxin was distributed in skin, brain, stomach, and liver with a concentration of 25, 7, 3, and 2 μg/g wet tissue, respectively. H. annectans lives on trees or other plants for its entire life cycle, and its skin contains the largest amount of anntoxin, which possibly helps defend against various aggressors or predators. A low dose of anntoxin was found to induce lethal toxicity for several potential predators, including the insect, snake, bird, and mouse. The tissue distribution and functional properties of the current toxin may provide insights into the ecological adaptation of tree-living amphibians. PMID:19535333

  9. The gene encoding proopiomelanocortin in the dog.

    PubMed

    Mol, J A; van Mansfeld, A D; Kwant, M M; van Wolferen, M; Rothuizen, J

    1991-01-01

    The regulation of the synthesis of ACTH in the dog is of interest for studies of the physiology of the pituitary-adrenocortical axis as well as for studies of the pathogenesis of pituitary-dependent hyperadrenocorticism. Despite this broad interest the nucleotide sequence encoding ACTH and its precursor proopiomelanocortin (POMC) is not known, nor is it clear whether there are differences in POMC mRNA from the anterior lobe or the intermediate lobe of the normal pituitary or from pituitary tumours causing ACTH excess. Following the preparation of a cDNA library from the canine intermediate lobe of the pituitary gland, the part of the mRNA that is translated into the proopiomelanocortin prohormone was amplified using a polymerase chain reaction. Sequence analysis revealed the highest homology with the porcine mRNA sequence. Translation in a single reading frame revealed highly homologous areas in the amino-terminal, carboxy-terminal, and ACTH part of the prohormone, whereas a high diversity was noticed at the sequence preceding ACTH and the beginning of beta-lipotropin. Northern blot analysis disclosed the presence of a POMC mRNA of approximately 1300 nucleotides. There were no size differences between the anterior lobe, intermediate lobe, and pituitary tumour derived POMC mRNA. The highest expression levels of POMC mRNA as related to the expression of the gene encoding glyceraldehyde-3-phosphate dehydrogenase were found in the intermediate lobe of the canine pituitary gland. It is concluded that excessive production of ACTH by pituitary tumours is not caused by relatively high expression levels or alterations in the size of mRNA.

  10. Characterization of the gene encoding mouse serum amyloid P component. Comparison with genes encoding other pentraxins.

    PubMed Central

    Whitehead, A S; Rits, M

    1989-01-01

    A CBA/J-strain mouse serum amyloid P component (SAP) genomic clone was isolated and analysed. The clone contains the entire SAP gene and specifies a primary transcript of 1065 nucleotide residues. This comprises a first exon of 206 nucleotide residues containing the mRNA 5'-untranslated region and sequence encoding the pre-SAP leader peptide and the first two amino acid residues of mature SAP separated by a single 110-base intron from a 749-nucleotide-residue second exon containing sequence encoding the bulk of the mature SAP and specifying the mRNA 3'-untranslated region. The overall organization is similar to that of the human SAP gene, and the coding region and intron sequences are highly conserved. The SAP RNA cap site was defined by primer extension analysis of polyadenylated acute-phase liver RNA. The 5'-region of the mouse SAP gene contains modified CAAT and TATA promoter elements preceded by a putative hepatocyte-nuclear-factor-1-recognition site; these structures are in a region that is highly homologous to the corresponding region of the human SAP gene. Comparisons of the mouse SAP gene structure and derived amino acid sequence with those of other mammalian pentraxins were made. Images Fig. 3. PMID:2481440

  11. Genes Encoding Enzymes Involved in Ethanol Metabolism

    PubMed Central

    Hurley, Thomas D.; Edenberg, Howard J.

    2012-01-01

    The effects of beverage alcohol (ethanol) on the body are determined largely by the rate at which it and its main breakdown product, acetaldehyde, are metabolized after consumption. The main metabolic pathway for ethanol involves the enzymes alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH). Seven different ADHs and three different ALDHs that metabolize ethanol have been identified. The genes encoding these enzymes exist in different variants (i.e., alleles), many of which differ by a single DNA building block (i.e., single nucleotide polymorphisms [SNPs]). Some of these SNPs result in enzymes with altered kinetic properties. For example, certain ADH1B and ADH1C variants that are commonly found in East Asian populations lead to more rapid ethanol breakdown and acetaldehyde accumulation in the body. Because acetaldehyde has harmful effects on the body, people carrying these alleles are less likely to drink and have a lower risk of alcohol dependence. Likewise, an ALDH2 variant with reduced activity results in acetaldehyde buildup and also has a protective effect against alcoholism. In addition to affecting drinking behaviors and risk for alcoholism, ADH and ALDH alleles impact the risk for esophageal cancer. PMID:23134050

  12. Associations of nicotinamide N-methyltransferase gene single nucleotide polymorphisms with sport performance and relative maximal oxygen uptake.

    PubMed

    Li, Jiang-Hua; Chen, Wei; Zhu, Xiao-Juan; Lin, Ya-Jun; Qiu, Li-Qiang; Cai, Can-Xin; Wang, Ya-Hui; Xiong, Qun; Chen, Fei; Chen, Li-Hui

    2016-11-30

    To observe the associations between single nucleotide polymorphisms (SNPs) of nicotinamide N-methyltransferase (NNMT) gene and sport performance and to analyse genotype associations of the associated SNPs with sport performance and relative maximal oxygen uptake ([Formula: see text]). Participants were selected from 685 Chinese Han male college students. The completion times of a 1000-m run and a 50-m run were used to reflect sport performance, respectively. Nineteen tagSNPs were genotyped with Polymerase chain reaction-ligase detection reaction. Relative [Formula: see text] was directly determined with a cardiopulmonary function analyser. A significant association was found between rs2256292 and 1000-m run performance, but no significant association was found between any tagSNPs and 50-m run performance. The genotype associations of rs2256292 with 1000-m run performance and with relative [Formula: see text] were both significant under the recessive model (CC vs. CG + GG). No tagSNP in NNMT is significantly associated with 50-m run performance but rs2256292 is significantly associated with 1000-m run performance. The genotype associations of rs2256292 with sport performance are significant under recessive model, and a higher relative [Formula: see text] may be the physiological reason for minor homozygote CC carriers being of the better 1000-m runners.

  13. Mosaic tetracycline resistance genes encoding ribosomal protection proteins.

    PubMed

    Warburton, Philip J; Amodeo, Nina; Roberts, Adam P

    2016-12-01

    First reported in 2003, mosaic tetracycline resistance genes are a subgroup of the genes encoding ribosomal protection proteins (RPPs). They are formed when two or more RPP-encoding genes recombine resulting in a functional chimera. To date, the majority of mosaic genes are derived from sections of three RPP genes, tet(O), tet(W) and tet(32), with others comprising tet(M) and tet(S). In this first review of mosaic genes, we report on their structure, diversity and prevalence, and suggest that these genes may be responsible for an under-reported contribution to tetracycline resistance in bacteria.

  14. Mosaic tetracycline resistance genes encoding ribosomal protection proteins

    PubMed Central

    Warburton, Philip J.; Amodeo, Nina; Roberts, Adam P.

    2016-01-01

    First reported in 2003, mosaic tetracycline resistance genes are a subgroup of the genes encoding ribosomal protection proteins (RPPs). They are formed when two or more RPP-encoding genes recombine resulting in a functional chimera. To date, the majority of mosaic genes are derived from sections of three RPP genes, tet(O), tet(W) and tet(32), with others comprising tet(M) and tet(S). In this first review of mosaic genes, we report on their structure, diversity and prevalence, and suggest that these genes may be responsible for an under-reported contribution to tetracycline resistance in bacteria. PMID:27494928

  15. Activation of nicotinamide N-methyltransferase gene promoter by hepatocyte nuclear factor-1beta in human papillary thyroid cancer cells.

    PubMed

    Xu, Jimin; Capezzone, Marco; Xu, Xiao; Hershman, Jerome M

    2005-02-01

    We previously demonstrated that the human nicotinamide N-methytransferase (NNMT) gene was highly expressed in many papillary thyroid cancers and cell lines. The expression in other papillary and follicular cancers or cell lines and normal thyroid cells was low or undetectable. To gain an understanding of the molecular mechanism of this cell-specific expression, the NNMT promoter was cloned and studied by luciferase reporter gene assay. The promoter construct was expressed highly in papillary cancer cell lines, including those with higher (e.g. BHP 2-7) and lower (e.g. BHP 14-9) NNMT gene expression, and expressed weakly in follicular thyroid cancer cell lines. Further study with 5'-deletion promoter construct suggested that the NNMT promoter was regulated differently in BHP 2-7 and BHP 14-9 cells. In BHP 2-7 cells, promoter activity was dependent on an upstream sequence. In BHP 14-9 cells, sequence in the basal promoter region contributed notably to the overall promoter activity. RT-PCR or Western blot analysis indicated that hepatocyte nuclear factor-1beta (HNF-1beta) was expressed in only papillary cancer cell lines with high NNMT gene expression. HNF-1beta was not expressed or expressed very weakly in other papillary, follicular, and Hurthle cancer cell lines and primary cultures of normal thyroid cells and benign thyroid conditions. A HNF-1 binding site was identified in the NNMT basal promoter region. Mutations in this site decreased NNMT promoter activity in the HNF-1beta-positive BHP 2-7 cells, but not in the HNF-1beta-negative BHP 14-9 cells. HNF-1beta bound to the HNF-1 site specifically as a homodimer as determined by gel retardation assays with HNF-1beta-specific antibody. Cotransfection of a HNF-1beta expression plasmid increased NNMT promoter activity significantly in both HNF-1beta-positive and -negative thyroid cancer cell lines and Hep G2 liver cancer cells. Furthermore, transient expression of HNF-1beta in BHP 14-9 cells increased endogenous NNMT

  16. Gene encoding herbicide safener binding protein

    SciTech Connect

    Walton, J.D.; Scott-Craig, J.S.

    1999-10-26

    The cDNA encoding safener binding protein (SafBP), also referred to as SBP1, is presented. The deduced amino acid sequence is provided. Methods of making and using SBP1 and SafBP to alter a plant's sensitivity to certain herbicides or a plant's responsiveness to certain safeners are also provided, as well as expression vectors, transgenic plants or other organisms transfected with vectors and seeds from the plants.

  17. Gene encoding herbicide safener binding protein

    SciTech Connect

    Walton, Jonathan D.; Scott-Craig, John S.

    1999-01-01

    The cDNA encoding safener binding protein (SafBP), also referred to as SBP1, is set forth in FIG. 5 and SEQ ID No. 1. The deduced amino acid sequence is provided in FIG. 5 and SEQ ID No. 2. Methods of making and using SBP1 and SafBP to alter a plant's sensitivity to certain herbicides or a plant's responsiveness to certain safeners are also provided, as well as expression vectors, transgenic plants or other organisms transfected with said vectors and seeds from said plants.

  18. The Riemerella anatipestifer AS87_01735 Gene Encodes Nicotinamidase PncA, an Important Virulence Factor

    PubMed Central

    Wang, Xiaolan; Liu, Beibei; Dou, Yafeng; Fan, Hongjie; Wang, Shaohui; Li, Tao; Ding, Chan

    2016-01-01

    ABSTRACT Riemerella anatipestifer is a major bacterial pathogen that causes septicemic and exudative diseases in domestic ducks. In our previous study, we found that deletion of the AS87_01735 gene significantly decreased the bacterial virulence of R. anatipestifer strain Yb2 (mutant RA625). The AS87_01735 gene was predicted to encode a nicotinamidase (PncA), a key enzyme that catalyzes the conversion of nicotinamide to nicotinic acid, which is an important reaction in the NAD+ salvage pathway. In this study, the AS87_01735 gene was expressed and identified as the PncA-encoding gene, using an enzymatic assay. Western blot analysis demonstrated that R. anatipestifer PncA was localized to the cytoplasm. The mutant strain RA625 (named Yb2ΔpncA in this study) showed a similar growth rate but decreased NAD+ quantities in both the exponential and stationary phases in tryptic soy broth culture, compared with the wild-type strain Yb2. In addition, Yb2ΔpncA-infected ducks showed much lower bacterial loads in their blood, and no visible histological changes were observed in the heart, liver, and spleen. Furthermore, Yb2ΔpncA immunization of ducks conferred effective protection against challenge with the virulent wild-type strain Yb2. Our results suggest that the R. anatipestifer AS87_01735 gene encodes PncA, which is an important virulence factor, and that the Yb2ΔpncA mutant can be used as a novel live vaccine candidate. IMPORTANCE Riemerella anatipestifer is reported worldwide as a cause of septicemic and exudative diseases of domestic ducks. The pncA gene encodes a nicotinamidase (PncA), a key enzyme that catalyzes the conversion of nicotinamide to nicotinic acid, which is an important reaction in the NAD+ salvage pathway. In this study, we identified and characterized the pncA-homologous gene AS87_01735 in R. anatipestifer strain Yb2. R. anatipestifer PncA is a cytoplasmic protein that possesses similar PncA activity, compared with other organisms. Generation of the

  19. Trichoderma asperellum Chi42 Genes Encode Chitinase

    PubMed Central

    Quang, Hoang Tan; Hung, Nguyen Bao; Huy, Nguyen Duc; Phuong, Truong Thi Bich; Ha, Tran Thi Thu

    2011-01-01

    Four Trichoderma strains (CH2, SH16, PQ34, and TN42) were isolated from soil samples collected from Quang Tri and Thua Thien Hue provinces in Vietnam. The strains exhibited high chitinolytic secretion. Strain PQ34 formed the largest zone of chitinase-mediated clearance (> 4 cm in diameter) in agar containing 1% (w/v) colloidal chitin. Analysis of the internal transcribed spacer regions of these strains indicated that they were Trichoderma asperellum. The molecular weights of the chitinases were approximately 42 kDa. Chitinase genes (chi42) of T. asperellum strains TN42, CH2, SH16, and PQ34 were 98~99% homologous to the ech42 gene of T. harzianum CB-Pin-01 (accession No. DQ166036). The deduced amino acid sequences of both T. asperellum strains SH16 and TN42 shared 100% similarity. PMID:22783101

  20. Bioinformatics analysis and detection of gelatinase encoded gene in Lysinibacillussphaericus

    NASA Astrophysics Data System (ADS)

    Repin, Rul Aisyah Mat; Mutalib, Sahilah Abdul; Shahimi, Safiyyah; Khalid, Rozida Mohd.; Ayob, Mohd. Khan; Bakar, Mohd. Faizal Abu; Isa, Mohd Noor Mat

    2016-11-01

    In this study, we performed bioinformatics analysis toward genome sequence of Lysinibacillussphaericus (L. sphaericus) to determine gene encoded for gelatinase. L. sphaericus was isolated from soil and gelatinase species-specific bacterium to porcine and bovine gelatin. This bacterium offers the possibility of enzymes production which is specific to both species of meat, respectively. The main focus of this research is to identify the gelatinase encoded gene within the bacteria of L. Sphaericus using bioinformatics analysis of partially sequence genome. From the research study, three candidate gene were identified which was, gelatinase candidate gene 1 (P1), NODE_71_length_93919_cov_158.931839_21 which containing 1563 base pair (bp) in size with 520 amino acids sequence; Secondly, gelatinase candidate gene 2 (P2), NODE_23_length_52851_cov_190.061386_17 which containing 1776 bp in size with 591 amino acids sequence; and Thirdly, gelatinase candidate gene 3 (P3), NODE_106_length_32943_cov_169.147919_8 containing 1701 bp in size with 566 amino acids sequence. Three pairs of oligonucleotide primers were designed and namely as, F1, R1, F2, R2, F3 and R3 were targeted short sequences of cDNA by PCR. The amplicons were reliably results in 1563 bp in size for candidate gene P1 and 1701 bp in size for candidate gene P3. Therefore, the results of bioinformatics analysis of L. Sphaericus resulting in gene encoded gelatinase were identified.

  1. Human germline antibody gene segments encode polyspecific antibodies.

    PubMed

    Willis, Jordan R; Briney, Bryan S; DeLuca, Samuel L; Crowe, James E; Meiler, Jens

    2013-04-01

    Structural flexibility in germline gene-encoded antibodies allows promiscuous binding to diverse antigens. The binding affinity and specificity for a particular epitope typically increase as antibody genes acquire somatic mutations in antigen-stimulated B cells. In this work, we investigated whether germline gene-encoded antibodies are optimal for polyspecificity by determining the basis for recognition of diverse antigens by antibodies encoded by three VH gene segments. Panels of somatically mutated antibodies encoded by a common VH gene, but each binding to a different antigen, were computationally redesigned to predict antibodies that could engage multiple antigens at once. The Rosetta multi-state design process predicted antibody sequences for the entire heavy chain variable region, including framework, CDR1, and CDR2 mutations. The predicted sequences matched the germline gene sequences to a remarkable degree, revealing by computational design the residues that are predicted to enable polyspecificity, i.e., binding of many unrelated antigens with a common sequence. The process thereby reverses antibody maturation in silico. In contrast, when designing antibodies to bind a single antigen, a sequence similar to that of the mature antibody sequence was returned, mimicking natural antibody maturation in silico. We demonstrated that the Rosetta computational design algorithm captures important aspects of antibody/antigen recognition. While the hypervariable region CDR3 often mediates much of the specificity of mature antibodies, we identified key positions in the VH gene encoding CDR1, CDR2, and the immunoglobulin framework that are critical contributors for polyspecificity in germline antibodies. Computational design of antibodies capable of binding multiple antigens may allow the rational design of antibodies that retain polyspecificity for diverse epitope binding.

  2. Selection for Genes Encoding Secreted Proteins and Receptors

    NASA Astrophysics Data System (ADS)

    Klein, Robert D.; Gu, Qimin; Goddard, Audrey; Rosenthal, Arnon

    1996-07-01

    Extracellular proteins play an essential role in the formation, differentiation, and maintenance of multicellular organisms. Despite that, the systematic identification of genes encoding these proteins has not been possible. We describe here a highly efficient method to isolate genes encoding secreted and membrane-bound proteins by using a single-step selection in yeast. Application of this method, termed signal peptide selection, to various tissues yielded 559 clones that appear to encode known or novel extracellular proteins. These include members of the transforming growth factor and epidermal growth factor protein families, endocrine hormones, tyrosine kinase receptors, serine/threonine kinase receptors, seven transmembrane receptors, cell adhesion molecules, extracellular matrix proteins, plasma proteins, and ion channels. The eventual identification of most, or all, extracellular signaling molecules will advance our understanding of fundamental biological processes and our ability to intervene in disease states.

  3. Gene Cluster Encoding Cholate Catabolism in Rhodococcus spp.

    PubMed Central

    Wilbrink, Maarten H.; Casabon, Israël; Stewart, Gordon R.; Liu, Jie; van der Geize, Robert; Eltis, Lindsay D.

    2012-01-01

    Bile acids are highly abundant steroids with important functions in vertebrate digestion. Their catabolism by bacteria is an important component of the carbon cycle, contributes to gut ecology, and has potential commercial applications. We found that Rhodococcus jostii RHA1 grows well on cholate, as well as on its conjugates, taurocholate and glycocholate. The transcriptome of RHA1 growing on cholate revealed 39 genes upregulated on cholate, occurring in a single gene cluster. Reverse transcriptase quantitative PCR confirmed that selected genes in the cluster were upregulated 10-fold on cholate versus on cholesterol. One of these genes, kshA3, encoding a putative 3-ketosteroid-9α-hydroxylase, was deleted and found essential for growth on cholate. Two coenzyme A (CoA) synthetases encoded in the cluster, CasG and CasI, were heterologously expressed. CasG was shown to transform cholate to cholyl-CoA, thus initiating side chain degradation. CasI was shown to form CoA derivatives of steroids with isopropanoyl side chains, likely occurring as degradation intermediates. Orthologous gene clusters were identified in all available Rhodococcus genomes, as well as that of Thermomonospora curvata. Moreover, Rhodococcus equi 103S, Rhodococcus ruber Chol-4 and Rhodococcus erythropolis SQ1 each grew on cholate. In contrast, several mycolic acid bacteria lacking the gene cluster were unable to grow on cholate. Our results demonstrate that the above-mentioned gene cluster encodes cholate catabolism and is distinct from a more widely occurring gene cluster encoding cholesterol catabolism. PMID:23024343

  4. Chlorella viruses contain genes encoding a complete polyamine biosynthetic pathway

    PubMed Central

    Baumann, Sascha; Sander, Adrianne; Gurnon, James R.; Yanai-Balser, Giane; VanEtten, James L.; Piotrowski, Markus

    2007-01-01

    Two genes encoding the putative polyamine biosynthetic enzymes agmatine iminohydrolase (AIH) and N-carbamoylputrescine amidohydrolase (CPA) were cloned from the chloroviruses PBCV-1, NY-2A and MT325. They were expressed in Escherichia coli to form C-terminal (His)6-tagged proteins and the recombinant proteins were purified by Ni2+- binding affinity chromatography. The biochemical properties of the two enzymes are similar to AIH and CPA enzymes from Arabidopsis thaliana and Pseudomonas aeruginosa. Together with the previously known virus genes encoding ornithine/arginine decarboxlyase (ODC/ADC) and homospermidine synthase, the chloroviruses have genes that encode a complete set of functional enzymes that synthesize the rare polyamine homospermidine from arginine via agmatine, N-carbamoylputrescine and putrescine. The PBCV-1 aih and cpa genes are expressed early during virus infection together with the odc/adc gene, suggesting that biosynthesis of putrescine is important in early stages of viral replication. The aih and cpa genes are widespread in the chlorella viruses. PMID:17101165

  5. NadN and e (P4) are essential for utilization of NAD and nicotinamide mononucleotide but not nicotinamide riboside in Haemophilus influenzae.

    PubMed

    Kemmer, G; Reilly, T J; Schmidt-Brauns, J; Zlotnik, G W; Green, B A; Fiske, M J; Herbert, M; Kraiss, A; Schlör, S; Smith, A; Reidl, J

    2001-07-01

    Haemophilus influenzae has an absolute requirement for NAD (factor V) because it lacks almost all the biosynthetic enzymes necessary for the de novo synthesis of that cofactor. Factor V can be provided as either nicotinamide adenosine dinucleotide (NAD), nicotinamide mononucleotide (NMN), or nicotinamide riboside (NR) in vitro, but little is known about the source or the mechanism of uptake of these substrates in vivo. As shown by us earlier, at least two gene products are involved in the uptake of NAD, the outer membrane lipoprotein e (P4), which has phosphatase activity and is encoded by hel, and a periplasmic NAD nucleotidase, encoded by nadN. It has also been observed that the latter gene product is essential for H. influenzae growth on media supplemented with NAD. In this report, we describe the functions and substrates of these two proteins as they act together in an NAD utilization pathway. Data are provided which indicate that NadN harbors not only NAD pyrophosphatase but also NMN 5'-nucleotidase activity. The e (P4) protein is also shown to have NMN 5'-nucleotidase activity, recognizing NMN as a substrate and releasing NR as its product. Insertion mutants of nadN or deletion and site-directed mutants of hel had attenuated growth and a reduced uptake phenotype when NMN served as substrate. A hel and nadN double mutant was only able to grow in the presence of NR, whereas no uptake of NMN was observed.

  6. The murine Sry gene encodes a nuclear transcriptional activator

    SciTech Connect

    Dubin, R.A.; Ostrer, H.

    1994-09-01

    The Sry gene functions as a genetic switch in gonadal ridge initiating testis determination. The murine Sry and human SRY open reading frames (ORF) share a conserved 79 amino acid motif, the HMG-box, that binds DNA. Outside this region the two genes share no additional homology. These studies were undertaken to determine whether the Sry/SRY genes encode nuclear transcriptional regulators. As judged by the accumulation of lacZ-SRY hybrid proteins in the nucleus, both the human and murine SRY ORFs contain a nuclear localization signal. The murine Sry HMG-box selectively binds the sequence NACAAT in vitro when presented with a random pool of oligonucleotides and binds AACAAT with the highest affinity. The murine Sry ORF, when expressed in HeLa cells, activates transcription of a reporter gene containing multiple copies of the AACAAT binding site. Activation was observed for a GAL4-responsive gene when the murine Sry ORF was linked to the DNA-binding domain of GAL4. Using this system, the activation function was mapped to a C-terminal glutamine/histidine-rich domain. In addition, LexA-Sry fusion genes activated a LexA-responsive gene in yeast. In contrast, a GAL4-human SRY fusion gene did not cause transcriptional activation. These studies suggest that both the human and mouse SRY ORFs encode nuclear, DNA-binding proteins, and that the mouse Sry ORF can function as a transcriptional activator with separable DNA-binding and activator domains.

  7. (Genetic engineering with a gene encoding a soybean storage protein)

    SciTech Connect

    Beachy, R.N.

    1985-12-18

    We have isolated and characterized a gene which encodes the alpha prime subunit of beta conglycinin. This gene was fully sequenced by DNA sequence analysis and a report of that work was prepared and submitted for publication in early November 1985. This represented the culmination of several years of research effort by several scientists. A preprint of that work is attached to this report and has been offered by Dr. J.J. Doyle, Dr. Mary A. Schuler and Dr. Jerry Slighton, as well as myself. This paper is a comparison of the alpha prime subunit gene with a similar gene from phaseolus vulgaris, the common garden bean. In this paper we compare the sequences that are 5' of the gene, and which would represent the transcriptional promoter, as well as the sequences within the structural region of the gene. The sequence paper also compares the amino acid sequence of these two genes with that of other genes from Phaseolus, peas and from soybeans. On the basis of this comparison, we predict evolutionary trends within the multigene families which encode these proteins in the various plants, as well as to look at the protein itself to try to predict regions of the protein that might have functional significance. All of this work was done on a prior DOE-BER grant and has simply been reported here for the first time.

  8. The Schizosaccharomyces pombe cho1+ gene encodes a phospholipid methyltransferase.

    PubMed Central

    Kanipes, M I; Hill, J E; Henry, S A

    1998-01-01

    The isolation of mutants of Schizosaccharomyces pombe defective in the synthesis of phosphatidylcholine via the methylation of phosphatidylethanolamine is reported. These mutants are choline auxotrophs and fall into two unlinked complementation groups, cho1 and cho2. We also report the analysis of the cho1+ gene, the first structural gene encoding a phospholipid biosynthetic enzyme from S. pombe to be cloned and characterized. The cho1+ gene disruption mutant (cho1Delta) is viable if choline is supplied and resembles the cho1 mutants isolated after mutagenesis. Sequence analysis of the cho1+ gene indicates that it encodes a protein closely related to phospholipid methyltransferases from Saccharomyces cerevisiae and rat. Phospholipid methyltransferases encoded by a rat liver cDNA and the S. cerevisiae OPI3 gene are both able to complement the choline auxotrophy of the S. pombe cho1 mutants. These results suggest that both the structure and function of the phospholipid N-methyltransferases are broadly conserved among eukaryotic organisms. PMID:9755189

  9. Discoveries of nicotinamide riboside as a nutrient and conserved NRK genes establish a Preiss-Handler independent route to NAD+ in fungi and humans.

    PubMed

    Bieganowski, Pawel; Brenner, Charles

    2004-05-14

    NAD+ is essential for life in all organisms, both as a coenzyme for oxidoreductases and as a source of ADPribosyl groups used in various reactions, including those that retard aging in experimental systems. Nicotinic acid and nicotinamide were defined as the vitamin precursors of NAD+ in Elvehjem's classic discoveries of the 1930s. The accepted view of eukaryotic NAD+ biosynthesis, that all anabolism flows through nicotinic acid mononucleotide, was challenged experimentally and revealed that nicotinamide riboside is an unanticipated NAD+ precursor in yeast. Nicotinamide riboside kinases from yeast and humans essential for this pathway were identified and found to be highly specific for phosphorylation of nicotinamide riboside and the cancer drug tiazofurin. Nicotinamide riboside was discovered as a nutrient in milk, suggesting that nicotinamide riboside is a useful compound for elevation of NAD+ levels in humans.

  10. Nicotinamide and the skin.

    PubMed

    Chen, Andrew C; Damian, Diona L

    2014-08-01

    Nicotinamide, an amide form of vitamin B3, boosts cellular energy and regulates poly-ADP-ribose-polymerase 1, an enzyme with important roles in DNA repair and the expression of inflammatory cytokines. Nicotinamide shows promise for the treatment of a wide range of dermatological conditions, including autoimmune blistering disorders, acne, rosacea, ageing skin and atopic dermatitis. In particular, recent studies have also shown it to be a potential agent for reducing actinic keratoses and preventing skin cancers.

  11. Identification and use of genes encoding amatoxin and phallotoxin

    SciTech Connect

    Hallen, Heather E.; Walton, Jonathan D.; Luo, Hong; Scott-Craig, John S.

    2016-12-13

    The present invention relates to compositions and methods comprising genes and peptides associated with cyclic peptide toxins and toxin production in mushrooms. In particular, the present invention relates to using genes and proteins from Amanita species encoding Amanita peptides, specifically relating to amatoxins and phallotoxins. In a preferred embodiment, the present invention also relates to methods for detecting Amanita peptide toxin genes for identifying Amanita peptide-producing mushrooms and for diagnosing suspected cases of mushroom poisoning. Further, the present inventions relate to providing kits for diagnosing and monitoring suspected cases of mushroom poisoning in patients.

  12. Clustered Genes Encoding the Methyltransferases of Methanogenesis from Monomethylamine

    PubMed Central

    Burke, Stephen A.; Lo, Sam L.; Krzycki, Joseph A.

    1998-01-01

    Coenzyme M (CoM) is methylated during methanogenesis from monomethyamine in a reaction catalyzed by three proteins. Using monomethylamine, a 52-kDa polypeptide termed monomethylamine methyltransferase (MMAMT) methylates the corrinoid cofactor bound to a second polypeptide, monomethylamine corrinoid protein (MMCP). Methylated MMCP then serves as a substrate for MT2-A, which methylates CoM. The genes for these proteins are clustered on 6.8 kb of DNA in Methanosarcina barkeri MS. The gene encoding MMCP (mtmC) is located directly upstream of the gene encoding MMAMT (mtmB). The gene encoding MT2-A (mtbA) was found 1.1 kb upstream of mtmC, but no obvious open reading frame was found in the intergenic region between mtbA and mtmC. A single monocistronic transcript was found for mtbA that initiated 76 bp from the translational start. Separate transcripts of 2.4 and 4.7 kb were detected, both of which carried mtmCB. The larger transcript also encoded mtmP, which is homologous to the APC family of cationic amine permeases and may therefore encode a methylamine permease. A single transcriptional start site was found 447 bp upstream of the translational start of mtmC. MtmC possesses the corrinoid binding motif found in corrinoid proteins involved in dimethylsulfide- and methanol-dependent methanogenesis, as well as in methionine synthase. The open reading frame of mtmB was interrupted by a single in-frame, midframe, UAG codon which was also found in mtmB from M. barkeri NIH. A mechanism that circumvents UAG-directed termination of translation must operate during expression of mtmB in this methanogen. PMID:9642198

  13. Clustered genes encoding the methyltransferases of methanogenesis from monomethylamine.

    PubMed

    Burke, S A; Lo, S L; Krzycki, J A

    1998-07-01

    Coenzyme M (CoM) is methylated during methanogenesis from monomethyamine in a reaction catalyzed by three proteins. Using monomethylamine, a 52-kDa polypeptide termed monomethylamine methyltransferase (MMAMT) methylates the corrinoid cofactor bound to a second polypeptide, monomethylamine corrinoid protein (MMCP). Methylated MMCP then serves as a substrate for MT2-A, which methylates CoM. The genes for these proteins are clustered on 6.8 kb of DNA in Methanosarcina barkeri MS. The gene encoding MMCP (mtmC) is located directly upstream of the gene encoding MMAMT (mtmB). The gene encoding MT2-A (mtbA) was found 1.1 kb upstream of mtmC, but no obvious open reading frame was found in the intergenic region between mtbA and mtmC. A single monocistronic transcript was found for mtbA that initiated 76 bp from the translational start. Separate transcripts of 2.4 and 4.7 kb were detected, both of which carried mtmCB. The larger transcript also encoded mtmP, which is homologous to the APC family of cationic amine permeases and may therefore encode a methylamine permease. A single transcriptional start site was found 447 bp upstream of the translational start of mtmC. MtmC possesses the corrinoid binding motif found in corrinoid proteins involved in dimethylsulfide- and methanol-dependent methanogenesis, as well as in methionine synthase. The open reading frame of mtmB was interrupted by a single in-frame, midframe, UAG codon which was also found in mtmB from M. barkeri NIH. A mechanism that circumvents UAG-directed termination of translation must operate during expression of mtmB in this methanogen.

  14. Identification of β-haemolysin-encoding genes in Streptococcus anginosus.

    PubMed

    Asam, D; Mauerer, S; Walheim, E; Spellerberg, B

    2013-08-01

    Streptococcus anginosus is an emerging pathogen, but little is known about its virulence factors. To detect the genes responsible for β-haemolysis we performed genomic mutagenesis of the β-haemolytic S. anginosus type strain ATCC 12395 using the vector pGhost9:ISS1. Integration site analysis of 15 non-haemolytic mutants identified a gene cluster with high homology to the genes of the streptolysin S (SLS) encoding sag gene cluster of S. pyogenes. The gene cluster harbours 10 open reading frames displaying significant similarities to the S. pyogenes genes sagA-sagI, with the identities on protein level ranging from 38 to 87%. Complementation assays of S. anginosus sagB and sagD integration mutants with the respective genes confirmed their importance for β-haemolysin production and suggest the presence of post-translational modifications in S. anginosus SLS similar to SLS of S. pyogenes. Characterization of the S. anginosus haemolysin in comparison to the S. pyogenes SLS showed that the haemolysin is surface bound, but in contrast to S. pyogenes neither fetal calf serum nor RNA was able to stabilize the haemolysin of S. anginosus in culture supernatants. Inhibition of β-haemolysis by polyethylene glycol of different sizes was carried out, giving no evidence of a pore-forming haemolytic mechanism. Analysis of a whole genome shotgun sequence of Streptococcus constellatus, a closely related streptococcal species that belongs to the S. anginosus group, revealed a similar sag gene cluster. Employing a genomic mutagenesis strategy we were able to determine an SLS encoding gene cluster in S. anginosus and demonstrate its importance for β-haemolysin production in S. anginosus.

  15. The sulfolobicin genes of Sulfolobus acidocaldarius encode novel antimicrobial proteins.

    PubMed

    Ellen, Albert F; Rohulya, Olha V; Fusetti, Fabrizia; Wagner, Michaela; Albers, Sonja-Verena; Driessen, Arnold J M

    2011-09-01

    Crenarchaea, such as Sulfolobus acidocaldarius and Sulfolobus tokodaii, produce antimicrobial proteins called sulfolobicins. These antimicrobial proteins inhibit the growth of closely related species. Here we report the identification of the sulfolobicin-encoding genes in S. acidocaldarius. The active sulfolobicin comprises two proteins that are equipped with a classical signal sequence. These proteins are secreted by the cells and found to be membrane vesicle associated. Gene inactivation studies demonstrate that both proteins are required for the bacteriostatic antimicrobial activity. Sulfolobicins constitute a novel class of antimicrobial proteins without detectable homology to any other protein.

  16. Construction of adiponectin-encoding plasmid DNA and gene therapy of non-obese type 2 diabetes mellitus.

    PubMed

    Nan, Mei Hua; Park, Jeong-Sook; Myung, Chang-Seon

    2010-01-01

    Adiponectin (ADN), an insulin-sensitizing adipokine, stimulates glucose uptake, inhibits gluconeogenesis, and plays an important role in improving insulin sensitivity. Since blood levels of ADN are low in type 2 diabetes mellitus (DM), this study was designed to investigate the therapeutic effectiveness of increasing the ADN level through injection of plasmid DNA encoding ADN in type 2 DM. A non-obese type 2 DM mouse model was established via combined administration of streptozotocin with nicotinamide and exhibited significantly higher plasma glucose concentration and insulin resistance compared with normal controls according to oral glucose tolerance and insulin challenge tests. Plasmid DNA encoding mouse ADN from differentiated NIH3T3 adipocytes was constructed in pVAX1 (pVAX/ADN). Transfection of pVAX/ADN into various cell lines including HeLa, HT22, HEK293, HepG2, and SK-Hep1 cells, increased ADN mRNA expression levels in a dose-dependent manner. The administration of pVAX/ADN into non-obese type 2 DM mice via tail vein significantly increased the blood level of ADN and decreased the plasma glucose concentration. Moreover, the parameters related to insulin resistance (HOMA-IR) and insulin sensitivity (QUICKI) were significantly improved. These results suggest that ADN gene therapy could be a clinically effective tool for the treatment of type 2 DM.

  17. Nicotinamide ribosyl uptake mutants in Haemophilus influenzae.

    PubMed

    Herbert, Mark; Sauer, Elizabeta; Smethurst, Graeme; Kraiss, Anita; Hilpert, Anna-Karina; Reidl, Joachim

    2003-09-01

    The gene for the nicotinamide riboside (NR) transporter (pnuC) was identified in Haemophilus influenzae. A pnuC mutant had only residual NR uptake and could survive in vitro with high concentrations of NR, but could not survive in vivo. PnuC may represent a target for the development of inhibitors for preventing H. influenzae disease.

  18. Photoprotective effects of nicotinamide.

    PubMed

    Damian, Diona L

    2010-04-01

    Sun protective measures can reduce numbers of both precancerous actinic keratoses and cutaneous squamous cell carcinomas within relatively short periods of time even in high-risk populations. Sunscreens, which tend to provide greater protection against shortwave UVB than against longer wavelength UVA radiation, can however provide only partial protection from the mutagenic and immune suppressive effects of sunlight. In large part, this reflects poor compliance with proper sunscreen application and reapplication. Skin cancer is by far the most common malignancy in Caucasian populations, and additional strategies to reduce the morbidity and economic burden of this disease are now urgently needed. Nicotinamide, the amide form of vitamin B3, is an inexpensive agent which is used for a variety of dermatological applications with little or no toxicity even at high doses. Nicotinamide has photoprotective effects against carcinogenesis and immune suppression in mice, and is photoimmunoprotective in humans when used as a lotion or orally. UV irradiation depletes keratinocytes of cellular energy and nicotinamide, which is a precursor of nicotinamide adenine dinucleotide, may act at least in part by providing energy repletion to irradiated cells.

  19. 'Green revolution' genes encode mutant gibberellin response modulators.

    PubMed

    Peng, J; Richards, D E; Hartley, N M; Murphy, G P; Devos, K M; Flintham, J E; Beales, J; Fish, L J; Worland, A J; Pelica, F; Sudhakar, D; Christou, P; Snape, J W; Gale, M D; Harberd, N P

    1999-07-15

    World wheat grain yields increased substantially in the 1960s and 1970s because farmers rapidly adopted the new varieties and cultivation methods of the so-called 'green revolution'. The new varieties are shorter, increase grain yield at the expense of straw biomass, and are more resistant to damage by wind and rain. These wheats are short because they respond abnormally to the plant growth hormone gibberellin. This reduced response to gibberellin is conferred by mutant dwarfing alleles at one of two Reduced height-1 (Rht-B1 and Rht-D1) loci. Here we show that Rht-B1/Rht-D1 and maize dwarf-8 (d8) are orthologues of the Arabidopsis Gibberellin Insensitive (GAI) gene. These genes encode proteins that resemble nuclear transcription factors and contain an SH2-like domain, indicating that phosphotyrosine may participate in gibberellin signalling. Six different orthologous dwarfing mutant alleles encode proteins that are altered in a conserved amino-terminal gibberellin signalling domain. Transgenic rice plants containing a mutant GAI allele give reduced responses to gibberellin and are dwarfed, indicating that mutant GAI orthologues could be used to increase yield in a wide range of crop species.

  20. Gene family encoding the major toxins of lethal Amanita mushrooms.

    PubMed

    Hallen, Heather E; Luo, Hong; Scott-Craig, John S; Walton, Jonathan D

    2007-11-27

    Amatoxins, the lethal constituents of poisonous mushrooms in the genus Amanita, are bicyclic octapeptides. Two genes in A. bisporigera, AMA1 and PHA1, directly encode alpha-amanitin, an amatoxin, and the related bicyclic heptapeptide phallacidin, a phallotoxin, indicating that these compounds are synthesized on ribosomes and not by nonribosomal peptide synthetases. alpha-Amanitin and phallacidin are synthesized as proproteins of 35 and 34 amino acids, respectively, from which they are predicted to be cleaved by a prolyl oligopeptidase. AMA1 and PHA1 are present in other toxic species of Amanita section Phalloidae but are absent from nontoxic species in other sections. The genomes of A. bisporigera and A. phalloides contain multiple sequences related to AMA1 and PHA1. The predicted protein products of this family of genes are characterized by a hypervariable "toxin" region capable of encoding a wide variety of peptides of 7-10 amino acids flanked by conserved sequences. Our results suggest that these fungi have a broad capacity to synthesize cyclic peptides on ribosomes.

  1. [Chronic nicotinamide overload and type 2 diabetes].

    PubMed

    Zhou, Shi-Sheng; Li, Da; Zhou, Yi-Ming; Sun, Wu-Ping; Liu, Xing-Xing; Lun, Yong-Zhi

    2010-02-25

    Type 2 diabetes is a major global health problem. It is generally accepted that type 2 diabetes is the result of gene-environmental interaction. However, the mechanism underlying the interaction is unclear. Diet change is known to play an important role in type 2 diabetes. The fact that the global high prevalence of type 2 diabetes has occurred following the spread of food fortification worldwide suggests a possible involvement of excess niacin intake. Our recent study found that nicotinamide overload and low nicotinamide detoxification may induce oxidative stress associated with insulin resistance. Based on the relevant facts, this review briefly summarized the relationship between the prevalence of type 2 diabetes and the nicotinamide metabolism changes induced by excess niacin intake, aldehyde oxidase inhibitors, liver diseases and functional defects of skin. We speculate that the gene-environmental interaction in type 2 diabetes may be a reflection of the outcome of the association of chronic nicotinamide overload-induced toxicity and the relatively low detoxification/excretion capacity of the body. Reducing the content of niacin in foods may be a promising strategy for the control of type 2 diabetes.

  2. Nicotinamide improves motor deficits and upregulates PGC-1α and BDNF gene expression in a mouse model of Huntington’s disease

    PubMed Central

    Hathorn, Tyisha; Snyder-Keller, Abigail; Messer, Anne

    2010-01-01

    Huntington’s disease (HD) is a fatal autosomal dominant neurodegenerative disorder caused by an expansion of the polyglutamine (polyQ) repeat in exon-1 in the Huntingtin gene (HTT). This results in misfolding and accumulation of the huntingtin (htt) protein, forming nuclear and cytoplasmic inclusions. HD is associated with dysregulation of gene expression as well as mitochondrial dysfunction. We hypothesized that by improving transcriptional regulation of genes necessary for energy metabolism, the HD motor phenotype would also improve. We therefore examined the protective effects of nicotinamide (NAM), a well-characterized water-soluble B vitamin that is an inhibitor of sirtuin1/class III NAD+-dependent histone deacetylase (HDAC). In this study, both mini-osmotic pumps and drinking water deliveries were tested at 250mg NAM/kg/day, using the B6.HDR6/1 transgenic mouse model. Results were similar for both modes of delivery, and there was no evidence of toxicity. We found that NAM treatment increased mRNA levels of brain-derived neurotrophic factor (BDNF), and Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), the master regulator of mitochondrial biogenesis. Protein levels of BDNF were also significantly increased. In addition, NAM treatment increased PGC-1α activation in HD mice, pointing to a possible mode of action as a therapeutic. Critically, NAM treatment was able to improve motor deficits associated with the HD phenotype, tested as time courses of open field, rotarod, and balance beam activities. These improvements were substantial, despite the fact that NAM did not appear to reduce htt aggregation, or to prevent late-stage weight loss. Our study therefore concludes that NAM or similar drugs may be beneficial in clinical treatment of the motor dysfunctions of HD, while additional therapeutic approaches must be added to combat the aggregation phenotype and overall physiological decline. PMID:20736066

  3. The Pea Gene LH Encodes ent-Kaurene Oxidase1

    PubMed Central

    Davidson, Sandra E.; Smith, Jennifer J.; Helliwell, Chris A.; Poole, Andrew T.; Reid, James B.

    2004-01-01

    The pea (Pisum sativum) homolog, PsKO1, of the Arabidopsis GA3 gene was isolated. It codes for a cytochrome P450 from the CYP701A subfamily and has ent-kaurene oxidase (KO) activity, catalyzing the three step oxidation of ent-kaurene to ent-kaurenoic acid in the gibberellin (GA) biosynthetic pathway when expressed in yeast (Saccharomyces cerevisiae). PsKO1 is encoded by the LH gene because in three independent mutant alleles, lh-1, lh-2, and lh-3, PsKO1 has altered sequence, and the lh-1 allele, when expressed in yeast, failed to metabolize ent-kaurene. The lh mutants of pea are GA deficient and have reduced internode elongation and root growth. One mutant (lh-2) also causes a large increase in seed abortion. PsKO1 (LH) is expressed in all tissues examined, including stems, roots, and seeds, and appears to be a single-copy gene. Differences in sensitivity to the GA synthesis inhibitor, paclobutrazol, between the mutants appear to result from the distinct nature of the genetic lesions. These differences may also explain the tissue-specific differences between the mutants. PMID:14988475

  4. The pea gene LH encodes ent-kaurene oxidase.

    PubMed

    Davidson, Sandra E; Smith, Jennifer J; Helliwell, Chris A; Poole, Andrew T; Reid, James B

    2004-03-01

    The pea (Pisum sativum) homolog, PsKO1, of the Arabidopsis GA3 gene was isolated. It codes for a cytochrome P450 from the CYP701A subfamily and has ent-kaurene oxidase (KO) activity, catalyzing the three step oxidation of ent-kaurene to ent-kaurenoic acid in the gibberellin (GA) biosynthetic pathway when expressed in yeast (Saccharomyces cerevisiae). PsKO1 is encoded by the LH gene because in three independent mutant alleles, lh-1, lh-2, and lh-3, PsKO1 has altered sequence, and the lh-1 allele, when expressed in yeast, failed to metabolize ent-kaurene. The lh mutants of pea are GA deficient and have reduced internode elongation and root growth. One mutant (lh-2) also causes a large increase in seed abortion. PsKO1 (LH) is expressed in all tissues examined, including stems, roots, and seeds, and appears to be a single-copy gene. Differences in sensitivity to the GA synthesis inhibitor, paclobutrazol, between the mutants appear to result from the distinct nature of the genetic lesions. These differences may also explain the tissue-specific differences between the mutants.

  5. The gene encoding proline dehydrogenase modulates sensorimotor gating in mice.

    PubMed

    Gogos, J A; Santha, M; Takacs, Z; Beck, K D; Luine, V; Lucas, L R; Nadler, J V; Karayiorgou, M

    1999-04-01

    Hemizygous cryptic deletions of the q11 band of human chromosome 22 have been associated with a number of psychiatric and behavioural phenotypes, including schizophrenia. Here we report the isolation and characterization of PRODH, a human homologue of Drosophila melanogaster sluggish-A (slgA), which encodes proline dehydrogenase responsible for the behavioural phenotype of the slgA mutant. PRODH is localized at chromosome 22q11 in a region deleted in some psychiatric patients. We also isolated the mouse homologue of slgA (Prodh), identified a mutation in this gene in the Pro/Re hyperprolinaemic mouse strain and found that these mice have a deficit in sensorimotor gating accompanied by regional neurochemical alterations in the brain. Sensorimotor gating is a neural filtering process that allows attention to be focused on a given stimulus, and is affected in patients with neuropsychiatric disorders. Furthermore, several lines of evidence suggest that proline may serve as a modulator of synaptic transmission in the mammalian brain. Our observations, in conjunction with the chromosomal location of PRODH, suggest a potential involvement of this gene in the 22q11-associated psychiatric and behavioural phenotypes.

  6. Characterization of a baculovirus gene encoding a small conotoxinlike polypeptide.

    PubMed Central

    Eldridge, R; Li, Y; Miller, L K

    1992-01-01

    We identified a gene of Autographa californica nuclear polyhedrosis virus (AcMNPV) that encodes a small cysteine-rich polypeptide which has size and sequence similarity to omega-conotoxins, a class of calcium ion (Ca2+) channel inhibitors, found in the venom of cone snails. Transcriptional analysis indicated that the 159-bp open reading frame, which we named ctl, and a downstream 984-bp open reading frame are transcribed as a single 1.3-kb bicistronic late RNA. The mature ctl gene product was identified as a small secreted protein by high-pressure liquid chromatography fractionation of extracellular fluid. Viruses with a site-specific deletion in ctl appeared normal with regard to the kinetics and virulence of infection, both in vitro and in vivo. Although we studied the behavior of wild-type and mutant virus-infected insects in some detail, a biological role for ctl in AcMNPV infection remains to be established. Images PMID:1404603

  7. RAG4 gene encodes a glucose sensor in Kluyveromyces lactis.

    PubMed Central

    Betina, S; Goffrini, P; Ferrero, I; Wésolowski-Louvel, M

    2001-01-01

    The rag4 mutant of Kluyveromyces lactis was previously isolated as a fermentation-deficient mutant, in which transcription of the major glucose transporter gene RAG1 was affected. The wild-type RAG4 was cloned by complementation of the rag4 mutation and found to encode a protein homologous to Snf3 and Rgt2 of Saccharomyces cerevisiae. These two proteins are thought to be sensors of low and high concentrations of glucose, respectively. Rag4, like Snf3 and Rgt2, is predicted to have the transmembrane structure of sugar transporter family proteins as well as a long C-terminal cytoplasmic tail possessing a characteristic 25-amino-acid sequence. Rag4 may therefore be expected to have a glucose-sensing function. However, the rag4 mutation was fully complemented by one copy of either SNF3 or RGT2. Since K. lactis appears to have no other genes of the SNF3/RGT2 type, we suggest that Rag4 of K. lactis may have a dual function of signaling high and low concentrations of glucose. In rag4 mutants, glucose repression of several inducible enzymes is abolished. PMID:11404320

  8. Isolated gene encoding an enzyme with UDP-glucose pyrophosphorylase and phosphoglucomutase activities from Cyclotella cryptica

    DOEpatents

    Jarvis, Eric E.; Roessler, Paul G.

    1999-01-01

    The present invention relates to a cloned gene which encodes an enzyme, the purified enzyme, and the applications and products resulting from the use of the gene and enzyme. The gene, isolated from Cyclotella cryptica, encodes a multifunctional enzyme that has both UDP-glucose pyrophosphorylase and phosphoglucomutase activities.

  9. Isolated gene encoding an enzyme with UDP-glucose pyrophosphorylase and phosphoglucomutase activities from Cyclotella cryptica

    DOEpatents

    Jarvis, E.E.; Roessler, P.G.

    1999-07-27

    The present invention relates to a cloned gene which encodes an enzyme, the purified enzyme, and the applications and products resulting from the use of the gene and enzyme. The gene, isolated from Cyclotella cryptica, encodes a multifunctional enzyme that has both UDP-glucose pyrophosphorylase and phosphoglucomutase activities. 8 figs.

  10. Nicotinamide prevents ultraviolet radiation-induced cellular energy loss.

    PubMed

    Park, Joohong; Halliday, Gary M; Surjana, Devita; Damian, Diona L

    2010-01-01

    UV radiation is carcinogenic by causing mutations in the skin and also by suppressing cutaneous antitumor immunity. We previously found nicotinamide (vitamin B3) to be highly effective at reducing UV-induced immunosuppression in human volunteers, with microarray studies on in vivo irradiated human skin suggesting that nicotinamide normalizes subsets of apoptosis, immune function and energy metabolism-related genes that are downregulated by UV exposure. Using human adult low calcium temperature keratinocytes, we further investigated nicotinamide's effects on cellular energy metabolism. We found that nicotinamide prevented UV-induced cellular ATP loss and protected against UV-induced glycolytic blockade. To determine whether nicotinamide alters the effects of UV-induced oxidative stress posttranslationally, we also measured UV-induced reactive oxygen species (ROS). Nicotinamide had no effect on ROS formation, and at the low UV doses used in these studies, equivalent to ambient daily sun exposure, there was no evidence of apoptosis. Hence, nicotinamide appears to exert its UV protective effects on the skin via its role in cellular energy pathways.

  11. Post transcriptional regulation of chloroplast gene expression by nuclear encoded gene products

    SciTech Connect

    Kuchka, M.R.

    1992-01-01

    Many individual chloroplast genes require the products of a collection of nuclear genes for their successful expression. These nuclear gene products apparently work with great specificity, each committed to the expression of a single chloroplast gene. We have chosen as a model nuclear mutants of Chlamydomonas affected in different stages in the expression of the chloroplast encoded Photosystem II polypeptide, D2. We have made the progress in understanding how nuclear gene products affect the translation of the D2 encoding MRNA. Two nuclear genes are required for this process which have been mapped genetically. In contrast to other examples of nuclear control of translation in the chloroplast, these nuclear gene products appear to be required either for specific stages in translation elongation or for the post-translational stabilization of the nascent D2 protein. Pseudoreversion analysis has led us to a locus which may be directly involved in D2 expression. We have made considerable progress in pursuing the molecular basis of psbd MRNA stabilization. psbD 5' UTR specific transcripts have been synthesized in vitro and used in gel mobility shift assays. UV-crosslinking studies are underway to identify the transacting factors which bind to these sequences. The continued examination of these mutants will help us to understand how nuclear gene products work in this specific case of chloroplast gene expression, and will elucidate how two distinct genomes can interact generally.

  12. Post transcriptional regulation of chloroplast gene expression by nuclear encoded gene products

    SciTech Connect

    Kuchka, M.R.

    1992-01-01

    The following is a review of research accomplished in the first two years of funding for the above mentioned project. The work performed is a molecular characterization of nuclear mutants of Chlamydomonas reinhardtii which are deficient in different stages in the post-transcriptional expression of a single chloroplast encoded polypeptide, the D2 protein of Photosystem II. Our long-term goals are to understand the molecular mechanisms by which nuclear gene products affect the expression of chloroplast genes. Specifically, we which to understand how specific nuclear gene products affect the turnover rate of the D2 encoding mRNA (psbD), how other nuclear encoded factors work to promote the translation of psbD mRNA and/or stabilize the D2 protein, and what the role of the D2 protein itself is in Photosystem II assembly and in the control of expression of other chloroplast genes. This progress report will be organized into four major sections concerning (I) The characterization of nuclear mutants affected in D2 translation/turnover, (II) The study of trans-acting factors which associate with the 5{prime} end of the psbD mRNA, (III) In vitro mutagenesis of the psbD gene, and (IV) Additional studies.

  13. Eukaryotic expression vectors bearing genes encoding cytotoxic proteins for cancer gene therapy.

    PubMed

    Glinka, Elena M

    2012-09-01

    Cancer gene therapy is a promising direction for the treatment of cancer patients. A primary goal of all cancer therapies is to selectively target and kill tumour cells. Such therapies are administered via different approaches, including both viral and non-viral delivery; however, both methods have advantages and disadvantages. Transcriptional targeting enables genes encoding toxic proteins to be expressed directly in cancer cells. Numerous vectors have been created with the purpose of killing cancer cells, and some have successfully suppressed malignant tumours. Data concerning the function of vectors bearing genes that encode cytotoxic proteins under the control of different promoters, including tissue/tumour specific and constitutive promoters, is summarised here. This review focuses on vectors that bear genes encoding diphtheria toxin, Pseudomonas exotoxin A, caspases, gef, streptolysin, and melittin. Data describing the efficacy of such vectors have been summarised. Notably, there are vectors that killed cancer cell lines originating from the same type of cancer with differential efficiency. Thus, there is differential inhibition of cancer cell growth dependent on the cell line. In this review, the constructs employing genes whose expression induces cell death and the efficiency with which they suppress cancer cell growth will be summarised.

  14. [Association of schizophrenia with variations in genes encoding transcription factors].

    PubMed

    Boyajyan, A S; Atshemyan, S A; Zakharyan, R V

    2015-01-01

    Alterations in neuronal plasticity and immune system play a key role in pathogenesis of schizophrenia. Identification of genetic factors contributing to these alterations will significantly encourage elucidation of molecular etiopathomechanisms of this disorder. Transcription factors c-Fos, c-Jun, and Ier5 are the important regulators of neuronal plasticity and immune response. In the present work we investigated a potential association of schizophrenia with a number of single nucleotide polymorphisms of c-Fos-,c-Jun and Ier5 encoding genes (FOS, JUN, and IER5 respectively). Genotyping of DNA samples of patients with schizophrenia and healthy individuals was performed using polymerase chain reaction with allele specific primers. The results obtained demonstrated association between schizophrenia and FOS rs1063169, FOS rs7101, JUN rs11688, and IER5 rs6425663 polymorphisms. Namely, it was found that the inheritance of FOS rs1063169*T, JUN rs11688*A, and IER5 rs6425663*T minor variants decreases risk for development of schizophrenia whereas the inheritance of FOS rs7101*T minor variant, especially its homozygous form, increases risk for development of this disorder.

  15. (Genetic engineering with a gene encoding a soybean storage protein). Progress report

    SciTech Connect

    Beachy, R.N.

    1985-01-01

    Progress is reported on research directed toward introducing a gene (Gmg 17.1) encoding the ..cap alpha..'-subunit of ..beta..-conglycinin, a soybean seed protein, into petunia plants using gene transfer mechanisms. (ACR)

  16. Biochemical evidence that secretor gene, Se, is a structural gene encoding a specific fucosyltransferase.

    PubMed Central

    Kumazaki, T; Yoshida, A

    1984-01-01

    Nonsecretors have no ABH blood group substances in their saliva and milk, but their erythrocytes contain the blood group substances. It has been generally believed that the secretor gene, Se, is a regulatory gene, not a structural gene, controlling the expression of (alpha 1----2)fucosyltransferase, which synthesizes the blood group H substance from its precursor, in secretions. To account for the existence of the blood type of "para Bombay" phenotype--i.e., H-negative in erythrocytes but H-positive in secretory fluids, another regulatory gene, Z, which would regulate the expression of the enzyme in the hematopoietic tissues, has been proposed. Contrary to this, a more simple model, in which the H gene and Se gene are both structural genes, encoding two separate fucosyltransferases in different tissues, was recently proposed. To settle the controversy, (alpha 1----2)fucosyltransferases were partially purified from human plasma and milk. The two enzymes differed from each other in the following respects: (i) the milk enzyme adsorbed to SP-Sephadex at pH 6.0, while the plasma enzyme did not; (ii) pH-activity profiles, with phenyl beta-D-galactoside as an acceptor, differed between the two enzymes; (iii) the milk enzyme exhibited lower thermal stability than the plasma enzyme; and (iv) Km values for several oligosaccharides with Gal(beta 1----3)GlcNAc and Gal(beta 1----4)GlcNAc as acceptors differed between the two enzymes. These results support the model that the Se gene is a structural gene encoding a distinctive (alpha 1----2)fucosyltransferase, refuting the classical regulatory gene model for the Se locus. The anomeric configuration of the fucosylated galactose residue produced by the action of enzyme was identified, thus establishing the specificity of the enzyme. Images PMID:6588382

  17. Ethylene-regulated gene expression: molecular cloning of the genes encoding an endochitinase from Phaseolus vulgaris.

    PubMed Central

    Broglie, K E; Gaynor, J J; Broglie, R M

    1986-01-01

    A full-length copy of bean leaf chitinase mRNA has been cloned. The 1146-base-pair insert of pCH18 encodes the 27-residue amino-terminal signal peptide of the precursor and 301 residues of the mature protein. Utilizing pCH18 as a hybridization probe, we have shown that the increase in translatable chitinase mRNA seen upon ethylene treatment of bean seedlings is due to a 75- to 100-fold increase in steady-state mRNA levels. Southern blot analysis of bean genomic DNA revealed that chitinase is encoded by a small, multigene family consisting of approximately four members. From our nucleotide sequence analysis of five additional chitinase cDNA clones, it appears that at least two of these genes are expressed. Three of the bean chitinase genes have been isolated from a Sau3A genomic library and partially characterized. Images PMID:2428042

  18. Species-specific duplications of NBS-encoding genes in Chinese chestnut (Castanea mollissima)

    PubMed Central

    Zhong, Yan; Li, Yingjun; Huang, Kaihui; Cheng, Zong-Ming

    2015-01-01

    The disease resistance (R) genes play an important role in protecting plants from infection by diverse pathogens in the environment. The nucleotide-binding site (NBS)-leucine-rich repeat (LRR) class of genes is one of the largest R gene families. Chinese chestnut (Castanea mollissima) is resistant to Chestnut Blight Disease, but relatively little is known about the resistance mechanism. We identified 519 NBS-encoding genes, including 374 NBS-LRR genes and 145 NBS-only genes. The majority of Ka/Ks were less than 1, suggesting the purifying selection operated during the evolutionary history of NBS-encoding genes. A minority (4/34) of Ka/Ks in non-TIR gene families were greater than 1, showing that some genes were under positive selection pressure. Furthermore, Ks peaked at a range of 0.4 to 0.5, indicating that ancient duplications arose during the evolution. The relationship between Ka/Ks and Ks indicated greater selective pressure on the newer and older genes with the critical value of Ks = 0.4–0.5. Notably, species-specific duplications were detected in NBS-encoding genes. In addition, the group of RPW8-NBS-encoding genes clustered together as an independent clade located at a relatively basal position in the phylogenetic tree. Many cis-acting elements related to plant defense responses were detected in promoters of NBS-encoding genes. PMID:26559332

  19. Combinatorial selection for replicable RNA by Qβ replicase while maintaining encoded gene function.

    PubMed

    Yumura, Mio; Yamamoto, Natsuko; Yokoyama, Katsushi; Mori, Hirotada; Yomo, Tetsuya; Ichihashi, Norikazu

    2017-01-01

    Construction of a complex artificial self-replication system is challenging in the field of in vitro synthetic biology. Recently, we developed a translation-coupled RNA replication system, wherein an artificial genomic RNA replicates with the Qβ RNA replicase gene encoded on itself. The challenge is to introduce additional genes into the RNA to develop a complex system that mimics natural living systems. However, most RNA sequence encoding genes are not replicable by the Qβ replicase owing to its requirement for strong secondary structures throughout the RNA sequence that are absent in most genes. In this study, we establish a new combinatorial selection method to find an RNA sequence with secondary structures and functional amino acid sequences of the encoded gene. We selected RNA sequences based on their in vitro replication and in vivo gene functions. First, we used the α-domain gene of β-galactosidase as a model-encoding gene, with functional selection based on blue-white screening. Through the combinatorial selection, we developed more replicable RNAs while maintaining the function of the encoded α-domain. The selected sequence improved the affinity between the minus strand RNA and Qβ replicase. Second, we established an in vivo selection method applicable to a broader range of genes by using an Escherichia coli strain with one of the essential genes complemented with a plasmid. We performed the combinatorial selection using an RNA encoding serS and obtained more replicable RNA encoding functional serS gene. These results suggest that combinatorial selection methods are useful for the development of RNA sequences replicable by Qβ replicase while maintaining the encoded gene function.

  20. Combinatorial selection for replicable RNA by Qβ replicase while maintaining encoded gene function

    PubMed Central

    Yumura, Mio; Yamamoto, Natsuko; Yokoyama, Katsushi; Mori, Hirotada; Yomo, Tetsuya

    2017-01-01

    Construction of a complex artificial self-replication system is challenging in the field of in vitro synthetic biology. Recently, we developed a translation-coupled RNA replication system, wherein an artificial genomic RNA replicates with the Qβ RNA replicase gene encoded on itself. The challenge is to introduce additional genes into the RNA to develop a complex system that mimics natural living systems. However, most RNA sequence encoding genes are not replicable by the Qβ replicase owing to its requirement for strong secondary structures throughout the RNA sequence that are absent in most genes. In this study, we establish a new combinatorial selection method to find an RNA sequence with secondary structures and functional amino acid sequences of the encoded gene. We selected RNA sequences based on their in vitro replication and in vivo gene functions. First, we used the α-domain gene of β-galactosidase as a model-encoding gene, with functional selection based on blue-white screening. Through the combinatorial selection, we developed more replicable RNAs while maintaining the function of the encoded α-domain. The selected sequence improved the affinity between the minus strand RNA and Qβ replicase. Second, we established an in vivo selection method applicable to a broader range of genes by using an Escherichia coli strain with one of the essential genes complemented with a plasmid. We performed the combinatorial selection using an RNA encoding serS and obtained more replicable RNA encoding functional serS gene. These results suggest that combinatorial selection methods are useful for the development of RNA sequences replicable by Qβ replicase while maintaining the encoded gene function. PMID:28328998

  1. Isolation of a gene encoding a putative polyamine transporter from Candida albicans, GPT1.

    PubMed

    McNemar, M D; Gorman, J A; Buckley, H R

    2001-04-01

    A gene encoding a transport protein from the pathogenic yeast, Candida albicans, has been isolated during a complementation experiment utilizing an ornithine decarboxylase-negative (spe1 Delta) strain of Saccharomyces cerevisiae. This gene restores gamma-aminobutyric acid (GABA) transport to a GABA transport-negative mutant of S. cerevisiae and encodes a protein which putatively allows transport of one or more of the polyamines. We have assigned the name GPT1 (GABA/polyamine transporter) to this gene.

  2. Crystallization of human nicotinamide phosphoribosyltransferase

    SciTech Connect

    Takahashi, Ryo; Nakamura, Shota; Yoshida, Takuya; Kobayashi, Yuji; Ohkubo, Tadayasu

    2007-05-01

    Human nicotinamide phosphoribosyltransferase has been crystallized using microseeding methods and X-ray diffraction data have been collected at 2.0 Å resolution. In the NAD biosynthetic pathway, nicotinamide phosphoribosyltransferase (NMPRTase; EC 2.4.2.12) plays an important role in catalyzing the synthesis of nicotinamide mononucleotide from nicotinamide and 5′-phosphoribosyl-1′-pyrophosphate. Because the diffraction pattern of the initally obtained crystals was not suitable for structure analysis, the crystal quality was improved by successive use of the microseeding technique. The resultant crystals diffracted to 2.0 Å resolution. These crystals belonged to space group P21, with unit-cell parameters a = 60.56, b = 106.40, c = 82.78 Å. Here, the crystallization of human NMPRTase is reported in the free form; the crystals should be useful for inhibitor-soaking experiments on the enzyme.

  3. Nicotinamide for skin cancer chemoprevention.

    PubMed

    Damian, Diona L

    2017-03-20

    Nicotinamide (vitamin B3 ) has a range of photoprotective effects in vitro and in vivo; it enhances DNA repair, reduces UV radiation-induced suppression of skin immune responses, modulates inflammatory cytokine production and skin barrier function and restores cellular energy levels after UV exposure. Pharmacological doses of nicotinamide have been shown to reduce actinic keratoses and nonmelanoma skin cancer incidence in high-risk individuals, making this a nontoxic and accessible option for skin cancer chemoprevention in this population.

  4. Enterotoxin-encoding genes in Staphylococcus spp. from bulk goat milk.

    PubMed

    Lyra, Daniele G; Sousa, Francisca G C; Borges, Maria F; Givisiez, Patrícia E N; Queiroga, Rita C R E; Souza, Evandro L; Gebreyes, Wondwossen A; Oliveira, Celso J B

    2013-02-01

    Although Staphylococcus aureus has been implicated as the main Staphylococcus species causing human food poisoning, recent studies have shown that coagulase-negative Staphylococcus could also harbor enterotoxin-encoding genes. Such organisms are often present in goat milk and are the most important mastitis-causing agents. Therefore, this study aimed to investigate the occurrence of enterotoxin-encoding genes among coagulase-positive (CoPS) and coagulase-negative (CoNS) staphylococci isolated from raw goat milk produced in the semi-arid region of Paraiba, the most important region for goat milk production in Brazil. Enterotoxin-encoding genes were screened in 74 staphylococci isolates (30 CoPS and 44 CoNS) by polymerase chain reaction targeting the genes sea, seb, sec, sed, see, seg, seh, and sei. Enterotoxin-encoding genes were found in nine (12.2%) isolates, and four different genes (sea, sec, seg, and sei) were identified amongst the isolates. The most frequent genes were seg and sei, which were often found simultaneously in 44.5% of the isolates. The gene sec was the most frequent among the classical genes, and sea was found only in one isolate. All CoPS isolates (n=7) harboring enterotoxigenic genes were identified as S. aureus. The two coagulase-negative isolates were S. haemolyticus and S. hominis subsp. hominis and they harbored sei and sec genes, respectively. A higher frequency of enterotoxin-encoding genes was observed amongst CoPS (23.3%) than CoNS (4.5%) isolates (p<0.05), reinforcing the importance of S. aureus as a potential foodborne agent. However, the potential risk posed by CoNS in goat milk should not be ignored because it has a higher occurrence in goat milk and enterotoxin-encoding genes were detected in some isolates.

  5. SURVEY AND SUMMARY: A survey of small RNA-encoding genes in Escherichia coli

    PubMed Central

    Hershberg, Ruth; Altuvia, Shoshy; Margalit, Hanah

    2003-01-01

    Small RNA (sRNA) molecules have gained much interest lately, as recent genome-wide studies have shown that they are widespread in a variety of organisms. The relatively small family of 10 known sRNA-encoding genes in Escherichia coli has been significantly expanded during the past two years with the discovery of 45 novel genes. Most of these genes are still uncharacterized and their cellular roles are unknown. In this survey we examined the sequence and genomic features of the 55 currently known sRNA-encoding genes in E.coli, attempting to identify their common characteristics. Such characterization is important for both expanding our understanding of this unique gene family and for improving the methods to predict and identify sRNA-encoding genes based on genomic information. PMID:12654996

  6. Two Fis Regulators Directly Repress the Expression of Numerous Effector-Encoding Genes in Legionella pneumophila

    PubMed Central

    Zusman, Tal; Speiser, Yariv

    2014-01-01

    Legionella pneumophila is an intracellular human pathogen that utilizes the Icm/Dot type IVB secretion system to translocate a large repertoire of effectors into host cells. For most of these effectors, there is no information regarding their regulation. Therefore, the aim of this study was to examine the involvement of the three L. pneumophila Fis homologs in the regulation of effector-encoding genes. Deletion mutants constructed in the genes encoding the three Fis regulators revealed that Fis1 (lpg0542 gene) and Fis3 (lpg1743) but not Fis2 (lpg1370) are partially required for intracellular growth of L. pneumophila in Acanthamoeba castellanii. To identify pathogenesis-related genes directly regulated by Fis, we established a novel in vivo system which resulted in the discovery of numerous effector-encoding genes directly regulated by Fis. Further examination of these genes revealed that Fis1 and Fis3 repress the level of expression of effector-encoding genes during exponential phase. Three groups of effector-encoding genes were identified: (i) effectors regulated mainly by Fis1, (ii) effectors regulated mainly by Fis3, and (iii) effectors regulated by both Fis1 and Fis3. Examination of the upstream regulatory region of all of these effector-encoding genes revealed multiple putative Fis regulatory elements, and site-directed mutagenesis confirmed that a few of these sites constitute part of a repressor binding element. Furthermore, gel mobility shift assays demonstrated the direct relation between the Fis1 and Fis3 regulators and these regulatory elements. Collectively, our results demonstrate for the first time that two of the three L. pneumophila Fis regulators directly repress the expression of Icm/Dot effector-encoding genes. PMID:25225276

  7. Isolation of a gene encoding a novel spectinomycin phosphotransferase from Legionella pneumophila.

    PubMed Central

    Suter, T M; Viswanathan, V K; Cianciotto, N P

    1997-01-01

    A gene capable of conferring spectinomycin resistance was isolated from Legionella pneumophila, the agent of Legionnaires' disease. The gene (aph) encoded a 36-kDa protein which has similarity to aminoglycoside phosphotransferases. Biochemical analysis confirmed that aph encodes a phosphotransferase which modifies spectinomycin but not hygromycin, kanamycin, or streptomycin. The strain that was the source of aph demonstrated resistance to spectinomycin, and Southern hybridizations determined that aph also exists in other legionellae. PMID:9174205

  8. Gene encoding human Ro-associated autoantigen Y5 RNA.

    PubMed Central

    Maraia, R; Sakulich, A L; Brinkmann, E; Green, E D

    1996-01-01

    Ro ribonucleoproteins are composed of Y RNAs and the Ro 60 kDa protein. While the Ro 60 kDa protein is implicated in an RNA discard pathway that recognizes 3'-extended 5S rRNAs, the function of Y RNAs remains unknown [O'Brien,C.A. and Wolin,S.L. (1995) Genes Dev. 8,2891-2903]. Y5 RNA occupies a large fraction of Ro 60 kDa protein in human Ro RNPs, contains an atypical 3'-extension not found on other Y RNAs, and constitutes an RNA antigen in certain autoimmune patients [Boulanger et al. (1995) Clin. Exp. Immunol. 99, 29-36]. An overabundance of Y RNA retroposed pseudogenes has previously complicated the isolation of mammalian Y RNA genes. The source gene for Y5 RNA was isolated from human DNA as well as from Galago senegalis DNA. Authenticity of the hY5 RNA gene was demonstrated in vivo and its activity was compared with the hY4 RNA gene that also uses a type 3 promoter for RNA polymerase III. The hY5 RNA gene was subsequently found to reside within a few hundred thousand base pairs of other Y RNA genes and the linear order of the four human Y RNA genes on chromosome 7q36 was determined. Phylogenetic comparative analyses of promoter and RNA structure indicate that the Y5 RNA gene has been subjected to positive selection during primate evolution. Consistent with the proposal of O'Brien and Harley [O'Brian,C.A. and Wolin,S.L. (1992) Gene 116, 285-289], analysis of flanking sequences suggest that the hY5 RNA gene may have originated as a retroposon. PMID:8836182

  9. Molecular and Genomic Analysis of Genes Encoding Surface-Anchored Proteins from Clostridium difficile

    PubMed Central

    Karjalainen, Tuomo; Waligora-Dupriet, Anne-Judith; Cerquetti, Marina; Spigaglia, Patrizia; Maggioni, Andrea; Mauri, Pierluigi; Mastrantonio, Paola

    2001-01-01

    The gene slpA, encoding the S-layer precursor protein in the virulent Clostridium difficile strains C253 and 79–685, was identified. The precursor protein carries a C-terminal highly conserved anchoring domain, similar to the one found in the Cwp66 adhesin (previously characterized in strain 79–685), an SLH domain, and a variable N-terminal domain mediating cell adherence. The genes encoding the S-layer precursor proteins and the Cwp66 adhesin are present in a genetic locus carrying 17 open reading frames, 11 of which encode a similar two-domain architecture, likely to include surface-anchored proteins. PMID:11292772

  10. Molecular cloning and characterization of the gene encoding rat submandibular gland apomucin, Mucsmg.

    PubMed

    Albone, E F; Hagen, F K; Szpirer, C; Tabak, L A

    1996-10-01

    Mucin glycoproteins are a major constituent of salivary secretions and play a primary role in the protection of the oral cavity. Rat submandibular glands (RSMG) synthesize and secrete a low molecular weight (114 kDa) mucin glycoprotein. We have isolated, partially sequenced, and characterized the gene which encodes the RSMG apomucin. The gene is encoded by three exons of 106 nt, 69 nt, and 991 nt, separated by introns of 921 nt and 12.5 kb. CAAT and TATA elements are present, at -68 and -26, respectively, in the 5' flanking sequence of the RSMG apomucin gene. The tandem repeat domain present in exon III consists of ten tandem repeats of 39 nt encoding the consensus sequence PTTDSTTPAPTTK. Sequence comparison and organization of the nucleic acid sequence encoding the tandem repeats of two alleles for this gene suggests that the apomucin gene has undergone recombinational events during its evolution. No significant sequence similarity was found with other mucin genes, or with other known salivary gland-specific genes. The gene was localized to rat chromosome 14 using somatic cell hybrids that segregate rat chromosomes. Since this, to our knowledge, represents the first RSMG mucin gene cloned, we have designated this gene Mucsmg.

  11. Nicotinamide in dermatology and photoprotection.

    PubMed

    Surjana, Devita; Damian, Diona L

    2011-01-01

    Nicotinamide (the amide form of vitamin B3) has been used in dermatology for more than 40 years for a diverse range of conditions including acne, rosacea, autoimmune bullous dermatoses, and now the treatment and prevention of photoaging and photoimmunosuppression. The broad clinical effects of nicotinamide may be explained by its role as a cellular energy precursor, a modulator of inflammatory cytokines, and an inhibitor of the nuclear enzyme poly(adenosine diphosphate-ribose) polymerase-1, which plays a significant role in DNA repair, maintenance ofgenomic stability, and cellular response to injury including inflammation and apoptosis. This review outlines the use of nicotinamide for inflammatory dermatoses and photoaging and focuses on its emerging role in photoprotection.

  12. An intron-encoded protein assists RNA splicing of multiple similar introns of different bacterial genes.

    PubMed

    Meng, Qing; Wang, Yanfei; Liu, Xiang-Qin

    2005-10-21

    Four group II introns were found in an unusually intron-rich dnaN gene (encoding the beta subunit of DNA polymerase III) of the cyanobacterium Trichodesmium erythraeum, and they have strong similarities to two introns of the RIR gene (encoding ribonucleotide reductase) of the same organism. Of these six introns, only the RIR-3 intron encodes a maturase protein and showed efficient RNA splicing when expressed in Escherichia coli cells. The other five introns do not encode a maturase protein and did not show RNA splicing in E. coli. But these maturase-less introns showed efficient RNA splicing when the RIR-3 intron-encoded maturase protein was co-expressed from a freestanding gene in the same cell. These findings demonstrated that an intron-encoded protein could function as a general maturase for multiple introns of different genes. Major implications may include an intron-mediated co-regulation of the different genes and a resemblance of the evolutionary origin of spliceosomal introns.

  13. Organization and control of genes encoding catabolic enzymes in Rhizobiaceae

    SciTech Connect

    Parke, D.; Ornston, L.N.

    1993-03-01

    Rhizobiaceae, a diverse bacterial group comprising rhizobia and agrobacteria, symbiotic partnership with plants form nitrogen-fixing nodules on plant roots or are plant pathogens. Phenolic compounds produced by plants serve as inducers of rhizobial nodulation genes and agrobacterial virulence genes reflect their capacity to utilize numerous aromatics, including phenolics, as a source of carbon and energy. In many microbes the aerobic degradation of numerous aromatic compounds to tricarboxylic acid cycle intermediates is achieved by the [beta]-ketoadipate pathway. Our initial studies focused on the organization and regulation of the ketoadipate pathway in Agrobacterium tumefaciens. We have cloned, identified and characterized a novel regulatory gene that modulates expression of an adjacent pca (protocatechuate) structural gene, pcaD. Regulation of pcaD is mediated by the regulatory gene, termed pcaQ, in concert with the intermediate [beta]-carboxy-cis,cis-muconate. [beta]-carboxy-cis,cismuconate is an unstable chemical, not marketed commercially, and it is unlikely to permeate Escherichia coli cells if supplied in media. Because of these factors, characterization of pcaQ in E. coli required an in vivo delivery system for [beta]-carboxycis,cis-muconate. This was accomplished by designing an E. coli strain that expressed an Acinetobacter calcoaceticus pcaA gene for conversion of protocatechuate to [beta]-carboxy-cis,cis-muconate.

  14. A highly divergent gene cluster in honey bees encodes a novel silk family.

    PubMed

    Sutherland, Tara D; Campbell, Peter M; Weisman, Sarah; Trueman, Holly E; Sriskantha, Alagacone; Wanjura, Wolfgang J; Haritos, Victoria S

    2006-11-01

    The pupal cocoon of the domesticated silk moth Bombyx mori is the best known and most extensively studied insect silk. It is not widely known that Apis mellifera larvae also produce silk. We have used a combination of genomic and proteomic techniques to identify four honey bee fiber genes (AmelFibroin1-4) and two silk-associated genes (AmelSA1 and 2). The four fiber genes are small, comprise a single exon each, and are clustered on a short genomic region where the open reading frames are GC-rich amid low GC intergenic regions. The genes encode similar proteins that are highly helical and predicted to form unusually tight coiled coils. Despite the similarity in size, structure, and composition of the encoded proteins, the genes have low primary sequence identity. We propose that the four fiber genes have arisen from gene duplication events but have subsequently diverged significantly. The silk-associated genes encode proteins likely to act as a glue (AmelSA1) and involved in silk processing (AmelSA2). Although the silks of honey bees and silkmoths both originate in larval labial glands, the silk proteins are completely different in their primary, secondary, and tertiary structures as well as the genomic arrangement of the genes encoding them. This implies independent evolutionary origins for these functionally related proteins.

  15. Isolation and characterization of genes encoding two chitinase enzymes from Serratia marcescens

    PubMed Central

    Jones, Jonathan D. G.; Grady, Karen L.; Suslow, Trevor V.; Bedbrook, John R.

    1986-01-01

    Analysis of clones isolated from a cosmid DNA library indicates that the Serratia marcescens chromosome contains at least two genes, chiA and chiB, which encode distinct secreted forms of the enzyme chitinase. These genes have been characterized by inspection of chitinase activity and secreted proteins in Escherichia coli strains containing subclones of these cosmids. The two chitinase genes show no detectable homology to each other. DNA sequence analysis of one of the genes predicts an amino acid sequence with an N-terminal signal peptide typical of genes encoding secreted bacterial proteins. This gene was mutagenized by cloning a neomycin phosphotransferase gene within its coding region, and the insertion mutation was recombined into the parental S. marcescens strain. The resulting chiA mutant transconjugant showed reduced chitinase production, reduced inhibition of fungal spore germination and reduced biological control of a fungal plant pathogen. ImagesFig. 2. PMID:16453672

  16. Overlapping gene expression profiles of cell migration and tumor invasion in human bladder cancer identify metallothionein E1 and nicotinamide N-methyltransferase as novel regulators of cell migration

    PubMed Central

    Wu, Y.; Siadaty, M. S.; Berens, M.E.; Hampton, G. M.

    2017-01-01

    Cell migration is essential to cancer invasion and metastasis and is spatially and temporally integrated through transcriptionally dependent and independent mechanisms. Since cell migration is studied in vitro, it is important to identify genes that both drive cell migration and are biologically relevant in promoting invasion and metastasis in patients with cancer. Here, gene expression profiling and a high throughput cell migration system answers this question in human bladder cancer. In vitro migration rates of 40 microarray profiled human bladder cancer cell lines were measured by radial migration assay (RMA). Genes whose expression was either directly or inversely associated with cell migration rate were identified and subsequently evaluated for their association with cancer stage in 61 patients. This analysis identified genes known to be associated with cell invasion such as versican, and novel ones, including metallothionein E1 (MTE1) and nicotinamide N-methyltransferase (NNMT), whose expression correlated positively with cancer cell migration and tumor stage. Using loss of function analysis, we show that MTE1 and NNMT are necessary for cancer cell migration. These studies provide a general approach to identify the clinically relevant genes in cancer cell migration and mechanistically implicate two novel genes in this process in human bladder cancer. PMID:18724390

  17. Nicotinamide riboside promotes Sir2 silencing and extends lifespan via Nrk and Urh1/Pnp1/Meu1 pathways to NAD+.

    PubMed

    Belenky, Peter; Racette, Frances G; Bogan, Katrina L; McClure, Julie M; Smith, Jeffrey S; Brenner, Charles

    2007-05-04

    Although NAD(+) biosynthesis is required for Sir2 functions and replicative lifespan in yeast, alterations in NAD(+) precursors have been reported to accelerate aging but not to extend lifespan. In eukaryotes, nicotinamide riboside is a newly discovered NAD(+) precursor that is converted to nicotinamide mononucleotide by specific nicotinamide riboside kinases, Nrk1 and Nrk2. In this study, we discovered that exogenous nicotinamide riboside promotes Sir2-dependent repression of recombination, improves gene silencing, and extends lifespan without calorie restriction. The mechanism of action of nicotinamide riboside is totally dependent on increased net NAD(+) synthesis through two pathways, the Nrk1 pathway and the Urh1/Pnp1/Meu1 pathway, which is Nrk1 independent. Additionally, the two nicotinamide riboside salvage pathways contribute to NAD(+) metabolism in the absence of nicotinamide-riboside supplementation. Thus, like calorie restriction in the mouse, nicotinamide riboside elevates NAD(+) and increases Sir2 function.

  18. NBS-LRR-Encoding genes in sorghum and their role in plant defense

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Nucleotide-binding site leucine-rich repeats (NBS-LRR) proteins are encoded by a large class of plant genes and many of them play an important role in plant defense against pest attack. Identification and characterization of the whole set of NBS-LRR genes in a plant genome will provide insights int...

  19. Characterization of the gene encoding a fibrinogen-related protein expressed in Crassostrea gigas hemocytes.

    PubMed

    Skazina, M A; Gorbushin, A M

    2016-07-01

    Four exons of the CgFrep1 gene (3333 bp long) encode a putative fibrinogen-related protein (324 aa) bearing a single C-terminal FBG domain. Transcripts of the gene obtained from hemocytes of different Pacific oysters show prominent individual variation based on SNP and indels of tandem repeats resulted in polymorphism of N-terminus of the putative CgFrep1 polypeptide. The polypeptide chain bears N-terminal coiled-coil region potentially acting as inter-subunit interface in the protein oligomerization. It is suggested that CgFrep1 gene encodes the oligomeric lectin composed of at least two subunits.

  20. Simultaneous quantitation of nicotinamide riboside, nicotinamide mononucleotide and nicotinamide adenine dinucleotide in milk by a novel enzyme-coupled assay.

    PubMed

    Ummarino, Simone; Mozzon, Massimo; Zamporlini, Federica; Amici, Adolfo; Mazzola, Francesca; Orsomando, Giuseppe; Ruggieri, Silverio; Raffaelli, Nadia

    2017-04-15

    Nicotinamide riboside, the most recently discovered form of vitamin B3, and its phosphorylated form nicotinamide mononucleotide, have been shown to be potent supplements boosting intracellular nicotinamide adenine dinucleotide (NAD) levels, thus preventing or ameliorating metabolic and mitochondrial diseases in mouse models. Here we report for the first time on the simultaneous quantitation of nicotinamide riboside, nicotinamide mononucleotide and NAD in milk by means of a fluorometric, enzyme-coupled assay. Application of this assay to milk from different species revealed that the three vitamers were present in human and donkey milk, while being selectively distributed in the other milks. Human milk was the richest source of nicotinamide mononucleotide. Overall, the three vitamers accounted for a significant fraction of total vitamin B3 content. Pasteurization did not affect the bovine milk content of nicotinamide riboside, whereas UHT processing fully destroyed the vitamin. In human milk, NAD levels were significantly affected by the lactation time.

  1. Cloning of a Serratia marcescens Gene Encoding Chitinase

    PubMed Central

    Fuchs, R. L.; McPherson, S. A.; Drahos, D. J.

    1986-01-01

    Serratia marcescens, a chitinase-producing microorganism, was shown to produce five unique chitinolytic proteins with subunit molecular masses of 21, 36, 48, 52, and 57 kilodaltons. A cosmid library of S. marcescens DNA was constructed in the broad-host-range cosmid pLAFR1 and screened in Escherichia coli for clones capable of degrading chitin. A total of four independent clones (22- to 27-kilobase inserts) were isolated, characterized by restriction endonuclease digestion, and shown to share a common 9.5-kilobase EcoR1 fragment apparently encoding the same 57-kilodalton chitinase, the most abundant chitinase produced by S. marcescens. Chitinase expression from these constructs in both E. coli and Pseudomonas fluorescens 701E1 is apparently driven by an S. marcescens promoter. The significantly higher chitinase levels produced in E. coli relative to those in P. fluorescens 701E1 suggest that E. coli may recognize this promoter sequence more efficiently than P. fluorescens. Images PMID:16347012

  2. Expression of hcp in freshwater Synechococcus spp., a gene encoding a hyperconserved protein in picocyanobacteria.

    PubMed

    Kutovaya, Olga A; McKay, R Michael L; Bullerjahn, George S

    2010-06-01

    Marine picoplankton of the genus Synechococcus and Prochlorococcus spp. are widely studied members of the picocyanobacterial clade, composed of unicellular cyanobacteria that dominate pelagic regions of the ocean. Less studied are the related freshwater Synechococcus spp. that similarly dominate the euphotic zone of oligotrophic lakes. Previous work has shown that marine picocyanobacteria harbor a small gene, hcp, that encodes a 62 amino acid protein 100% conserved among all strains examined. The gene is restricted exclusively to the picocyanobacterial lineage. The current study reveals that hcp is also 100% conserved in four freshwater Synechococcus spp. strains isolated from the Laurentian Great Lakes, and that the gene constitutively expressed with genes encoding a ribosomal protein and two tRNA genes. The synteny of the hcp region is also conserved between the marine and freshwater strains. Last, the hcp gene and the organization of the surrounding genetic region has been retained in the reduced genome of a picocyanobacterial endosymbiont of the amoeba Paulinella sp.

  3. The ZIC gene family encodes multi-functional proteins essential for patterning and morphogenesis.

    PubMed

    Houtmeyers, Rob; Souopgui, Jacob; Tejpar, Sabine; Arkell, Ruth

    2013-10-01

    The zinc finger of the cerebellum gene (ZIC) discovered in Drosophila melanogaster (odd-paired) has five homologs in Xenopus, chicken, mice, and humans, and seven in zebrafish. This pattern of gene copy expansion is accompanied by a divergence in gene and protein structure, suggesting that Zic family members share some, but not all, functions. ZIC genes are implicated in neuroectodermal development and neural crest cell induction. All share conserved regions encoding zinc finger domains, however their heterogeneity and specification remain unexplained. In this review, the evolution, structure, and expression patterns of the ZIC homologs are described; specific functions attributable to individual family members are supported. A review of data from functional studies in Xenopus and murine models suggest that ZIC genes encode multifunctional proteins operating in a context-specific manner to drive critical events during embryogenesis. The identification of ZIC mutations in congenital syndromes highlights the relevance of these genes in human development.

  4. Prevalence and characteristics of nim genes encoding 5-nitroimidazole resistance among Bacteroides strains isolated in Morocco.

    PubMed

    Haggoud, A; M'Hand, R A; Reysset, G; El M'Daghri, N; Benbachir, M; Moumni, M

    2001-01-01

    We report here an evaluation of the dissemination of nim genes, encoding 5-nitroimidazoles resistance, among Bacteroides clinical strains isolated in Morocco. This study was done using a PCR method. Among 60 strains studied, nine contain a copy of a nim gene. The sequence determination of these genes showed that they are homologous to three nim genes previously characterized in strains isolated in France: nimB (five genes), nimC (three genes), and nimA (one gene). Although the nimA and nimC genes were previously identified on plasmids pIP417 and pIP419, respectively, we found here that they have a chromosomal location. The MICs of three 5-nitroimidazole antibiotics (metronidazole, ornidazole, and tinidazole) of the nim gene-containing strains were very low (0.5-2 microg/ml), indicating that the nim genes were not efficiently expressed in these clinical isolates.

  5. Cloning and sequencing of the gene encoding cytochrome c sub 553 from Desulfovibrio vulgaris Hildenborough

    SciTech Connect

    van Rooijen, G.J.H.; Voordouw, G. ); Bruschi, M. )

    1989-06-01

    The gene encoding cytochrome c{sub 553} from Desulfovibrio vulgaris Hildenborough was cloned by using two synthetic deoxyoligonucleotide probes. The amino acid sequence derived from the sequence of the gene differs from that reported by Bruschi and LeGall. Renewed protein sequencing confirmed the correctness of the DNA-derived sequence. The gene sequence indicates cytochrome c{sub 553} to be synthesized as a precursor protein with an NH{sub 2}-terminal signal sequence of 24 residues.

  6. Cloning and expression of prion protein encoding gene of flounder ( Paralichthys olivaceus)

    NASA Astrophysics Data System (ADS)

    Zhang, Zhiwen; Sun, Xiuqin; Zhang, Jinxing; Zan, Jindong

    2008-02-01

    The prion protein (PrP) encoding gene of flounder ( Paralichthys olivaceus) was cloned. It was not interrupted by an intron. This gene has two promoters in its 5' upstream, indicating that its transcription may be intensive, and should have an important function. It was expressed in all 14 tissues tested, demonstrating that it is a house-keeping gene. Its expression in digestion and reproduction systems implies that the possible prions of fish may transfer horizontally.

  7. The Saccharomyces cerevisiae YMR315W Gene Encodes an NADP(H)-Specific Oxidoreductase Regulated by the Transcription Factor Stb5p in Response to NADPH Limitation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Engineered xylose-metabolizing cells grown on xylose show increased expression of YMR315W at both the mRNA and protein levels. Additionally, the YMR315W promoter contains a putative binding site for the transcription factor Stb5p, which has been shown to regulate genes involved in nicotinamide aden...

  8. Characterization of transcript processing of the gene encoding precerebellin-1.

    PubMed

    Kavety, B; Morgan, J I

    1998-12-10

    Precerebellin-1 (Cbln1) is a cerebellum-specific protein that shares significant sequence identity with the globular domains of the complement components C1qA, B and C, suggesting some common aspects of function and/or structure. As the C1q complex is composed of heterotrimers of C1qA, B and C it was hypothesized that multiple precerebellins may exist in a ternary complex. Northern blotting for cbln1 revealed multiple bands that could represent further family members or alternatively spliced variants. To discriminate these alternatives, probes derived from different regions of the cbln1 gene were used to identify and clone the transcripts detected on Northern blots. Four independent transcripts were repeatedly cloned from an adult mouse cerebellum cDNA library. Upon sequencing, all of these clones were found to be derived from the cbln1 gene and no additional precerebellin-related genes were isolated. Moreover, these clones accounted for the four cbln1-hybridizing bands (1.9, 2. 2, 3.2 and 5.5 kb) detected on Northern blots of adult cerebellum RNA. With one possible exception, these clones were all derived through alterations in the 3'-untranslated region (3'-UTR) of cbln1 that did not affect the coding sequence. This was achieved by the use of two polyadenylation sites and alternative (non-canonical) splicing in the 3'-UTR. Some additional variation in mRNA structure is provided by the use of alternative transcription start sites in cbln1. The possible significance of this level of diversity in the 3'-UTR is discussed.

  9. A neurotransmitter transporter encoded by the Drosophila inebriated gene

    PubMed Central

    Soehnge, Holly; Huang, Xi; Becker, Marie; Whitley, Penn; Conover, Diana; Stern, Michael

    1996-01-01

    Behavioral and electrophysiological studies on mutants defective in the Drosophila inebriated (ine) gene demonstrated increased excitability of the motor neuron. In this paper, we describe the cloning and sequence analysis of ine. Mutations in ine were localized on cloned DNA by restriction mapping and restriction fragment length polymorphism (RFLP) mapping of ine mutants. DNA from the ine region was then used to isolate an ine cDNA. In situ hybridization of ine transcripts to developing embryos revealed expression of this gene in several cell types, including the posterior hindgut, Malpighian tubules, anal plate, garland cells, and a subset of cells in the central nervous system. The ine cDNA contains an open reading frame of 658 amino acids with a high degree of sequence similarity to members of the Na+/Cl−-dependent neurotransmitter transporter family. Members of this family catalyze the rapid reuptake of neurotransmitters released into the synapse and thereby play key roles in controlling neuronal function. We conclude that ine mutations cause increased excitability of the Drosophila motor neuron by causing the defective reuptake of the substrate neurotransmitter of the ine transporter and thus overstimulation of the motor neuron by this neurotransmitter. From this observation comes a unique opportunity to perform a genetic dissection of the regulation of excitability of the Drosophila motor neuron. PMID:8917579

  10. The evolution of genes encoding for green fluorescent proteins: insights from cephalochordates (amphioxus)

    NASA Astrophysics Data System (ADS)

    Yue, Jia-Xing; Holland, Nicholas D.; Holland, Linda Z.; Deheyn, Dimitri D.

    2016-06-01

    Green Fluorescent Protein (GFP) was originally found in cnidarians, and later in copepods and cephalochordates (amphioxus) (Branchiostoma spp). Here, we looked for GFP-encoding genes in Asymmetron, an early-diverged cephalochordate lineage, and found two such genes closely related to some of the Branchiostoma GFPs. Dim fluorescence was found throughout the body in adults of Asymmetron lucayanum, and, as in Branchiostoma floridae, was especially intense in the ripe ovaries. Spectra of the fluorescence were similar between Asymmetron and Branchiostoma. Lineage-specific expansion of GFP-encoding genes in the genus Branchiostoma was observed, largely driven by tandem duplications. Despite such expansion, purifying selection has strongly shaped the evolution of GFP-encoding genes in cephalochordates, with apparent relaxation for highly duplicated clades. All cephalochordate GFP-encoding genes are quite different from those of copepods and cnidarians. Thus, the ancestral cephalochordates probably had GFP, but since GFP appears to be lacking in more early-diverged deuterostomes (echinoderms, hemichordates), it is uncertain whether the ancestral cephalochordates (i.e. the common ancestor of Asymmetron and Branchiostoma) acquired GFP by horizontal gene transfer (HGT) from copepods or cnidarians or inherited it from the common ancestor of copepods and deuterostomes, i.e. the ancestral bilaterians.

  11. The evolution of genes encoding for green fluorescent proteins: insights from cephalochordates (amphioxus)

    PubMed Central

    Yue, Jia-Xing; Holland, Nicholas D.; Holland, Linda Z.; Deheyn, Dimitri D.

    2016-01-01

    Green Fluorescent Protein (GFP) was originally found in cnidarians, and later in copepods and cephalochordates (amphioxus) (Branchiostoma spp). Here, we looked for GFP-encoding genes in Asymmetron, an early-diverged cephalochordate lineage, and found two such genes closely related to some of the Branchiostoma GFPs. Dim fluorescence was found throughout the body in adults of Asymmetron lucayanum, and, as in Branchiostoma floridae, was especially intense in the ripe ovaries. Spectra of the fluorescence were similar between Asymmetron and Branchiostoma. Lineage-specific expansion of GFP-encoding genes in the genus Branchiostoma was observed, largely driven by tandem duplications. Despite such expansion, purifying selection has strongly shaped the evolution of GFP-encoding genes in cephalochordates, with apparent relaxation for highly duplicated clades. All cephalochordate GFP-encoding genes are quite different from those of copepods and cnidarians. Thus, the ancestral cephalochordates probably had GFP, but since GFP appears to be lacking in more early-diverged deuterostomes (echinoderms, hemichordates), it is uncertain whether the ancestral cephalochordates (i.e. the common ancestor of Asymmetron and Branchiostoma) acquired GFP by horizontal gene transfer (HGT) from copepods or cnidarians or inherited it from the common ancestor of copepods and deuterostomes, i.e. the ancestral bilaterians. PMID:27311567

  12. Characterization of two genes, Impa1 and Impa2 encoding mouse myo-inositol monophosphatases.

    PubMed

    Shamir, A; Sjøholt, G; Ebstein, R P; Agam, G; Steen, V M

    2001-06-27

    The enzyme myo-inositol monophosphatase (Impa) catalyzes the synthesis of free myo-inositol from various myo-inositol monophosphates in the phosphatidylinositol signaling system. Impa is a lithium-blockable enzyme that has been hypothesized to be the biological target for lithium-salts used as mood-stabilizing drugs in the treatment of manic-depressive (bipolar) illness. As an initial step to explore the functional consequences of reduced or absent Impa activity in an animal model we here report the isolation of two Impa-encoding mouse genes, Impa1 and Impa2. Impa1 spans approximately 17.5 kb and contains nine exons of 46--1354 bp encoding a protein of 277 amino acids. Impa2 spans at least 19.5 kb and contains eight exons of 46--444 bp size encoding a protein of 290 amino acids. The genomic structure including the positions of the exon-intron splice sites seems to be conserved among myo-inositol monophosphatase genes in mammalian species. One or more Impa-like genes do also exist in evolutionary more distant species like invertebrates, plants and bacteria. The proteins encoded by the non-vertebrate genes seem to be equally related to Impa1 and Impa2. We therefore suggest that the Impa1 and Impa2 genes duplicated from a common ancestral gene after the evolutionary divergence of vertebrates.

  13. The same Arabidopsis gene encodes both cytosolic and mitochondrial alanyl-tRNA synthetases.

    PubMed Central

    Mireau, H; Lancelin, D; Small, I D

    1996-01-01

    In plants, all aminoacyl-tRNA synthetases are nuclearly encoded, despite the fact that their activities are required in the three protein-synthesizing cell compartments (cytosol, mitochondria, and chloroplasts). To investigate targeting of these enzymes, we cloned cDNAs encoding alanyl-tRNA synthetase (AlaRS) and the corresponding nuclear gene, ALATS, from Arabidopsis by using degenerate polymerase chain reaction primers based on highly conserved regions shared between known AlaRSs from other organisms. Analysis of the transcription of the gene showed the presence of two potential translation initiation codons in some ALATS mRNAs. Translation from the upstream AUG would generate an N-terminal extension with features characteristic of mitochondrial targeting peptides. A polyclonal antibody raised against part of the Arabidopsis AlaRS revealed that the Arabidopsis cytosolic and mitochondrial AlaRSs are immunologically similar, suggesting that both isoforms are encoded by the ALATS gene. In vitro experiments confirmed that two polypeptides can be translated from AlATS transcripts, with most ribosomes initiating on the downstream AUG to give the shorter polypeptide corresponding in size to the cytosolic enzyme. The ability of the presequence encoded between the two initiation codons to direct polypeptides to mitochondria was demonstrated by expression of fusion proteins in tobacco protoplasts and in yeast. We conclude that the ALATS gene encodes both the cytosolic and the mitochondrial forms of AlaRS, depending on which of the two AUG codons is used to initiate translation. PMID:8672889

  14. Genes encoding calmodulin-binding proteins in the Arabidopsis genome

    NASA Technical Reports Server (NTRS)

    Reddy, Vaka S.; Ali, Gul S.; Reddy, Anireddy S N.

    2002-01-01

    Analysis of the recently completed Arabidopsis genome sequence indicates that approximately 31% of the predicted genes could not be assigned to functional categories, as they do not show any sequence similarity with proteins of known function from other organisms. Calmodulin (CaM), a ubiquitous and multifunctional Ca(2+) sensor, interacts with a wide variety of cellular proteins and modulates their activity/function in regulating diverse cellular processes. However, the primary amino acid sequence of the CaM-binding domain in different CaM-binding proteins (CBPs) is not conserved. One way to identify most of the CBPs in the Arabidopsis genome is by protein-protein interaction-based screening of expression libraries with CaM. Here, using a mixture of radiolabeled CaM isoforms from Arabidopsis, we screened several expression libraries prepared from flower meristem, seedlings, or tissues treated with hormones, an elicitor, or a pathogen. Sequence analysis of 77 positive clones that interact with CaM in a Ca(2+)-dependent manner revealed 20 CBPs, including 14 previously unknown CBPs. In addition, by searching the Arabidopsis genome sequence with the newly identified and known plant or animal CBPs, we identified a total of 27 CBPs. Among these, 16 CBPs are represented by families with 2-20 members in each family. Gene expression analysis revealed that CBPs and CBP paralogs are expressed differentially. Our data suggest that Arabidopsis has a large number of CBPs including several plant-specific ones. Although CaM is highly conserved between plants and animals, only a few CBPs are common to both plants and animals. Analysis of Arabidopsis CBPs revealed the presence of a variety of interesting domains. Our analyses identified several hypothetical proteins in the Arabidopsis genome as CaM targets, suggesting their involvement in Ca(2+)-mediated signaling networks.

  15. Genes encoding calmodulin-binding proteins in the Arabidopsis genome.

    PubMed

    Reddy, Vaka S; Ali, Gul S; Reddy, Anireddy S N

    2002-03-22

    Analysis of the recently completed Arabidopsis genome sequence indicates that approximately 31% of the predicted genes could not be assigned to functional categories, as they do not show any sequence similarity with proteins of known function from other organisms. Calmodulin (CaM), a ubiquitous and multifunctional Ca(2+) sensor, interacts with a wide variety of cellular proteins and modulates their activity/function in regulating diverse cellular processes. However, the primary amino acid sequence of the CaM-binding domain in different CaM-binding proteins (CBPs) is not conserved. One way to identify most of the CBPs in the Arabidopsis genome is by protein-protein interaction-based screening of expression libraries with CaM. Here, using a mixture of radiolabeled CaM isoforms from Arabidopsis, we screened several expression libraries prepared from flower meristem, seedlings, or tissues treated with hormones, an elicitor, or a pathogen. Sequence analysis of 77 positive clones that interact with CaM in a Ca(2+)-dependent manner revealed 20 CBPs, including 14 previously unknown CBPs. In addition, by searching the Arabidopsis genome sequence with the newly identified and known plant or animal CBPs, we identified a total of 27 CBPs. Among these, 16 CBPs are represented by families with 2-20 members in each family. Gene expression analysis revealed that CBPs and CBP paralogs are expressed differentially. Our data suggest that Arabidopsis has a large number of CBPs including several plant-specific ones. Although CaM is highly conserved between plants and animals, only a few CBPs are common to both plants and animals. Analysis of Arabidopsis CBPs revealed the presence of a variety of interesting domains. Our analyses identified several hypothetical proteins in the Arabidopsis genome as CaM targets, suggesting their involvement in Ca(2+)-mediated signaling networks.

  16. Transcriptional regulation of genes encoding arabinan-degrading enzymes in Bacillus subtilis.

    PubMed

    Raposo, Maria Paiva; Inácio, José Manuel; Mota, Luís Jaime; de Sá-Nogueira, Isabel

    2004-03-01

    Bacillus subtilis produces hemicellulases capable of releasing arabinosyl oligomers and arabinose from plant cell walls. In this work, we characterize the transcriptional regulation of three genes encoding arabinan-degrading enzymes that are clustered with genes encoding enzymes that further catabolize arabinose. The abfA gene comprised in the metabolic operon araABDLMNPQ-abfA and the xsa gene located 23 kb downstream most probably encode alpha-L-arabinofuranosidases (EC 3.2.1.55). Here, we show that the abnA gene, positioned immediately upstream from the metabolic operon, encodes an endo-alpha-1,5-arabinanase (EC 3.2.1.99). Furthermore, by in vivo RNA studies, we inferred that abnA and xsa are monocistronic and are transcribed from sigma(A)-like promoters. Transcriptional fusion analysis revealed that the expression of the three arabinases is induced by arabinose and arabinan and is repressed by glucose. The levels of induction by arabinose and arabinan are higher during early postexponential growth, suggesting a temporal regulation. Moreover, the induction mechanism of these genes is mediated through negative control by the key regulator of arabinose metabolism, AraR. Thus, we analyzed AraR-DNA interactions by in vitro quantitative DNase I footprinting and in vivo analysis of single-base-pair substitutions within the promoter regions of xsa and abnA. The results indicate that transcriptional repression of the abfA and xsa genes is achieved by a tightly controlled mechanism but that the regulation of abnA is more flexible. We suggest that the expression of genes encoding extracellular degrading enzymes of arabinose-containing polysaccharides, transport systems, and intracellular enzymes involved in further catabolism is regulated by a coordinate mechanism triggered by arabinose via AraR.

  17. A hydrogenase-linked gene in Methanobacterium thermoautotrophicum strain delta H encodes a polyferredoxin.

    PubMed Central

    Reeve, J N; Beckler, G S; Cram, D S; Hamilton, P T; Brown, J W; Krzycki, J A; Kolodziej, A F; Alex, L; Orme-Johnson, W H; Walsh, C T

    1989-01-01

    The genes mvhDGA, which encode the subunit polypeptides of the methyl viologen-reducing hydrogenase in Methanobacterium thermoautotrophicum strain delta H, have been cloned and sequenced. These genes, together with a fourth open reading frame designated mvhB, are tightly linked and appear to form an operon that is transcribed starting 42 base pairs upstream of mvhD. The organization and sequences of the mvhG and mvhA genes indicate a common evolutionary ancestry with genes encoding the small and large subunits of hydrogenases in eubacterial species. The product of the mvhB gene is predicted to contain six tandomly repeated bacterial-ferredoxin-like domains and, therefore, is predicted to be a polyferredoxin that could contain as many as 48 iron atoms in 12 Fe4S4 clusters. PMID:2654933

  18. Role of sirtuins in lifespan regulation is linked to methylation of nicotinamide.

    PubMed

    Schmeisser, Kathrin; Mansfeld, Johannes; Kuhlow, Doreen; Weimer, Sandra; Priebe, Steffen; Heiland, Ines; Birringer, Marc; Groth, Marco; Segref, Alexandra; Kanfi, Yariv; Price, Nathan L; Schmeisser, Sebastian; Schuster, Stefan; Pfeiffer, Andreas F H; Guthke, Reinhard; Platzer, Matthias; Hoppe, Thorsten; Cohen, Haim Y; Zarse, Kim; Sinclair, David A; Ristow, Michael

    2013-11-01

    Sirtuins, a family of histone deacetylases, have a fiercely debated role in regulating lifespan. In contrast with recent observations, here we find that overexpression of sir-2.1, the ortholog of mammalian SirT1, does extend Caenorhabditis elegans lifespan. Sirtuins mandatorily convert NAD(+) into nicotinamide (NAM). We here find that NAM and its metabolite, 1-methylnicotinamide (MNA), extend C. elegans lifespan, even in the absence of sir-2.1. We identify a previously unknown C. elegans nicotinamide-N-methyltransferase, encoded by a gene now named anmt-1, to generate MNA from NAM. Disruption and overexpression of anmt-1 have opposing effects on lifespan independent of sirtuins, with loss of anmt-1 fully inhibiting sir-2.1-mediated lifespan extension. MNA serves as a substrate for a newly identified aldehyde oxidase, GAD-3, to generate hydrogen peroxide, which acts as a mitohormetic reactive oxygen species signal to promote C. elegans longevity. Taken together, sirtuin-mediated lifespan extension depends on methylation of NAM, providing an unexpected mechanistic role for sirtuins beyond histone deacetylation.

  19. The euryhaline yeast Debaryomyces hansenii has two catalase genes encoding enzymes with differential activity profile.

    PubMed

    Segal-Kischinevzky, Claudia; Rodarte-Murguía, Beatriz; Valdés-López, Victor; Mendoza-Hernández, Guillermo; González, Alicia; Alba-Lois, Luisa

    2011-03-01

    Debaryomyces hansenii is a spoilage yeast able to grow in a variety of ecological niches, from seawater to dairy products. Results presented in this article show that (i) D. hansenii has an inherent resistance to H2O2 which could be attributed to the fact that this yeast has a basal catalase activity which is several-fold higher than that observed in Saccharomyces cerevisiae under the same culture conditions, (ii) D. hansenii has two genes (DhCTA1 and DhCTT1) encoding two catalase isozymes with a differential enzymatic activity profile which is not strictly correlated with a differential expression profile of the encoding genes.

  20. Human Genetic Disorders Caused by Mutations in Genes Encoding Biosynthetic Enzymes for Sulfated Glycosaminoglycans*

    PubMed Central

    Mizumoto, Shuji; Ikegawa, Shiro; Sugahara, Kazuyuki

    2013-01-01

    A number of genetic disorders are caused by mutations in the genes encoding glycosyltransferases and sulfotransferases, enzymes responsible for the synthesis of sulfated glycosaminoglycan (GAG) side chains of proteoglycans, including chondroitin sulfate, dermatan sulfate, and heparan sulfate. The phenotypes of these genetic disorders reflect disturbances in crucial biological functions of GAGs in human. Recent studies have revealed that mutations in genes encoding chondroitin sulfate and dermatan sulfate biosynthetic enzymes cause various disorders of connective tissues. This minireview focuses on growing glycobiological studies of recently described genetic diseases caused by disturbances in biosynthetic enzymes for sulfated GAGs. PMID:23457301

  1. Characterization of the FKBP12-Encoding Genes in Aspergillus fumigatus

    PubMed Central

    Richards, Amber D.; Vargas-Muñiz, José M.; Renshaw, Hilary; Steinbach, William J.

    2015-01-01

    Invasive aspergillosis, largely caused by Aspergillus fumigatus, is responsible for a growing number of deaths among immunosuppressed patients. Immunosuppressants such as FK506 (tacrolimus) that target calcineurin have shown promise for antifungal drug development. FK506-binding proteins (FKBPs) form a complex with calcineurin in the presence of FK506 (FKBP12-FK506) and inhibit calcineurin activity. Research on FKBPs in fungi is limited, and none of the FKBPs have been previously characterized in A. fumigatus. We identified four orthologous genes of FKBP12, the human FK506 binding partner, in A. fumigatus and designated them fkbp12-1, fkbp12-2, fkbp12-3, and fkbp12-4. Deletional analysis of the four genes revealed that the Δfkbp12-1 strain was resistant to FK506, indicating FKBP12-1 as the key mediator of FK506-binding to calcineurin. The endogenously expressed FKBP12-1-EGFP fusion protein localized to the cytoplasm and nuclei under normal growth conditions but also to the hyphal septa following FK506 treatment, revealing its interaction with calcineurin. The FKBP12-1-EGFP fusion protein didn’t localize at the septa in the presence of FK506 in the cnaA deletion background, confirming its interaction with calcineurin. Testing of all deletion strains in the Galleria mellonella model of aspergillosis suggested that these proteins don’t play an important role in virulence. While the Δfkbp12-2 and Δfkbp12-3 strains didn’t show any discernable phenotype, the Δfkbp12-4 strain displayed slight growth defect under normal growth conditions and inhibition of the caspofungin-mediated “paradoxical growth effect” at higher concentrations of the antifungal caspofungin. Together, these results indicate that while only FKBP12-1 is the bona fide binding partner of FK506, leading to the inhibition of calcineurin in A. fumigatus, FKBP12-4 may play a role in basal growth and the caspofungin-mediated paradoxical growth response. Exploitation of differences between A

  2. Association of the gene encoding neurogranin with schizophrenia in males.

    PubMed

    Ruano, Dina; Aulchenko, Yurii S; Macedo, António; Soares, Maria J; Valente, José; Azevedo, Maria H; Hutz, Mara H; Gama, Clarissa S; Lobato, Maria I; Belmonte-de-Abreu, Paulo; Goodman, Ann B; Pato, Carlos; Heutink, Peter; Palha, Joana A

    2008-01-01

    The neurogranin (NRGN) gene produces a postsynaptic brain-specific protein that regulates calmodulin-Ca(2+) availability in neurons. Acting downstream of the NMDA receptor and upstream of calcineurin and other proteins implicated in schizophrenia, NRGN is a good candidate for association studies in schizophrenia. NRGN expression is regulated during development and is modulated by thyroid hormones and retinoids, molecules essential for the proper development of the central nervous system. Given the genetic complexity of schizophrenia and the potential genetic heterogeneity in different populations, we studied a possible association of NRGN with schizophrenia in 73 Azorean proband-parent triads and in two independent case-control samples from the Portuguese-mainland (244 schizophrenic and 210 controls) and Brazil (69 schizophrenic and 85 mentally healthy individuals). Genotype distribution showed association of the rs7113041 SNP with schizophrenia in males of Portuguese origin, which was confirmed by the analysis of the proband-parent triads. This evidence, implicating NRGN in schizophrenia, introduces another player into the glutamatergic hypothesis of schizophrenia.

  3. Cloning and expression of genes encoding Haemophilus somnus antigens.

    PubMed Central

    Corbeil, L B; Chikami, G; Yarnall, M; Smith, J; Guiney, D G

    1988-01-01

    A genomic library of Haemophilus somnus 2336, a virulent isolate from a calf with pneumonia (later used to reproduce H. somnus experimental pneumonia), was constructed in the cosmid vector pHC79. The gene bank in Escherichia coli DH1 was screened by filter immunoassay with convalescent-phase serum, which reacted with several outer membrane antigens of H. somnus. On Western blotting (immunoblotting) of immunoreactive colonies, five clones were found to express proteins which comigrated with H. somnus surface antigens. Three clones (DH1 pHS1, pHS3, and pHS4) expressed both a 120-kilodalton (kDa) antigen and a 76-kDa antigen, one clone (DH1 pHS2) expressed only the 76-kDa antigen, and the fifth clone (DH1 pHS5) expressed a 60-kDa antigen. The 120-kDa and 76-kDa antigens were found internally, whereas the 60-kDa protein was detected in the DH1 pHS5 culture supernatant as membrane blebs or insoluble protein. Both the H. somnus 120-kDa antigen and the recombinant 120-kDa antigen had immunoglobulin Fc-binding activity. Restriction endonuclease mapping demonstrated that the genomic DNA inserts of clones expressing the 76-kDa antigen shared a common 28.4-kilobase-pair region, and the three clones also expressing the 120-kDa antigen shared an additional 7.0-kilobase-pair region. The restriction endonuclease map of pHS5, which expressed the 60-kDa antigen, was not similar to the maps of the other four plasmids. Since these three H. somnus antigens reacted with protective convalescent-phase serum, the recombinants which express these proteins should be useful in further studies of protective immunity in bovine H. somnus disease. Images PMID:2843469

  4. Structure, Expression, Chromosomal Location and Product of the Gene Encoding Adh2 in Petunia

    PubMed Central

    Gregerson, R. G.; Cameron, L.; McLean, M.; Dennis, P.; Strommer, J.

    1993-01-01

    In most higher plants the genes encoding alcohol dehydrogenase comprise a small gene family, usually with two members. The Adh1 gene of Petunia has been cloned and analyzed, but a second identifiable gene was not recovered from any of three genomic libraries. We have therefore employed the polymerase chain reaction to obtain the major portion of a second Adh gene. From sequence, mapping and northern data we conclude this gene encodes ADH2, the major anaerobically inducible Adh gene of Petunia. The availability of both Adh1 and Adh2 from Petunia has permitted us to compare their structures and patterns of expression to those of the well-studied Adh genes of maize, of which one is highly expressed developmentally, while both are induced in response to hypoxia. Despite their evolutionary distance, evidenced by deduced amino acid sequence as well as taxonomic classification, the pairs of genes are regulated in strikingly similar ways in maize and Petunia. Our findings suggest a significant biological basis for the regulatory strategy employed by these distant species for differential expression of multiple Adh genes. PMID:8096485

  5. Characteristics analysis of the luzA gene encoding chaperone from Photobacterium leiognathi related to bioluminescence.

    PubMed

    Lin, J W; Lin, B J; Chen, H Y; Weng, S F

    1998-03-27

    Nucleotide sequence of the luzA gene (GenBank accession No. AF039303) from Photobacterium leiognathi ATCC 25521 (NCIMB 2193) has been determined, and the chaperone encoded by the luzA gene was deduced. The LuzA chaperone has a calculated M(r) 26,295 and comprises 230 amino acid residues; the hydrophobic alpha-helix N-terminal 21 amino acid residues MKKTIFALLFMSVFI SYPSFA is the leader peptide, therefore the matured LuzA chaperone has a calculated M(r) 23,871 and comprises 209 amino acid residues only. The periplasmic LuzA chaperone is the protein concerned with the protein folding, assembly and stability. The luzA gene and the related genes are closely linked to the sod gene, that encoding Cu/Zn superoxide dismutase enables to enhance bioluminescence of the lux operon; the gene order of the luzA gene and related genes is -ufo'-luzA-ufoI-ufoII-ter->-R&R'-sod-ufo-- >. In trans complementation bioluminoassays in vivo elicit that the LuzA chaperone might be not directly concerned with bioluminescence of the lux operon from P. leiognathi in E. coli, but might enable to stabilize the proteins related to bioluminescence. The unidentified ufoII gene closely linked to the luzA gene is able to enhance bioluminescence.

  6. Intra- and inter-generic transfer of pathogenicity island-encoded virulence genes by cos phages.

    PubMed

    Chen, John; Carpena, Nuria; Quiles-Puchalt, Nuria; Ram, Geeta; Novick, Richard P; Penadés, José R

    2015-05-01

    Bacteriophage-mediated horizontal gene transfer is one of the primary driving forces of bacterial evolution. The pac-type phages are generally thought to facilitate most of the phage-mediated gene transfer between closely related bacteria, including that of mobile genetic elements-encoded virulence genes. In this study, we report that staphylococcal cos-type phages transferred the Staphylococcus aureus pathogenicity island SaPIbov5 to non-aureus staphylococcal species and also to different genera. Our results describe the first intra- and intergeneric transfer of a pathogenicity island by a cos phage, and highlight a gene transfer mechanism that may have important implications for pathogen evolution.

  7. A Gene Selection Method for Microarray Data Based on Binary PSO Encoding Gene-to-Class Sensitivity Information.

    PubMed

    Han, Fei; Yang, Chun; Wu, Ya-Qi; Zhu, Jian-Sheng; Ling, Qing-Hua; Song, Yu-Qing; Huang, De-Shuang

    2017-01-01

    Traditional gene selection methods for microarray data mainly considered the features' relevance by evaluating their utility for achieving accurate predication or exploiting data variance and distribution, and the selected genes were usually poorly explicable. To improve the interpretability of the selected genes as well as prediction accuracy, an improved gene selection method based on binary particle swarm optimization (BPSO) and prior information is proposed in this paper. In the proposed method, BPSO encoding gene-to-class sensitivity (GCS) information is used to perform gene selection. The gene-to-class sensitivity information, extracted from the samples by extreme learning machine (ELM), is encoded into the selection process in four aspects: initializing particles, updating the particles, modifying maximum velocity, and adopting mutation operation adaptively. Constrained by the gene-to-class sensitivity information, the new method can select functional gene subsets which are significantly sensitive to the samples' classes. With the few discriminative genes selected by the proposed method, ELM, K-nearest neighbor and support vector machine classifiers achieve much high prediction accuracy on five public microarray data, which in turn verifies the efficiency and effectiveness of the proposed gene selection method.

  8. Genes encoding Cher-TPR fusion proteins are predominantly found in gene clusters encoding chemosensory pathways with alternative cellular functions.

    PubMed

    Muñoz-Martínez, Francisco; García-Fontana, Cristina; Rico-Jiménez, Miriam; Alfonso, Carlos; Krell, Tino

    2012-01-01

    Chemosensory pathways correspond to major signal transduction mechanisms and can be classified into the functional families flagellum-mediated taxis, type four pili-mediated taxis or pathways with alternative cellular functions (ACF). CheR methyltransferases are core enzymes in all of these families. CheR proteins fused to tetratricopeptide repeat (TPR) domains have been reported and we present an analysis of this uncharacterized family. We show that CheR-TPRs are widely distributed in GRAM-negative but almost absent from GRAM-positive bacteria. Most strains contain a single CheR-TPR and its abundance does not correlate with the number of chemoreceptors. The TPR domain fused to CheR is comparatively short and frequently composed of 2 repeats. The majority of CheR-TPR genes were found in gene clusters that harbor multidomain response regulators in which the REC domain is fused to different output domains like HK, GGDEF, EAL, HPT, AAA, PAS, GAF, additional REC, HTH, phosphatase or combinations thereof. The response regulator architectures coincide with those reported for the ACF family of pathways. Since the presence of multidomain response regulators is a distinctive feature of this pathway family, we conclude that CheR-TPR proteins form part of ACF type pathways. The diversity of response regulator output domains suggests that the ACF pathways form a superfamily which regroups many different regulatory mechanisms, in which all CheR-TPR proteins appear to participate. In the second part we characterize WspC of Pseudomonas putida, a representative example of CheR-TPR. The affinities of WspC-Pp for S-adenosylmethionine and S-adenosylhomocysteine were comparable to those of prototypal CheR, indicating that WspC-Pp activity is in analogy to prototypal CheRs controlled by product feed-back inhibition. The removal of the TPR domain did not impact significantly on the binding constants and consequently not on the product feed-back inhibition. WspC-Pp was found to be

  9. Identification of nuclear genes encoding chloroplast-localized proteins required for embryo development in Arabidopsis.

    PubMed

    Bryant, Nicole; Lloyd, Johnny; Sweeney, Colleen; Myouga, Fumiyoshi; Meinke, David

    2011-04-01

    We describe here the diversity of chloroplast proteins required for embryo development in Arabidopsis (Arabidopsis thaliana). Interfering with certain chloroplast functions has long been known to result in embryo lethality. What has not been reported before is a comprehensive screen for embryo-defective (emb) mutants altered in chloroplast proteins. From a collection of transposon and T-DNA insertion lines at the RIKEN chloroplast function database (http://rarge.psc.riken.jp/chloroplast/) that initially appeared to lack homozygotes and segregate for defective seeds, we identified 23 additional examples of EMB genes that likely encode chloroplast-localized proteins. Fourteen gene identities were confirmed with allelism tests involving duplicate mutant alleles. We then queried journal publications and the SeedGenes database (www.seedgenes.org) to establish a comprehensive dataset of 381 nuclear genes encoding chloroplast proteins of Arabidopsis associated with embryo-defective (119 genes), plant pigment (121 genes), gametophyte (three genes), and alternate (138 genes) phenotypes. Loci were ranked based on the level of certainty that the gene responsible for the phenotype had been identified and the protein product localized to chloroplasts. Embryo development is frequently arrested when amino acid, vitamin, or nucleotide biosynthesis is disrupted but proceeds when photosynthesis is compromised and when levels of chlorophyll, carotenoids, or terpenoids are reduced. Chloroplast translation is also required for embryo development, with genes encoding chloroplast ribosomal and pentatricopeptide repeat proteins well represented among EMB datasets. The chloroplast accD locus, which is necessary for fatty acid biosynthesis, is essential in Arabidopsis but not in Brassica napus or maize (Zea mays), where duplicated nuclear genes compensate for its absence or loss of function.

  10. Characterization of Genes Encoding for Acquired Bacitracin Resistance in Clostridium perfringens

    PubMed Central

    Charlebois, Audrey; Jalbert, Louis-Alexandre; Harel, Josée; Masson, Luke; Archambault, Marie

    2012-01-01

    Phenotypic bacitracin resistance has been reported in Clostridium perfringens. However, the genes responsible for the resistance have not yet been characterized. Ninety-nine C. perfringens isolates recovered from broilers and turkeys were tested for phenotypic bacitracin resistance. Bacitracin MIC90 (>256 µg/ml) was identical for both turkey and chicken isolates; whereas MIC50 was higher in turkey isolates (6 µg/ml) than in chicken isolates (3 µg/ml). Twenty-four of the 99 isolates showed high-level bacitracin resistance (MIC breakpoint >256 µg/ml) and the genes encoding for this resistance were characterized in C. perfringens c1261_A strain using primer walking. Sequence analysis and percentages of amino acid identity revealed putative genes encoding for both an ABC transporter and an overproduced undecaprenol kinase in C. perfringens c1261_A strain. These two mechanisms were shown to be both encoded by the putative bcrABD operon under the control of a regulatory gene, bcrR. Efflux pump inhibitor thioridazine was shown to increase significantly the susceptibility of strain c1261_A to bacitracin. Upstream and downstream from the bcr cluster was an IS1216-like element, which may play a role in the dissemination of this resistance determinant. Pulsed-field gel electrophoresis with prior double digestion with I-CeuI/MluI enzymes followed by hybridization analyses revealed that the bacitracin resistance genes bcrABDR were located on the chromosome. Semi-quantitative RT-PCR demonstrated that this gene cluster is expressed under bacitracin stress. Microarray analysis revealed the presence of these genes in all bacitracin resistant strains. This study reports the discovery of genes encoding for a putative ABC transporter and an overproduced undecaprenol kinase associated with high-level bacitracin resistance in C. perfringens isolates from turkeys and broiler chickens. PMID:22970221

  11. Bacillus subtilis acyl carrier protein is encoded in a cluster of lipid biosynthesis genes.

    PubMed Central

    Morbidoni, H R; de Mendoza, D; Cronan, J E

    1996-01-01

    A cluster of Bacillus subtilis fatty acid synthetic genes was isolated by complementation of an Escherichia coli fabD mutant encoding a thermosensitive malonyl coenzyme A-acyl carrier protein transacylase. The B. subtilis genomic segment contains genes that encode three fatty acid synthetic proteins, malonyl coenzyme A-acyl carrier protein transacylase (fabD), 3-ketoacyl-acyl carrier protein reductase (fabG), and the N-terminal 14 amino acid residues of acyl carrier protein (acpP). Also present is a sequence that encodes a homolog of E. coli plsX, a gene that plays a poorly understood role in phospholipid synthesis. The B. subtilis plsX gene weakly complemented an E. coli plsX mutant. The order of genes in the cluster is plsX fabD fabG acpP, the same order found in E. coli, except that in E. coli the fabH gene lies between plsX and fabD. The absence of fabH in the B. subtilis cluster is consistent with the different fatty acid compositions of the two organisms. The amino acid sequence of B. subtilis acyl carrier protein was obtained by sequencing the purified protein, and the sequence obtained strongly resembled that of E. coli acyl carrier protein, except that most of the protein retained the initiating methionine residue. The B. subtilis fab cluster was mapped to the 135 to 145 degrees region of the chromosome. PMID:8759840

  12. Characterization of Genes Encoding Key Enzymes Involved in Anthocyanin Metabolism of Kiwifruit during Storage Period

    PubMed Central

    Li, Boqiang; Xia, Yongxiu; Wang, Yuying; Qin, Guozheng; Tian, Shiping

    2017-01-01

    ‘Hongyang’ is a red fleshed kiwifruit with high anthocyanin content. In this study, we mainly investigated effects of different temperatures (25 and 0°C) on anthocyanin biosynthesis in harvested kiwifruit, and characterized the genes encoding key enzymes involved in anthocyanin metabolism, as well as evaluated the mode of the action, by which low temperature regulates anthocyanin accumulation in ‘Hongyang’ kiwifruit during storage period. The results showed that low temperature could effectively enhance the anthocyanin accumulation of kiwifruit in the end of storage period (90 days), which related to the increase in mRNA levels of ANS1, ANS2, DRF1, DRF2, and UGFT2. Moreover, the transcript abundance of MYBA1-1 and MYB5-1, the genes encoding an important component of MYB–bHLH–WD40 (MBW) complex, was up-regulated, possibly contributing to the induction of specific anthocyanin biosynthesis genes under the low temperature. To further investigate the roles of AcMYB5-1/5-2/A1-1 in regulation of anthocyanin biosynthesis, genes encoding the three transcription factors were transiently transformed in Nicotiana benthamiana leaves. Overexpression of AcMYB5-1/5-2/A1-1 activated the gene expression of NtANS and NtDFR in tobacco. Our results suggested that low temperature storage could stimulate the anthocyanin accumulation in harvested kiwifruit via regulating several structural and regulatory genes involved in anthocyanin biosynthesis. PMID:28344589

  13. Unusually high frequency of genes encoding vegetative insecticidal proteins in an Australian Bacillus thuringiensis collection.

    PubMed

    Beard, Cheryl E; Court, Leon; Boets, Annemie; Mourant, Roslyn; Van Rie, Jeroen; Akhurst, Raymond J

    2008-09-01

    Of 188 Australian Bacillus thuringiensis strains screened for genes encoding soluble insecticidal proteins by polymerase chain reaction/restriction-length fragment polymorphism (RFLP) analysis, 87% showed the presence of such genes. Although 135 isolates (72%) produced an RFLP pattern identical to that expected for vip3A genes, 29 isolates possessed a novel vip-like gene. The novel vip-like gene was cloned from B. thuringiensis isolate C81, and sequence analysis demonstrated that it was 94% identical to the vip3Ba1 gene. The new gene was designated vip3Bb2. Cell-free supernatants from both the B. thuringiensis strain C81 and from Escherichia coli expressing the Vip3Bb2 protein were toxic for the cotton bollworm, Helicoverpa armigera.

  14. Analyses of nuclearly encoded mitochondrial genes suggest gene duplication as a mechanism for resolving intralocus sexually antagonistic conflict in Drosophila.

    PubMed

    Gallach, Miguel; Chandrasekaran, Chitra; Betrán, Esther

    2010-01-01

    Gene duplication is probably the most important mechanism for generating new gene functions. However, gene duplication has been overlooked as a potentially effective way to resolve genetic conflicts. Here, we analyze the entire set of Drosophila melanogaster nuclearly encoded mitochondrial duplicate genes and show that both RNA- and DNA-mediated mitochondrial gene duplications exhibit an unexpectedly high rate of relocation (change in location between parental and duplicated gene) as well as an extreme tendency to avoid the X chromosome. These trends are likely related to our observation that relocated genes tend to have testis-specific expression. We also infer that these trends hold across the entire Drosophila genus. Importantly, analyses of gene ontology and functional interaction networks show that there is an overrepresentation of energy production-related functions in these mitochondrial duplicates. We discuss different hypotheses to explain our results and conclude that our findings substantiate the hypothesis that gene duplication for male germline function is likely a mechanism to resolve intralocus sexually antagonistic conflicts that we propose are common in testis. In the case of nuclearly encoded mitochondrial duplicates, our hypothesis is that past sexually antagonistic conflict related to mitochondrial energy function in Drosophila was resolved by gene duplication.

  15. Screening of the Enterocin-Encoding Genes and Antimicrobial Activity in Enterococcus Species.

    PubMed

    Ogaki, Mayara Baptistucci; Rocha, Katia Real; Terra, MÁrcia Regina; Furlaneto, MÁrcia Cristina; Maia, Luciana Furlaneto

    2016-06-28

    In the current study, a total of 135 enterococci strains from different sources were screened for the presence of the enterocin-encoding genes entA, entP, entB, entL50A, and entL50B. The enterocin genes were present at different frequencies, with entA occurring the most frequently, followed by entP and entB; entL50A and L50B were not detected. The occurrence of single enterocin genes was higher than the occurrence of multiple enterocin gene combinations. The 80 isolates that harbor at least one enterocin-encoding gene (denoted "Gene(+) strains") were screened for antimicrobial activity. A total of 82.5% of the Gene(+) strains inhibited at least one of the indicator strains, and the isolates harboring multiple enterocin-encoding genes inhibited a larger number of indicator strains than isolates harboring a single gene. The indicator strains that exhibited growth inhibition included Listeria innocua strain CLIP 12612 (ATCC BAA-680), Listeria monocytogenes strain CDC 4555, Enterococcus faecalis ATCC 29212, Staphylococcus aureus ATCC 25923, S. aureus ATCC 29213, S. aureus ATCC 6538, Salmonella enteritidis ATCC 13076, Salmonella typhimurium strain UK-1 (ATCC 68169), and Escherichia coli BAC 49LT ETEC. Inhibition due to either bacteriophage lysis or cytolysin activity was excluded. The growth inhibition of antilisterial Gene+ strains was further tested under different culture conditions. Among the culture media formulations, the MRS agar medium supplemented with 2% (w/v) yeast extract was the best solidified medium for enterocin production. Our findings extend the current knowledge of enterocin-producing enterococci, which may have potential applications as biopreservatives in the food industry due to their capability of controlling food spoilage pathogens.

  16. A Single Gene May Encode Differentially Localized Ca2+-ATPases in Tomato.

    PubMed Central

    Ferrol, N; Bennett, AB

    1996-01-01

    Previously, a partial-length cDNA and a complete genomic clone encoding a putative sarcoplasmic reticulum-type Ca2+-ATPase (LCA, Lycopersicon Ca2+-ATPase) were isolated from tomato. To determine the subcellular localization of this Ca2+-ATPase, specific polyclonal antibodies raised against a fusion protein encoding a portion of the LCA polypeptide were generated. Based on hybridization of the LCA cDNA and of the nucleotide sequence encoding the fusion protein to genomic DNA, it appears that LCA and the fusion protein domain are encoded by a single gene in tomato. Antibodies raised against the LCA domain fusion protein reacted specifically with two polypeptides of 116 and 120 kD that are localized in the vacuolar and plasma membranes, respectively. The distribution of vanadate-sensitive ATP-dependent Ca2+ transport activities in sucrose gradients coincided with the distribution of the immunodetected proteins. The ATP-dependent Ca2+ transport activities associated with tonoplast and plasma membrane fractions shared similar properties, because both fractions were inhibited by vanadate but insensitive to carbonyl cyanide m-chlorophenylhydrazone, nitrate, and calmodulin. Moreover, antibodies raised against the LCA domain fusion protein inhibited ATP-dependent Ca2+ uptake activity associated with both the tonoplast and plasma membrane fractions. These data suggest that a single gene (LCA) may encode two P-type Ca2+-ATPase isoforms that are differentially localized in the tonoplast and plasma membrane of tomato roots. PMID:12239413

  17. Nicotinamide riboside restores cognition through an upregulation of proliferator-activated receptor-γ coactivator 1α regulated β-secretase 1 degradation and mitochondrial gene expression in Alzheimer's mouse models.

    PubMed

    Gong, Bing; Pan, Yong; Vempati, Prashant; Zhao, Wei; Knable, Lindsay; Ho, Lap; Wang, Jun; Sastre, Magdalena; Ono, Kenjiro; Sauve, Anthony A; Pasinetti, Giulio M

    2013-06-01

    Nicotinamide adenine dinucleotide (NAD)(+), a coenzyme involved in redox activities in the mitochondrial electron transport chain, has been identified as a key regulator of the lifespan-extending effects, and the activation of NAD(+) expression has been linked with a decrease in beta-amyloid (Aβ) toxicity in Alzheimer's disease (AD). Nicotinamide riboside (NR) is a NAD(+) precursor, it promotes peroxisome proliferator-activated receptor-γ coactivator 1 (PGC)-1α expression in the brain. Evidence has shown that PGC-1α is a crucial regulator of Aβ generation because it affects β-secretase (BACE1) degradation. In this study we tested the hypothesis that NR treatment in an AD mouse model could attenuate Aβ toxicity through the activation of PGC-1α-mediated BACE1 degradation. Using the Tg2576 AD mouse model, using in vivo behavioral analyses, biochemistry assays, small hairpin RNA (shRNA) gene silencing and electrophysiological recording, we found (1) dietary treatment of Tg2576 mice with 250 mg/kg/day of NR for 3 months significantly attenuates cognitive deterioration in Tg2576 mice and coincides with an increase in the steady-state levels of NAD(+) in the cerebral cortex; (2) application of NR to hippocampal slices (10 μM) for 4 hours abolishes the deficits in long-term potentiation recorded in the CA1 region of Tg2576 mice; (3) NR treatment promotes PGC-1α expression in the brain coinciding with enhanced degradation of BACE1 and the reduction of Aβ production in Tg2576 mice. Further in vitro studies confirmed that BACE1 protein content is decreased by NR treatment in primary neuronal cultures derived from Tg2576 embryos, in which BACE1 degradation was prevented by PGC-1α-shRNA gene silencing; and (4) NR treatment and PGC-1α overexpression enhance BACE1 ubiquitination and proteasomal degradation. Our studies suggest that dietary treatment with NR might benefit AD cognitive function and synaptic plasticity, in part by promoting PGC-1α-mediated BACE1

  18. Organization of the genes encoding [Fe] hydrogenase in Desulfovibrio vulgaris subsp. oxamicus Monticello.

    PubMed Central

    Voordouw, G; Strang, J D; Wilson, F R

    1989-01-01

    The genes encoding the periplasmic [Fe] hydrogenase from Desulfovibrio vulgaris subsp. oxamicus Monticello were cloned by exploiting their homology with the hydAB genes from D. vulgaris subsp. vulgaris Hildenborough, in which this enzyme is present as a heterologous dimer of alpha and beta subunits. Nucleotide sequencing showed that the enzyme is encoded by an operon in which the gene for the 46-kilodalton (kDa) alpha subunit precedes that of the 13.5-kDa beta subunit, exactly as in the Hildenborough strain. The pairs of hydA and hydB genes are highly homologous; both alpha subunits (420 amino acid residues) share 79% sequence identity, while the unprocessed beta subunits (124 and 123 amino acid residues, respectively) share 71% sequence identity. In contrast, there appears to be no sequence homology outside these coding regions, with the exception of a possible promoter element, which was found approximately 90 base pairs upstream from the translational start of the hydA gene. The recently discovered hydC gene, which may code for a 65.8-kDa fusion protein (gamma) of the alpha and beta subunits and is present immediately downstream from the hydAB genes in the Hildenborough strain, was found to be absent from the Monticello strain. The implication of this result for the possible function of the hydC gene product in Desulfovibrio species is discussed. Images PMID:2661538

  19. The maize brown midrib4 (bm4) gene encodes a functional folylpolyglutamate synthase

    PubMed Central

    Li, Li; Hill-Skinner, Sarah; Liu, Sanzhen; Beuchle, Danielle; Tang, Ho Man; Yeh, Cheng-Ting; Nettleton, Dan; Schnable, Patrick S

    2015-01-01

    Mutations in the brown midrib4 (bm4) gene affect the accumulation and composition of lignin in maize. Fine-mapping analysis of bm4 narrowed the candidate region to an approximately 105 kb interval on chromosome 9 containing six genes. Only one of these six genes, GRMZM2G393334, showed decreased expression in mutants. At least four of 10 Mu-induced bm4 mutant alleles contain a Mu insertion in the GRMZM2G393334 gene. Based on these results, we concluded that GRMZM2G393334 is the bm4 gene. GRMZM2G393334 encodes a putative folylpolyglutamate synthase (FPGS), which functions in one-carbon (C1) metabolism to polyglutamylate substrates of folate-dependent enzymes. Yeast complementation experiments demonstrated that expression of the maize bm4 gene in FPGS-deficient met7 yeast is able to rescue the yeast mutant phenotype, thus demonstrating that bm4 encodes a functional FPGS. Consistent with earlier studies, bm4 mutants exhibit a modest decrease in lignin concentration and an overall increase in the S:G lignin ratio relative to wild-type. Orthologs of bm4 include at least one paralogous gene in maize and various homologs in other grasses and dicots. Discovery of the gene underlying the bm4 maize phenotype illustrates a role for FPGS in lignin biosynthesis. PMID:25495051

  20. Structure and expression of nuclear genes encoding rubisco activase. Final technical report

    SciTech Connect

    Zielinski, R.E.

    1994-06-01

    Rubisco activase (Rca) is a soluble chloroplast protein that catalyzes the activation of rubisco, the enzyme that initiates the photosynthetic carbon reduction cycle, to catalytic competency. Rca in barley consists of three polypeptides, one of 46- and two of 42-kDa, but the quaternary structure of the protein is not known. The authors have isolated and completely sequenced 8.8 kb of barley genomic DNA containing two, tandemly oriented activase genes (RcaA and RcaB) and three different cDNAs encoding the 42- and 46-kDa Rca polypeptide isoforms. Genomic Southern blot assays indicate that these sequences represent the entire Rca gene family in barley. Pre-mRNAs transcribed from the RcaA gene are alternatively spliced to give mRNAs encoding both 46- (RcaA1) and 42-kDa (RcaA2) Rca isoforms. The RcaB gene encodes a single polypeptide of 42 kDa. Primer extension and northern blot assays indicate that RcaB mRNA is expressed at a level that is 10- to 100-fold lower than RcaA mRNA. Analyses at the mRNA and protein level showed that Rca gene expression is coordinated by that of the rubisco subunits during barley leaf development.

  1. Systematic Identification and Characterization of Novel Human Skin-Associated Genes Encoding Membrane and Secreted Proteins

    PubMed Central

    Buhren, Bettina Alexandra; Martinez, Cynthia; Schrumpf, Holger; Gasis, Marcia; Grether-Beck, Susanne; Krutmann, Jean

    2013-01-01

    Through bioinformatics analyses of a human gene expression database representing 105 different tissues and cell types, we identified 687 skin-associated genes that are selectively and highly expressed in human skin. Over 50 of these represent uncharacterized genes not previously associated with skin and include a subset that encode novel secreted and plasma membrane proteins. The high levels of skin-associated expression for eight of these novel therapeutic target genes were confirmed by semi-quantitative real time PCR, western blot and immunohistochemical analyses of normal skin and skin-derived cell lines. Four of these are expressed specifically by epidermal keratinocytes; two that encode G-protein-coupled receptors (GPR87 and GPR115), and two that encode secreted proteins (WFDC5 and SERPINB7). Further analyses using cytokine-activated and terminally differentiated human primary keratinocytes or a panel of common inflammatory, autoimmune or malignant skin diseases revealed distinct patterns of regulation as well as disease associations that point to important roles in cutaneous homeostasis and disease. Some of these novel uncharacterized skin genes may represent potential biomarkers or drug targets for the development of future diagnostics or therapeutics. PMID:23840300

  2. Identification and Functional Analysis of Trypanosoma cruzi Genes That Encode Proteins of the Glycosylphosphatidylinositol Biosynthetic Pathway

    PubMed Central

    Cardoso, Mariana S.; Junqueira, Caroline; Trigueiro, Ricardo C.; Shams-Eldin, Hosam; Macedo, Cristiana S.; Araújo, Patrícia R.; Gomes, Dawidson A.; Martinelli, Patrícia M.; Kimmel, Jürgen; Stahl, Philipp; Niehus, Sebastian; Schwarz, Ralph T.; Previato, José O.; Mendonça-Previato, Lucia; Gazzinelli, Ricardo T.; Teixeira, Santuza M. R.

    2013-01-01

    Background Trypanosoma cruzi is a protist parasite that causes Chagas disease. Several proteins that are essential for parasite virulence and involved in host immune responses are anchored to the membrane through glycosylphosphatidylinositol (GPI) molecules. In addition, T. cruzi GPI anchors have immunostimulatory activities, including the ability to stimulate the synthesis of cytokines by innate immune cells. Therefore, T. cruzi genes related to GPI anchor biosynthesis constitute potential new targets for the development of better therapies against Chagas disease. Methodology/Principal Findings In silico analysis of the T. cruzi genome resulted in the identification of 18 genes encoding proteins of the GPI biosynthetic pathway as well as the inositolphosphorylceramide (IPC) synthase gene. Expression of GFP fusions of some of these proteins in T. cruzi epimastigotes showed that they localize in the endoplasmic reticulum (ER). Expression analyses of two genes indicated that they are constitutively expressed in all stages of the parasite life cycle. T. cruzi genes TcDPM1, TcGPI10 and TcGPI12 complement conditional yeast mutants in GPI biosynthesis. Attempts to generate T. cruzi knockouts for three genes were unsuccessful, suggesting that GPI may be an essential component of the parasite. Regarding TcGPI8, which encodes the catalytic subunit of the transamidase complex, although we were able to generate single allele knockout mutants, attempts to disrupt both alleles failed, resulting instead in parasites that have undergone genomic recombination and maintained at least one active copy of the gene. Conclusions/Significance Analyses of T. cruzi sequences encoding components of the GPI biosynthetic pathway indicated that they are essential genes involved in key aspects of host-parasite interactions. Complementation assays of yeast mutants with these T. cruzi genes resulted in yeast cell lines that can now be employed in high throughput screenings of drugs against this

  3. UBA 1: an essential yeast gene encoding ubiquitin-activating enzyme.

    PubMed Central

    McGrath, J P; Jentsch, S; Varshavsky, A

    1991-01-01

    All known functions of ubiquitin are mediated through its covalent attachment to other proteins. The post-translational formation of ubiquitin--protein conjugates is preceded by an ATP-requiring step in which the carboxyl terminus of ubiquitin is adenylated and subsequently joined, through a thiolester bond, to a cysteine residue in the ubiquitin-activating enzyme, also known as E1. We report the isolation and functional analysis of the gene (UBA1) for the ubiquitin-activating enzyme of the yeast Saccharomyces cerevisiae. UBA1 encodes a 114 kd protein whose amino acid sequence contains motifs characteristic of nucleotide-binding sites. Expression of catalytically active UBA1 protein in E. coli, which lacks the ubiquitin system, confirmed that the yeast UBA1 gene encodes a ubiquitin-activating enzyme. Deletion of the UBA1 gene is lethal, demonstrating that the formation of ubiquitin--protein conjugates is essential for cell viability. Images PMID:1989885

  4. Filamentous-haemagglutinin-like protein genes encoded on a plasmid of Moraxella bovis.

    PubMed

    Kakuda, Tsutomu; Sarataphan, Nopporn; Tanaka, Tetsuya; Takai, Shinji

    2006-11-26

    The complete nucleotide sequence of a plasmid, pMBO-1, from Moraxella bovis strain Epp63 was determined. We identified 30 open reading frames (ORFs) encoded by the 44,215bp molecule. Two large ORFs, flpA and flpB, encoding proteins with similarity to Bordetella pertussis filamentous haemagglutinin (FHA), were identified on the same plasmid. The gene for a specific accessory protein (Fap), which may play a role in the secretion of Flp protein, was also identified. Reverse transcriptase PCR analysis of total RNA isolated from M. bovis Epp63 indicated that the flpA, flpB, and fap genes are all transcribed. Southern blot analysis indicated that the flp and fap genes are present in other clinical isolates of geographically diverse M. bovis.

  5. Expression patterns of genes encoding plasma membrane aquaporins during fruit development in cucumber (Cucumis sativus L.).

    PubMed

    Shi, Jin; Wang, Jinfang; Li, Ren; Li, Dianbo; Xu, Fengfeng; Sun, Qianqian; Zhao, Bin; Mao, Ai-Jun; Guo, Yang-Dong

    2015-11-01

    Aquaporins are membrane channels precisely regulating water movement through cell membranes in most living organisms. Despite the advances in the physiology of fruit development, their participation during fruit development in cucumber still barely understood. In this paper, the expressions of 12 genes encoding plasma membrane intrinsic proteins (PIPs) were analyzed during cucumber fruit development in our work. Based on the homology search with known PIPs from rice, Arabidopsis and strawberry, 12 cucumber PIP genes subfamily members were identified. Cellular localization assays indicated that CsPIPs were localized in the plasma membrane. The qRT-PCR analysis of CsPIPs showed that 12 CsPIPs were differentially expressed during fruit development. These results suggest that 12 genes encoding plasma membrane intrinsic proteins (CsPIPs) play very important roles in cucumber life cycle and the data generated will be helpful in understanding their precise roles during fruit development in cucumber.

  6. Cloning and expression analysis of a prion protein encoding gene in guppy ( Poecilia reticulata)

    NASA Astrophysics Data System (ADS)

    Wu, Suihan; Wei, Qiwei; Yang, Guanpin; Wang, Dengqiang; Zou, Guiwei; Chen, Daqing

    2008-11-01

    The full length cDNA of a prion protein (PrP) encoding gene of guppy ( Poecilia reticulata) and the corresponding genomic DNA were cloned. The cDNA was 2245 bp in length and contained an open reading frame (ORF) of 1545 bp encoding a protein of 515 amino acids, which held all typical structural characteristics of the functional PrP. The cloned genomic DNA fragment corresponding to the cDNA was 3720 bp in length, consisting of 2 introns and 2 exons. The 5' untranslated region of cDNA originated from the 2 exons, while the ORF originated from the second exon. Although the gene was transcribed in diverse tissues including brain, eye, liver, intestine, muscle and tail, its transcript was most abundant in the brain. In addition, the transcription of the gene was enhanced by 5 salinity, implying that it was associated with the response of guppy to saline stress.

  7. Campylobacter jejuni gene cj0511 encodes a serine peptidase essential for colonisation

    PubMed Central

    Karlyshev, A.V.; Thacker, G.; Jones, M.A.; Clements, M.O.; Wren, B.W.

    2014-01-01

    According to MEROPS peptidase database, Campylobacter species encode 64 predicted peptidases. However, proteolytic properties of only a few of these proteins have been confirmed experimentally. In this study we identified and characterised a Campylobacter jejuni gene cj0511 encoding a novel peptidase. The proteolytic activity associated with this enzyme was demonstrated in cell lysates. Moreover, enzymatic studies conducted with a purified protein confirmed a prediction of it being a serine peptidase. Furthermore, cj0511 mutant was found to be severely attenuated in chicken colonisation model, suggesting a role of the Cj0511 protein in infection. PMID:24918062

  8. Horse cDNA clones encoding two MHC class I genes

    SciTech Connect

    Barbis, D.P.; Maher, J.K.; Stanek, J.; Klaunberg, B.A.; Antczak, D.F.

    1994-12-31

    Two full-length clones encoding MHC class I genes were isolated by screening a horse cDNA library, using a probe encoding in human HLA-A2.2Y allele. The library was made in the pcDNA1 vector (Invitrogen, San Diego, CA), using mRNA from peripheral blood lymphocytes obtained from a Thoroughbred stallion (No. 0834) homozygous for a common horse MHC haplotype (ELA-A2, -B2, -D2; Antczak et al. 1984; Donaldson et al. 1988). The clones were sequenced, using SP6 and T7 universal primers and horse-specific oligonucleotides designed to extend previously determined sequences.

  9. A mouse speciation gene encodes a meiotic histone H3 methyltransferase.

    PubMed

    Mihola, Ondrej; Trachtulec, Zdenek; Vlcek, Cestmir; Schimenti, John C; Forejt, Jiri

    2009-01-16

    Speciation genes restrict gene flow between the incipient species and related taxa. Three decades ago, we mapped a mammalian speciation gene, hybrid sterility 1 (Hst1), in the intersubspecific hybrids of house mouse. Here, we identify this gene as Prdm9, encoding a histone H3 lysine 4 trimethyltransferase. We rescued infertility in male hybrids with bacterial artificial chromosomes carrying Prdm9 from a strain with the "fertility" Hst1(f) allele. Sterile hybrids display down-regulated microrchidia 2B (Morc2b) and fail to compartmentalize gammaH2AX into the pachynema sex (XY) body. These defects, seen also in Prdm9-null mutants, are rescued by the Prdm9 transgene. Identification of a vertebrate hybrid sterility gene reveals a role for epigenetics in speciation and opens a window to a hybrid sterility gene network.

  10. Analysis of Genes Encoding Penicillin-Binding Proteins in Clinical Isolates of Acinetobacter baumannii ▿ †

    PubMed Central

    Cayô, Rodrigo; Rodríguez, María-Cruz; Espinal, Paula; Fernández-Cuenca, Felipe; Ocampo-Sosa, Alain A.; Pascual, Álvaro; Ayala, Juan A.; Vila, Jordi; Martínez-Martínez, Luis

    2011-01-01

    There is limited information on the role of penicillin-binding proteins (PBPs) in the resistance of Acinetobacter baumannii to β-lactams. This study presents an analysis of the allelic variations of PBP genes in A. baumannii isolates. Twenty-six A. baumannii clinical isolates (susceptible or resistant to carbapenems) from three teaching hospitals in Spain were included. The antimicrobial susceptibility profile, clonal pattern, and genomic species identification were also evaluated. Based on the six complete genomes of A. baumannii, the PBP genes were identified, and primers were designed for each gene. The nucleotide sequences of the genes identified that encode PBPs and the corresponding amino acid sequences were compared with those of ATCC 17978. Seven PBP genes and one monofunctional transglycosylase (MGT) gene were identified in the six genomes, encoding (i) four high-molecular-mass proteins (two of class A, PBP1a [ponA] and PBP1b [mrcB], and two of class B, PBP2 [pbpA or mrdA] and PBP3 [ftsI]), (ii) three low-molecular-mass proteins (two of type 5, PBP5/6 [dacC] and PBP6b [dacD], and one of type 7 (PBP7/8 [pbpG]), and (iii) a monofunctional enzyme (MtgA [mtgA]). Hot spot mutation regions were observed, although most of the allelic changes found translated into silent mutations. The amino acid consensus sequences corresponding to the PBP genes in the genomes and the clinical isolates were highly conserved. The changes found in amino acid sequences were associated with concrete clonal patterns but were not directly related to susceptibility or resistance to β-lactams. An insertion sequence disrupting the gene encoding PBP6b was identified in an endemic carbapenem-resistant clone in one of the participant hospitals. PMID:21947403

  11. Analysis of genes encoding penicillin-binding proteins in clinical isolates of Acinetobacter baumannii.

    PubMed

    Cayô, Rodrigo; Rodríguez, María-Cruz; Espinal, Paula; Fernández-Cuenca, Felipe; Ocampo-Sosa, Alain A; Pascual, Alvaro; Ayala, Juan A; Vila, Jordi; Martínez-Martínez, Luis

    2011-12-01

    There is limited information on the role of penicillin-binding proteins (PBPs) in the resistance of Acinetobacter baumannii to β-lactams. This study presents an analysis of the allelic variations of PBP genes in A. baumannii isolates. Twenty-six A. baumannii clinical isolates (susceptible or resistant to carbapenems) from three teaching hospitals in Spain were included. The antimicrobial susceptibility profile, clonal pattern, and genomic species identification were also evaluated. Based on the six complete genomes of A. baumannii, the PBP genes were identified, and primers were designed for each gene. The nucleotide sequences of the genes identified that encode PBPs and the corresponding amino acid sequences were compared with those of ATCC 17978. Seven PBP genes and one monofunctional transglycosylase (MGT) gene were identified in the six genomes, encoding (i) four high-molecular-mass proteins (two of class A, PBP1a [ponA] and PBP1b [mrcB], and two of class B, PBP2 [pbpA or mrdA] and PBP3 [ftsI]), (ii) three low-molecular-mass proteins (two of type 5, PBP5/6 [dacC] and PBP6b [dacD], and one of type 7 (PBP7/8 [pbpG]), and (iii) a monofunctional enzyme (MtgA [mtgA]). Hot spot mutation regions were observed, although most of the allelic changes found translated into silent mutations. The amino acid consensus sequences corresponding to the PBP genes in the genomes and the clinical isolates were highly conserved. The changes found in amino acid sequences were associated with concrete clonal patterns but were not directly related to susceptibility or resistance to β-lactams. An insertion sequence disrupting the gene encoding PBP6b was identified in an endemic carbapenem-resistant clone in one of the participant hospitals.

  12. Expression of neuropeptide- and hormone-encoding genes in the Ciona intestinalis larval brain.

    PubMed

    Hamada, Mayuko; Shimozono, Naoki; Ohta, Naoyuki; Satou, Yutaka; Horie, Takeo; Kawada, Tsuyoshi; Satake, Honoo; Sasakura, Yasunori; Satoh, Nori

    2011-04-15

    Despite containing only approximately 330 cells, the central nervous system (CNS) of Ciona intestinalis larvae has an architecture that is similar to the vertebrate CNS. Although only vertebrates have a distinct hypothalamus-the source of numerous neurohormone peptides that play pivotal roles in the development, function, and maintenance of various neuronal and endocrine systems, it is suggested that the Ciona brain contains a region that corresponds to the vertebrate hypothalamus. To identify genes expressed in the brain, we isolated brain vesicles using transgenic embryos carrying Ci-β-tubulin(promoter)::Kaede, which resulted in robust Kaede expression in the larval CNS. The associated transcriptome was investigated using microarray analysis. We identified 565 genes that were preferentially expressed in the larval brain. Among these genes, 11 encoded neurohormone peptides including such hypothalamic peptides as gonadotropin-releasing hormone and oxytocin/vasopressin. Six of the identified peptide genes had not been previously described. We also found that genes encoding receptors for some of the peptides were expressed in the brain. Interestingly, whole-mount in situ hybridization showed that most of the peptide genes were expressed in the ventral brain. This catalog of the genes expressed in the larval brain should help elucidate the evolution, development, and functioning of the chordate brain.

  13. A gene encoding a new cold-active lipase from an Antarctic isolate of Penicillium expansum.

    PubMed

    Mohammed, Suja; Te'o, Junior; Nevalainen, Helena

    2013-08-01

    Cold-active lipases are of significant interest as biocatalysts in industrial processes. We have identified a lipase that displayed activity towards long carbon-chain-p-nitrophenyl substrates (C12-C18) at 25 °C from the culture supernatant of an Antarctic Penicillium expansum strain assigned P. expansum SM3. Zymography revealed a protein band of around 30 kDa with activity towards olive oil. DNA fragments of a lipase gene designated as lipPE were isolated from the genomic DNA of P. expansum SM3 by genomic walking PCR. Subsequently, the complete genomic lipPE gene was amplified using gene-specific primers designed from the 5'- and 3'-regions. Reverse transcription PCR was used to amplify the lipPE cDNA. The deduced amino acid sequence consisted of 285 residues that included a predicted signal peptide. Three peptides identified by LC/MS/MS analysis of the proteins in the culture supernatant of P. expansum were also present in the deduced amino acid sequence of the lipPE gene suggesting that this gene encoded the lipase identified by initial zymogram activity analysis. Full analysis of the nucleotide and the deduced amino acid sequences indicated that the lipPE gene encodes a novel P. expansum lipase. The lipPE gene was expressed in E. coli for further characterization of the enzyme with a view of assessing its suitability for industrial applications.

  14. The Saccharomyces Cerevisiae Spt7 Gene Encodes a Very Acidic Protein Important for Transcription in Vivo

    PubMed Central

    Gansheroff, L. J.; Dollard, C.; Tan, P.; Winston, F.

    1995-01-01

    Mutations in the SPT7 gene of Saccharomyces cerevisiae originally were identified as suppressors of Ty and {delta small} insertion mutations in the 5' regions of the HIS4 and LYS2 genes. Other genes that have been identified in mutant hunts of this type have been shown to play a role in transcription. In this work we show that SPT7 is also important for proper transcription in vivo. We have cloned and sequenced the SPT7 gene and have shown that it encodes a large, acidic protein that is localized to the nucleus. The SPT7 protein contains a bromodomain sequence; a deletion that removes the bromodomain from the SPT7 protein causes no detectable mutant phenotype. Strains that contain an spt7 null mutation are viable but grow very slowly and have transcriptional defects at many loci including insertion mutations, Ty elements, the INO1 gene and the MFA1 gene. These transcriptional defects and other mutant phenotypes are similar to those caused by certain mutations in SPT15, which encodes the TATA binding protein (TBP). The similarity of the phenotypes of spt7 and spt15 mutants, including effects of spt7 mutations on the transcription start site of certain genes, suggests that SPT7 plays an important role in transcription initiation in vivo. PMID:7713415

  15. MYB98 positively regulates a battery of synergid-expressed genes encoding filiform apparatus localized proteins.

    PubMed

    Punwani, Jayson A; Rabiger, David S; Drews, Gary N

    2007-08-01

    The synergid cells within the female gametophyte are essential for reproduction in angiosperms. MYB98 encodes an R2R3-MYB protein required for pollen tube guidance and filiform apparatus formation by the synergid cells. To test the predicted function of MYB98 as a transcriptional regulator, we determined its subcellular localization and examined its DNA binding properties. We show that MYB98 binds to a specific DNA sequence (TAAC) and that a MYB98-green fluorescent protein fusion protein localizes to the nucleus, consistent with a role in transcriptional regulation. To identify genes regulated by MYB98, we tested previously identified synergid-expressed genes for reduced expression in myb98 female gametophytes and identified 16 such genes. We dissected the promoter of one of the downstream genes, DD11, and show that it contains a MYB98 binding site required for synergid expression, suggesting that DD11 is regulated directly by MYB98. To gain insight into the functions of the downstream genes, we chose five genes and determined the subcellular localization of the encoded proteins. We show that these five proteins are secreted into the filiform apparatus, suggesting that they play a role in either the formation or the function of this unique structure. Together, these data suggest that MYB98 functions as a transcriptional regulator in the synergid cells and activates the expression of genes required for pollen tube guidance and filiform apparatus formation.

  16. Coevolution between Nuclear-Encoded DNA Replication, Recombination, and Repair Genes and Plastid Genome Complexity.

    PubMed

    Zhang, Jin; Ruhlman, Tracey A; Sabir, Jamal S M; Blazier, John Chris; Weng, Mao-Lun; Park, Seongjun; Jansen, Robert K

    2016-02-17

    Disruption of DNA replication, recombination, and repair (DNA-RRR) systems has been hypothesized to cause highly elevated nucleotide substitution rates and genome rearrangements in the plastids of angiosperms, but this theory remains untested. To investigate nuclear-plastid genome (plastome) coevolution in Geraniaceae, four different measures of plastome complexity (rearrangements, repeats, nucleotide insertions/deletions, and substitution rates) were evaluated along with substitution rates of 12 nuclear-encoded, plastid-targeted DNA-RRR genes from 27 Geraniales species. Significant correlations were detected for nonsynonymous (dN) but not synonymous (dS) substitution rates for three DNA-RRR genes (uvrB/C, why1, and gyrA) supporting a role for these genes in accelerated plastid genome evolution in Geraniaceae. Furthermore, correlation between dN of uvrB/C and plastome complexity suggests the presence of nucleotide excision repair system in plastids. Significant correlations were also detected between plastome complexity and 13 of the 90 nuclear-encoded organelle-targeted genes investigated. Comparisons revealed significant acceleration of dN in plastid-targeted genes of Geraniales relative to Brassicales suggesting this correlation may be an artifact of elevated rates in this gene set in Geraniaceae. Correlation between dN of plastid-targeted DNA-RRR genes and plastome complexity supports the hypothesis that the aberrant patterns in angiosperm plastome evolution could be caused by dysfunction in DNA-RRR systems.

  17. Coevolution between Nuclear-Encoded DNA Replication, Recombination, and Repair Genes and Plastid Genome Complexity

    PubMed Central

    Zhang, Jin; Ruhlman, Tracey A.; Sabir, Jamal S. M.; Blazier, John Chris; Weng, Mao-Lun; Park, Seongjun; Jansen, Robert K.

    2016-01-01

    Disruption of DNA replication, recombination, and repair (DNA-RRR) systems has been hypothesized to cause highly elevated nucleotide substitution rates and genome rearrangements in the plastids of angiosperms, but this theory remains untested. To investigate nuclear–plastid genome (plastome) coevolution in Geraniaceae, four different measures of plastome complexity (rearrangements, repeats, nucleotide insertions/deletions, and substitution rates) were evaluated along with substitution rates of 12 nuclear-encoded, plastid-targeted DNA-RRR genes from 27 Geraniales species. Significant correlations were detected for nonsynonymous (dN) but not synonymous (dS) substitution rates for three DNA-RRR genes (uvrB/C, why1, and gyrA) supporting a role for these genes in accelerated plastid genome evolution in Geraniaceae. Furthermore, correlation between dN of uvrB/C and plastome complexity suggests the presence of nucleotide excision repair system in plastids. Significant correlations were also detected between plastome complexity and 13 of the 90 nuclear-encoded organelle-targeted genes investigated. Comparisons revealed significant acceleration of dN in plastid-targeted genes of Geraniales relative to Brassicales suggesting this correlation may be an artifact of elevated rates in this gene set in Geraniaceae. Correlation between dN of plastid-targeted DNA-RRR genes and plastome complexity supports the hypothesis that the aberrant patterns in angiosperm plastome evolution could be caused by dysfunction in DNA-RRR systems. PMID:26893456

  18. Isolation and expression of two aquaporin-encoding genes from the marine phanerogam Posidonia oceanica.

    PubMed

    Maestrini, Pierluigi; Giordani, Tommaso; Lunardi, Andrea; Cavallini, Andrea; Natali, Lucia

    2004-12-01

    Seagrasses such as Posidonia oceanica (L.) Delile are marine phanerogams, widespread in various seas, where they form large prairies representing dynamic substrates exceeding the area of the sediment surface several times over and allowing settlement of epiphyte organisms. Studying mechanisms involved in water transport in marine plants, we isolated two aquaporin-encoding genes, PoPIP1;1 and PoTIP1;1, showing high similarity to plasma membrane- and tonoplast-intrinsic protein-encoding genes, respectively. PoPIP1;1 is unique in the genome of P. oceanica, while PoTIP1;1 belongs to an aquaporin subfamily of at least four members. PoPIP1;1 and PoTIP1;1 encode functional proteins, as indicated by expression experiments in Xenopus oocytes. Both genes are constitutively expressed in the leaves, with higher levels of transcripts in young than in differentiated leaf tissues. Variations of salt concentration in aquarium determined different PoPIP1;1 and PoTIP1;1 transcript accumulation, indicating the existence of adaptation mechanisms related to gene expression also in marine plants, i.e. adapted to very high salt concentrations. Hyposalinity induced lower levels of PIP1 transcripts, while hypersalinity determined more PIP1 transcripts than normal salinity. TIP1 transcripts increased in response to both hypo- and hypersalinity after 2 days of treatment and went back to control levels after 5 d.

  19. Overexpression of a Harpin-encoding gene hrf1 in rice enhances drought tolerance

    PubMed Central

    Zhang, Lei; Xiao, Shanshan; Feng, Wei; Wu, Zhidan; Gao, Xuewen; Liu, Fengquan; Shao, Min

    2011-01-01

    Harpin proteins are well known as eliciters that induce multiple responses in plants, such as systemic acquired resistance, hypersensitive response, enhancement of growth, resistance to the green peach aphid, and tolerance to drought. Overexpression of Harpin-encoding genes enhances plant resistance to diseases in tobacco, rice, rape, and cotton; however, it is not yet known whether the expression of Harpin-encoding genes in vivo improves plant tolerance to abiotic stresses. The results of this study showed that overexpression of a Harpin-encoding gene hrf1 in rice increased drought tolerance through abscisic acid (ABA) signalling. hrf1- overexpression induces an increase in ABA content and promotes stomatal closure in rice. The hrf1 transgenic rice lines exhibited a significant increase in water retention ability, levels of free proline and soluble sugars, tolerance to oxidative stress, reactive oxygen species-scavenging ability, and expression levels of four stress-related genes, OsLEA3-1, OsP5CS, Mn-SOD, and NM_001074345, under drought stress. The study confirmed that hrf1 conferred enhanced tolerance to drought stress on transgenic crops. These results suggest that Harpins may offer new opportunities for generating drought resistance in other crops. PMID:21527628

  20. Cloning and characterization of largemouth bass ( Micropterus salmoides) myostatin encoding gene and its promoter

    NASA Astrophysics Data System (ADS)

    Li, Shengjie; Bai, Junjie; Wang, Lin

    2008-08-01

    Myostatin or GDF-8, a member of the transforming growth factor-β (TGF-β) superfamily, has been demonstrated to be a negative regulator of skeletal muscle mass in mammals. In the present study, we obtained a 5.64 kb sequence of myostatin encoding gene and its promoter from largemouth bass ( Micropterus salmoides). The myostatin encoding gene consisted of three exons (488 bp, 371 bp and 1779 bp, respectively) and two introns (390 bp and 855 bp, respectively). The intron-exon boundaries were conservative in comparison with those of mammalian myostatin encoding genes, whereas the size of introns was smaller than that of mammals. Sequence analysis of 1.569 kb of the largemouth bass myostatin gene promoter region revealed that it contained two TATA boxes, one CAAT box and nine putative E-boxes. Putative muscle growth response elements for myocyte enhancer factor 2 (MEF2), serum response factor (SRF), activator protein 1 (AP1), etc., and muscle-specific Mt binding site (MTBF) were also detected. Some of the transcription factor binding sites were conserved among five teleost species. This information will be useful for studying the transcriptional regulation of myostatin in fish.

  1. Identification and transcriptional control of Caulobacter crescentus genes encoding proteins containing a cold shock domain.

    PubMed

    Lang, Elza A S; Marques, Marilis V

    2004-09-01

    The cold shock proteins are small peptides that share a conserved domain, called the cold shock domain (CSD), that is important for nucleic acid binding. The Caulobacter crescentus genome has four csp genes that encode proteins containing CSDs. Three of these (cspA, cspB, and cspC) encode peptides of about 7 kDa and are very similar to the cold shock proteins of other bacteria. Analysis by reverse transcription-PCR of the fourth gene (cspD), which was previously annotated as encoding a 7-kDa protein, revealed that the mRNA is larger and probably encodes a putative 21-kDa protein, containing two CSDs. A search in protein sequences databases revealed that this new domain arrangement has thus far only been found among deduced peptides of alpha-proteobacteria. Expression of each Caulobacter csp gene was studied both in response to cold shock and to growth phase, and we have found that only cspA and cspB are induced by cold shock, whereas cspC and cspD are induced at stationary phase, with different induction rates. The transcription start sites were determined for each gene, and a deletion mapping of the cspD promoter region defined a sequence required for maximal levels of expression, indicating that regulation of this gene occurs at the transcriptional level. Deletion of cspA, but not cspD, caused a reduction in viability when cells were incubated at 10 degrees C for prolonged times, suggesting that cspA is important for adaptation to a low temperature.

  2. Isolation of a gene encoding a glycosylated cytokinin oxidase from maize.

    PubMed

    Morris, R O; Bilyeu, K D; Laskey, J G; Cheikh, N N

    1999-02-16

    The major cytokinin oxidase in immature maize kernels was purified to homogeneity. Selected tryptic peptides were used to design degenerate oligonucleotide primers for PCR isolation of a fragment of the oxidase gene. Hybridization of the PCR fragment to a maize genomic library allowed isolation of a full-length cytokinin oxidase gene (ckx1). The gene encodes a protein of approximately 57 kDa that possesses a signal peptide, eight consensus N-glycosylation sequences and a consensus FAD binding sequence. Expression of ckx1 in Pichia caused secretion of active glycosylated cytokinin oxidase that contains a substrate-reducible FAD. The gene displays sequence homology with a putative oxidoreductase from Arabidopsis thaliana and with the fas5 gene from Rhodococcus fascians.

  3. A negative element involved in Kaposi's sarcoma-associated herpesvirus-encoded ORF11 gene expression

    SciTech Connect

    Chen, Lei

    2009-01-01

    The ORF11 of the Kaposi's sarcoma-associated herpesvirus (KSHV) is a lytic viral gene with delayed-early expression kinetics. How the ORF11 gene expression is regulated in the KSHV lytic cascade is largely unknown. Here we report that the deletion of the KSHV viral IL-6 gene from the viral genome leads to deregulated ORF11 gene expression. The KSHV-encoded viral IL-6 protein was found not to be essentially involved in the regulation of ORF11, suggesting a potential transcriptional cis-regulation. A negative element was identified downstream of the ORF11 gene, which suppresses the ORF11 basal promoter activity in a position-independent manner.

  4. Distribution of genes encoding aminoglycoside-modifying enzymes among clinical isolates of methicillin-resistant staphylococci.

    PubMed

    Perumal, N; Murugesan, S; Krishnan, P

    2016-01-01

    The objective of this study was to determine the distribution of genes encoding aminoglycoside-modifying enzymes (AMEs) and staphylococcal cassette chromosome mec (SCCmec) elements among clinical isolates of methicillin-resistant staphylococci (MRS). Antibiotic susceptibility test was done using Kirby-Bauer disk diffusion method. The presence of SCCmec types and AME genes, namely, aac (6')-Ie-aph (2''), aph (3')-IIIa and ant (4')-Ia was determined using two different multiplex polymerase chain reaction. The most encountered AME genes were aac (6')-Ie-aph (2'') (55.4%) followed by aph (3')-IIIa (32.3%) and ant (4')-Ia gene (9%). SCCmec type I (34%) was predominant in this study. In conclusion, the aac (6')-Ie-aph (2'') was the most common AME gene and SCCmec type I was most predominant among the MRS isolates.

  5. Mammalian ets-1 and ets-2 genes encode highly conserved proteins.

    PubMed Central

    Watson, D K; McWilliams, M J; Lapis, P; Lautenberger, J A; Schweinfest, C W; Papas, T S

    1988-01-01

    Cellular ets sequences homologous to v-ets of the avian leukemia virus E26 are highly conserved. In mammals the ets sequences are dispersed on two separate chromosomal loci, called ets-1 and ets-2. To determine the structure of these two genes and identify the open reading frames that code for the putative proteins, we have sequenced human ets-1 cDNAs and ets-2 cDNA clones obtained from both human and mouse. The human ETS1 gene is capable of encoding a protein of 441 amino acids. This protein is greater than 95% identical to the chicken c-ets-1 gene product. Thus, the human ETS1 gene is homologous to the chicken c-ets-1 gene, the protooncogene that the E26 virus transduced. Human and mouse ets-2 cDNA clones are closely related and contain open reading frames capable of encoding proteins of 469 and 468 residues, respectively. Direct comparison of these data with previously published findings indicates that ets is a family of genes whose members share distinct domains. PMID:2847145

  6. The ctnG gene encodes carbonic anhydrase involved in mycotoxin citrinin biosynthesis from Monascus aurantiacus.

    PubMed

    Li, Yan-Ping; Tang, Xiao; Wu, Wei; Xu, Yang; Huang, Zhi-Bing; He, Qing-Hua

    2015-01-01

    Citrinin, a fungal secondary metabolite of polyketide origin, is moderately nephrotoxic to vertebrates, including humans. Citrinin is synthesised by condensation of acetyl-CoA and malonyl-CoA. Six genes involved in the citrinin biosynthesis, including pksCT, ctnA and ctnB, have been cloned in Monascus purpureus. The pksCT gene encodes a polyketide synthase; ctnA is a regulatory factor; and ctnB encodes an oxidoreductase. When the three genes were respectively disrupted, the disruption strains drastically decreased citrinin production or barely produced citrinin. Ten new genes have been discovered in Monascus aurantiacus besides the above six genes. One of these gene displayed the highest similarity to the β-carbonic anhydrase gene from Aspergillus oryzae (74% similarity) and was designated ctnG. To learn more about the citrinin biosynthetic pathway, a ctnG-replacement vector was constructed to disrupt ctnG with the hygromycin resistance gene as the selection marker, then transformed into M. aurantiacus Li AS3.4384 by a protoplast-PEG method. The citrinin content of three disruptants was reduced to about 50%, meanwhile pigment production decreased by 23%, respectively, over those of the wild-type strains. ctnG was deduced to be involved in the formation of malonyl-CoA as a common precursor of red pigments and citrinin. Therefore, the disruption of the ctnG gene decreased citrinin and pigment production. M. aurantiacus Li AS3.4384 can produce higher concentrations of citrinin than other strains such as M. purpureus and M. ruber. Establishing the function of citrinin biosynthetic genes in M. aurantiacus is helpful in understanding the citrinin synthetic pathway and adopting some strategies to control contamination.

  7. The Evolutionary Fate of the Genes Encoding the Purine Catabolic Enzymes in Hominoids, Birds, and Reptiles

    PubMed Central

    Keebaugh, Alaine C.; Thomas, James W.

    2010-01-01

    Gene loss has been proposed to play a major role in adaptive evolution, and recent studies are beginning to reveal its importance in human evolution. However, the potential consequence of a single gene-loss event upon the fates of functionally interrelated genes is poorly understood. Here, we use the purine metabolic pathway as a model system in which to explore this important question. The loss of urate oxidase (UOX) activity, a necessary step in this pathway, has occurred independently in the hominoid and bird/reptile lineages. Because the loss of UOX would have removed the functional constraint upon downstream genes in this pathway, these downstream genes are generally assumed to have subsequently deteriorated. In this study, we used a comparative genomics approach to empirically determine the fate of UOX itself and the downstream genes in five hominoids, two birds, and a reptile. Although we found that the loss of UOX likely triggered the genetic deterioration of the immediate downstream genes in the hominoids, surprisingly in the birds and reptiles, the UOX locus itself and some of the downstream genes were present in the genome and predicted to encode proteins. To account for the variable pattern of gene retention and loss after the inactivation of UOX, we hypothesize that although gene loss is a common fate for genes that have been rendered obsolete due to the upstream loss of an enzyme a metabolic pathway, it is also possible that same lack of constraint will foster the evolution of new functions or allow the optimization of preexisting alternative functions in the downstream genes, thereby resulting in gene retention. Thus, adaptive single-gene losses have the potential to influence the long-term evolutionary fate of functionally interrelated genes. PMID:20106906

  8. Cloning of the gene encoding streptococcal immunoglobulin A protease and its expression in Escherichia coli.

    PubMed Central

    Gilbert, J V; Plaut, A G; Fishman, Y; Wright, A

    1988-01-01

    We have identified and cloned a 6-kilobase-pair segment of chromosomal DNA from Streptococcus sanguis ATCC 10556 that encodes immunoglobulin A (IgA) protease activity when cloned into Escherichia coli. The enzyme specified by the iga gene in plasmid pJG1 accumulates in the periplasm of E. coli MM294 cells and has a substrate specificity for human IgA1 identical to that of native S. sanguis protease. Hybridization experiments with probes from within the encoding DNA showed no detectable homology at the nucleotide sequence level with chromosomal DNA of gram-negative bacteria that excrete IgA protease. Moreover, the S. sanguis iga gene probes showed no detectable hybridization with chromosomal DNA of S. pneumoniae, although the IgA proteases of these two streptococcal species cleaved the identical peptide bond in the human IgA1 heavy-chain hinge region. Images PMID:3294181

  9. Characterization of emb, a Gene Encoding the Major Adhesin of Streptococcus defectivus

    PubMed Central

    Manganelli, Riccardo; van de Rijn, Ivo

    1999-01-01

    Streptococcus defectivus is one of the nutritionally variant streptococci, a class of viridans group streptococci first isolated from patients with endocarditis and otitis media. In previous studies, NVS-47, a clinical isolate of S. defectivus, was shown to bind to the extracellular matrix. A high-molecular-weight surface protein was identified and proposed to be responsible for mediating this binding. In the present study, the gene encoding this protein was identified by transposon mutagenesis and characterized. The gene (emb) was found to be larger than 14 kb and was partially sequenced. It encodes a protein containing at least 50 repeats of 77 amino acids predicted to assume an alternating coiled-coil conformation. The domain responsible for extracellular matrix binding was mapped to the N terminus of the protein. From sequence analysis, Emb is proposed to be the prototype of a new family of streptococcal fibrillar proteins. PMID:9864195

  10. Bacteriophage T5 gene D10 encodes a branch-migration protein

    PubMed Central

    Wong, Io Nam; Sayers, Jon R.; Sanders, Cyril M.

    2016-01-01

    Helicases catalyze the unwinding of double-stranded nucleic acids where structure and phosphate backbone contacts, rather than nucleobase sequence, usually determines substrate specificity. We have expressed and purified a putative helicase encoded by the D10 gene of bacteriophage T5. Here we report that this hitherto uncharacterized protein possesses branch migration and DNA unwinding activity. The initiation of substrate unwinding showed some sequence dependency, while DNA binding and DNA-dependent ATPase activity did not. DNA footprinting and purine-base interference assays demonstrated that D10 engages these substrates with a defined polarity that may be established by protein-nucleobase contacts. Bioinformatic analysis of the nucleotide databases revealed genes predicted to encode proteins related to D10 in archaebacteria, bacteriophages and in viruses known to infect a range of eukaryotic organisms. PMID:28009009

  11. Human TOP3: a single-copy gene encoding DNA topoisomerase III.

    PubMed Central

    Hanai, R; Caron, P R; Wang, J C

    1996-01-01

    A human cDNA encoding a protein homologous to the Escherichia coli DNA topoisomerase I subfamily of enzymes has been identified through cloning and sequencing. Expressing the cloned human cDNA in yeast (delta)top1 cells lacking endogenous DNA topoisomerase I yielded an activity in cell extracts that specifically reduces the number of supercoils in a highly negatively supercoiled DNA. On the basis of these results, the human gene containing the cDNA sequence has been denoted TOP3, and the protein it encodes has been denoted DNA topoisomerase III. Screening of a panel of human-rodent somatic hybrids and fluorescence in situ hybridization of cloned TOP3 genomic DNA to metaphase chromosomes indicate that human TOP3 is a single-copy gene located at chromosome 17p11.2-12. Images Fig. 2 PMID:8622991

  12. The udhA Gene of Escherichia coli Encodes a Soluble Pyridine Nucleotide Transhydrogenase

    PubMed Central

    Boonstra, Birgitte; French, Christopher E.; Wainwright, Ian; Bruce, Neil C.

    1999-01-01

    The udhA gene of Escherichia coli was cloned and expressed in E. coli and found to encode an enzyme with soluble pyridine nucleotide transhydrogenase activity. The N-terminal end of the enzyme contains the fingerprint motif of a dinucleotide binding domain, not present in published E. coli genome sequences due to a sequencing error. E. coli is hereby the first organism reported to possess both a soluble and a membrane-bound pyridine nucleotide transhydrogenase. PMID:9922271

  13. Enterotoxin-Encoding Genes in Staphylococcus spp. from Food Handlers in a University Restaurant.

    PubMed

    da Silva, Sabina Dos Santos Paulino; Cidral, Thiago André; Soares, Maria José dos Santos; de Melo, Maria Celeste Nunes

    2015-11-01

    Food handlers carrying enterotoxin-producing Staphylococcus are a potential source of food poisoning. The aim of this study was to analyze genes encoding enterotoxins in coagulase-positive Staphylococcus (CoPS) and coagulase-negative Staphylococcus (CoNS) isolated from the anterior nostrils and hands of food handlers at a university restaurant in the city of Natal, Northeast Brazil. Thirty food handlers were screened for the study. The isolates were subjected to Gram staining, a bacitracin sensitivity test, mannitol fermentation, and catalase and coagulase tests. CoNS and CoPS strains were subsequently identified by a Vitek 2 System (BioMerieux, France) and various biochemical tests. Polymerase chain reaction was used to detect genes for enterotoxins A, B, C, D, E, G, H, and I (sea, seb, sec, sed, see, seg, seh, and sei) and a disc-diffusion method was used to determine susceptibility to several classes of antimicrobials. All food handlers presented staphylococci on their hands and/or noses. The study found 58 Staphylococcus spp., of which 20.7% were CoPS and 79.3% were CoNS. S. epidermidis was the most prevalent species. Twenty-nine staphylococci (50%) were positive for one or more enterotoxin genes, and the most prevalent genes were seg and sei, each with a frequency of 29.3%. Indeed, CoNS encoded a high percentage of enterotoxin genes (43.5%). However, S. aureus encoded even more enterotoxin genes (75%). Most isolates showed sensitivity to the antibiotics used for testing, except for penicillin (only 35% sensitive). The results from this study reinforce that coagulase-negative as well as coagulase-positive staphylococci isolated from food handlers are capable of genotypic enterotoxigenicity.

  14. Sieve element occlusion (SEO) genes encode structural phloem proteins involved in wound sealing of the phloem.

    PubMed

    Ernst, Antonia M; Jekat, Stephan B; Zielonka, Sascia; Müller, Boje; Neumann, Ulla; Rüping, Boris; Twyman, Richard M; Krzyzanek, Vladislav; Prüfer, Dirk; Noll, Gundula A

    2012-07-10

    The sieve element occlusion (SEO) gene family originally was delimited to genes encoding structural components of forisomes, which are specialized crystalloid phloem proteins found solely in the Fabaceae. More recently, SEO genes discovered in various non-Fabaceae plants were proposed to encode the common phloem proteins (P-proteins) that plug sieve plates after wounding. We carried out a comprehensive characterization of two tobacco (Nicotiana tabacum) SEO genes (NtSEO). Reporter genes controlled by the NtSEO promoters were expressed specifically in immature sieve elements, and GFP-SEO fusion proteins formed parietal agglomerates in intact sieve elements as well as sieve plate plugs after wounding. NtSEO proteins with and without fluorescent protein tags formed agglomerates similar in structure to native P-protein bodies when transiently coexpressed in Nicotiana benthamiana, and the analysis of these protein complexes by electron microscopy revealed ultrastructural features resembling those of native P-proteins. NtSEO-RNA interference lines were essentially devoid of P-protein structures and lost photoassimilates more rapidly after injury than control plants, thus confirming the role of P-proteins in sieve tube sealing. We therefore provide direct evidence that SEO genes in tobacco encode P-protein subunits that affect translocation. We also found that peptides recently identified in fascicular phloem P-protein plugs from squash (Cucurbita maxima) represent cucurbit members of the SEO family. Our results therefore suggest a common evolutionary origin for P-proteins found in the sieve elements of all dicotyledonous plants and demonstrate the exceptional status of extrafascicular P-proteins in cucurbits.

  15. Structure and function of the DNA ligases encoded by the mammalian LIG3 gene.

    PubMed

    Tomkinson, Alan E; Sallmyr, Annahita

    2013-12-01

    Among the mammalian genes encoding DNA ligases (LIG), the LIG3 gene is unique in that it encodes multiple DNA ligase polypeptides with different cellular functions. Notably, this nuclear gene encodes the only mitochondrial DNA ligase and so is essential for this organelle. In the nucleus, there is significant functional redundancy between DNA ligase IIIα and DNA ligase I in excision repair. In addition, DNA ligase IIIα is essential for DNA replication in the absence of the replicative DNA ligase, DNA ligase I. DNA ligase IIIα is a component of an alternative non-homologous end joining (NHEJ) pathway for DNA double-strand break (DSB) repair that is more active when the major DNA ligase IV-dependent pathway is defective. Unlike its other nuclear functions, the role of DNA ligase IIIα in alternative NHEJ is independent of its nuclear partner protein, X-ray repair cross-complementing protein 1 (XRCC1). DNA ligase IIIα is frequently overexpressed in cancer cells, acting as a biomarker for increased dependence upon alternative NHEJ for DSB repair and it is a promising novel therapeutic target.

  16. Citrate synthase encoded by the CIT2 gene of Saccharomyces cerevisiae is peroxisomal.

    PubMed Central

    Lewin, A S; Hines, V; Small, G M

    1990-01-01

    The product of the CIT2 gene has the tripeptide SKL at its carboxyl terminus. This amino acid sequence has been shown to act as a peroxisomal targeting signal in mammalian cells. We examined the subcellular site of this extramitochondrial citrate synthase. Cells of Saccharomyces cerevisiae were grown on oleate medium to induce peroxisome proliferation. A fraction containing membrane-enclosed vesicles and organelles was analyzed by sedimentation on density gradients. In wild-type cells, the major peak of citrate synthase activity was recovered in the mitochondrial fraction, but a second peak of activity cosedimented with peroxisomes. The peroxisomal activity, but not the mitochondrial activity, was inhibited by incubation at pH 8.1, a characteristic of the extramitochondrial citrate synthase encoded by the CIT2 gene. In a strain in which the CIT1 gene encoding mitochondrial citrate synthase had been disrupted, the major peak of citrate synthase activity was peroxisomal, and all of the activity was sensitive to incubation at pH 8.1. Yeast cells bearing a cit2 disruption were unable to mobilize stored lipids and did not form stable peroxisomes in oleate. We conclude that citrate synthase encoded by CIT2 is peroxisomal and participates in the glyoxylate cycle. Images PMID:2181273

  17. Molecular cloning, nucleotide sequence and expression of a Sulfolobus solfataricus gene encoding a class II fumarase.

    PubMed

    Colombo, S; Grisa, M; Tortora, P; Vanoni, M

    1994-01-03

    Fumarase catalyzes the interconversion of L-malate and fumarate. A Sulfolobus solfataricus fumarase gene (fumC) was cloned and sequenced. Typical archaebacterial regulatory sites were identified in the region flanking the fumC open reading frame. The fumC gene encodes a protein of 438 amino acids (47,899 Da) which shows several significant similarities with class II fumarases from both eubacterial and eukariotic sources as well as with aspartases. S. solfataricus fumarase expressed in Escherichia coli retains enzymatic activity and its thermostability is comparable to that of S. solfataricus purified enzyme despite a 11 amino acid C-terminal deletion.

  18. Localization of the human genes encoding the two subunits of general transcription factor TFIIE.

    PubMed

    Purrello, M; Di Pietro, C; Rapisarda, A; Motta, S; Pavone, L; Grzeschik, K H; Sichel, G

    1994-09-01

    TFIIE is a general transcription factor for class II genes composed of two types of subunits, a large one of 56 kDa and a small of 34 kDa. By Southern analysis at high and at low stringency of a panel of mouse/human hybrid cell lines and by in situ chromosomal hybridization, we have demonstrated that both polypeptides are encoded by genes that are single copy in the human genome and are localized at 3q13-q21 and at 8p12, respectively. A TaqI RFLP (heterozygosity index of 0.07) was detected at the locus for the 56-kDa subunit.

  19. Chromosomal localization of the gene encoding the human DNA helicase RECQL and its mouse homologue

    SciTech Connect

    Puranam, K.L.; Kennington, E.; Blackshear, P.J.

    1995-04-10

    We have determined the chromosomal location of the human and mouse genes encoding the RECQL protein, a putative DNA helicase homologous to the bacterial DNA helicase, RecQ. RECQL was localized to human chromosome 12 by analysis of human-rodent somatic cell hybrid DNA, fine mapping of RECQL by fluorescence in situ hybridization revealed its chromosomal location to be 12p11-p12. The corresponding mouse gene, Recql, was mapped to the telomeric end of mouse chromosome 6 by analysis of DNA from an interspecific cross. 19 refs., 2 figs.

  20. Molecular characterization of genes encoding leucoanthocyanidin reductase involved in proanthocyanidin biosynthesis in apple

    PubMed Central

    Liao, Liao; Vimolmangkang, Sornkanok; Wei, Guochao; Zhou, Hui; Korban, Schuyler S.; Han, Yuepeng

    2015-01-01

    Proanthocyanidins (PAs) are the major component of phenolics in apple, but mechanisms involved in PA biosynthesis remain unclear. Here, the relationship between the PA biosynthesis and the expression of genes encoding leucoanthocyanidin reductase (LAR) and anthocyanidin reductase (ANR) was investigated in fruit skin of one apple cultivar and three crabapples. Transcript levels of LAR1 and ANR2 genes were significantly correlated with the contents of catechin and epicatechin, respectively, which suggests their active roles in PA synthesis. Surprisingly, transcript levels for both LAR1 and LAR2 genes were almost undetectable in two crabapples that accumulated both flavan-3-ols and PAs. This contradicts the previous finding that LAR1 gene is a strong candidate regulating the accumulation of metabolites such as epicatechin and PAs in apple. Ectopic expression of apple MdLAR1 gene in tobacco suppresses expression of the late genes in anthocyanin biosynthetic pathway, resulting in loss of anthocyanin in flowers. Interestingly, a decrease in PA biosynthesis was also observed in flowers of transgenic tobacco plants overexpressing the MdLAR1 gene, which could be attributed to decreased expression of both the NtANR1 and NtANR2 genes. Our study not only confirms the in vivo function of apple LAR1 gene, but it is also helpful for understanding the mechanism of PA biosynthesis. PMID:25914714

  1. Structure and evolutionary origin of the gene encoding a human serum mannose-binding protein.

    PubMed Central

    Taylor, M E; Brickell, P M; Craig, R K; Summerfield, J A

    1989-01-01

    The N-terminal sequence of the major human serum mannose-binding protein (MBP1) was shown to be identical at all positions determined with the amino acid sequence predicted from a cDNA clone of a human liver MBP mRNA. An oligonucleotide corresponding to part of the sequence of this cDNA clone was used to isolate a cosmid genomic clone containing a homologous gene. The intron/exon structure of this gene was found to closely resemble that of the gene encoding a rat liver MBP (MBP A). The nucleotide sequence of the exons differed in several places from that of the human cDNA clone published by Ezekowitz, Day & Herman [(1988) J. Exp. Med. 167, 1034-1046]. The MBP molecule comprises a signal peptide, a cysteine-rich domain, a collagen-like domain, a 'neck' region and a carbohydrate-binding domain. Each domain is encoded by a separate exon. This genomic organization lends support to the hypothesis that the gene arose during evolution by a process of exon shuffling. Several consensus sequences that may be involved in controlling the expression of human serum MBP have been identified in the promoter region of the gene. The consensus sequences are consistent with the suggestion that this mammalian serum lectin is regulated as an acute-phase protein synthesized by the liver. PMID:2590164

  2. Two homologous low-temperature-inducible genes from Arabidopsis encode highly hydrophobic proteins.

    PubMed Central

    Capel, J; Jarillo, J A; Salinas, J; Martínez-Zapater, J M

    1997-01-01

    We have characterized two related cDNAs (RCI2A and RCI2B) corresponding to genes from Arabidopsis thaliana, the expression of which is transiently induced by low, nonfreezing temperatures. RCI2A and RCI2B encode small (54 amino acids), highly hydrophobic proteins that bear two potential transmembrane domains. They show similarity to proteins encoded by genes from barley (Hordeum vulgare L.) and wheatgrass (Lophophyrum elongatum) that are regulated by different stress conditions. Their high level of sequence homology (78%) and their genomic location in a single restriction fragment suggest that both genes originated as a result of a tandem duplication. However, their regulatory sequences have diverged enough to confer on them different expression patterns. Like most of the cold-inducible plant genes characterized, the expression of RCI2A and RCI2B is also promoted by abscisic acid (ABA) and dehydration but is not a general response to stress conditions, since it is not induced by salt stress or by anaerobiosis. Furthermore, low temperatures are able to induce RCI2A and RCI2B expression in ABA-deficient and -insensitive genetic backgrounds, indicating that both ABA-dependent and -independent pathways regulate the low-temperature responsiveness of these two genes. PMID:9342870

  3. Genome analysis and identification of gelatinase encoded gene in Enterobacter aerogenes

    NASA Astrophysics Data System (ADS)

    Shahimi, Safiyyah; Mutalib, Sahilah Abdul; Khalid, Rozida Abdul; Repin, Rul Aisyah Mat; Lamri, Mohd Fadly; Bakar, Mohd Faizal Abu; Isa, Mohd Noor Mat

    2016-11-01

    In this study, bioinformatic analysis towards genome sequence of E. aerogenes was done to determine gene encoded for gelatinase. Enterobacter aerogenes was isolated from hot spring water and gelatinase species-specific bacterium to porcine and fish gelatin. This bacterium offers the possibility of enzymes production which is specific to both species gelatine, respectively. Enterobacter aerogenes was partially genome sequenced resulting in 5.0 mega basepair (Mbp) total size of sequence. From pre-process pipeline, 87.6 Mbp of total reads, 68.8 Mbp of total high quality reads and 78.58 percent of high quality percentage was determined. Genome assembly produced 120 contigs with 67.5% of contigs over 1 kilo base pair (kbp), 124856 bp of N50 contig length and 55.17 % of GC base content percentage. About 4705 protein gene was identified from protein prediction analysis. Two candidate genes selected have highest similarity identity percentage against gelatinase enzyme available in Swiss-Prot and NCBI online database. They were NODE_9_length_26866_cov_148.013245_12 containing 1029 base pair (bp) sequence with 342 amino acid sequence and NODE_24_length_155103_cov_177.082458_62 which containing 717 bp sequence with 238 amino acid sequence, respectively. Thus, two paired of primers (forward and reverse) were designed, based on the open reading frame (ORF) of selected genes. Genome analysis of E. aerogenes resulting genes encoded gelatinase were identified.

  4. Separate nuclear genes encode cytosolic and mitochondrial nucleoside diphosphate kinase in Dictyostelium discoideum.

    PubMed

    Troll, H; Winckler, T; Lascu, I; Müller, N; Saurin, W; Véron, M; Mutzel, R

    1993-12-05

    We have previously isolated cDNA clones for the gip17 gene encoding the cytosolic nucleoside diphosphate (NDP) kinase from Dictyostelium discoideum, and partial cDNAs for guk, a second member of the NDP kinase gene family (Wallet, V., Mutzel, R., Troll, H., Barzu, O., Wurster, B., Véron, M., and Lacombe, M. L. (1990) J. Natl. Cancer Inst. 80, 1199-1202). We now characterize genomic DNA clones for both NDP kinase genes, and we show that guk defines a nuclear-encoded mitochondrial NDP kinase. Isolated D. discoideum mitochondria contain 3% of the total cellular NDP kinase activity. Antibodies which specifically recognize and inhibit the activity of either cytosolic or mitochondrial NDP kinase unambiguously distinguish between these activities. The nascent mitochondrial NDP kinase contains a presequence of 57 amino acids that is removed during import into the organelle as shown by determination of the NH2 terminus of the mature protein from mitochondria. The genes for mitochondrial and cytosolic NDP kinases contain four and two introns, respectively. The positions of the of the introns in the gene for the cytosolic enzyme match exactly the positions of the second and fourth introns in the coding region of its mitochondrial homologue. From these results we conclude that the isozymes diverged from a common ancestor, and we discuss possible phylogenetic pathways for the evolution of cytosolic and organelle NDP kinases.

  5. Characterization of the lys2 gene of Penicillium chrysogenum encoding alpha-aminoadipic acid reductase.

    PubMed

    Casqueiro, J; Gutiérrez, S; Bañuelos, O; Fierro, F; Velasco, J; Martín, J F

    1998-09-01

    A DNA fragment containing a gene homologous to LYS2 gene of Saccharomyces cerevisiae was cloned from a genomic DNA library of Penicillium chrysogenum AS-P-78. It encodes a protein of 1409 amino acids (Mr 154859) with strong similarity to the S. cerevisiae (49.9% identity) Schizosaccharomyces pombe (51.3% identity) and Candida albicans (48.12% identity) alpha-aminoadipate reductases and a lesser degree of identity to the amino acid-activating domains of the non-ribosomal peptide synthetases, including the alpha-aminoadipate-activating domain of the alpha-aminoadipyl-cysteinyl-valine synthetase of P. chrysogenum (12.4% identical amino acids). The lys2 gene contained one intron in the 5'-region and other in the 3'-region, as shown by comparing the nucleotide sequences of the cDNA and genomic DNA, and was transcribed as a 4.7-kb monocistronic mRNA. The lys2 gene was localized on chromosome III (7.5 Mb) in P. chrysogenum AS-P-78 and on chromosome IV (5.6 Mb) in strain P2, whereas the penicillin gene cluster is known to be located in chromosome I in both strains. The lys2-encoded protein is a member of the aminoacyladenylate-forming enzyme family with a reductase domain in its C-terminal region.

  6. The dhnA gene of Escherichia coli encodes a class I fructose bisphosphate aldolase.

    PubMed Central

    Thomson, G J; Howlett, G J; Ashcroft, A E; Berry, A

    1998-01-01

    The gene encoding the Escherichia coli Class I fructose-1, 6-bisphosphate aldolase (FBP aldolase) has been cloned and the protein overproduced in high amounts. This gene sequence has previously been identified as encoding an E. coli dehydrin in the GenBanktrade mark database [gene dhnA; entry code U73760; Close and Choi (1996) Submission to GenBanktrade mark]. However, the purified protein overproduced from the dhnA gene shares all its properties with those known for the E. coli Class I FBP aldolase. The protein is an 8-10-mer with a native molecular mass of approx. 340 kDa, each subunit consisting of 349 amino acids. The Class I enzyme shows low sequence identity with other known FBP aldolases, both Class I and Class II (in the order of 20%), which may be reflected by some novel properties of this FBP aldolase. The active-site peptide has been isolated and the Schiff-base-forming lysine residue (Lys236) has been identified by a combination of site-directed mutagenesis, kinetics and electrospray-ionization MS. A second lysine residue (Lys238) has been implicated in substrate binding. The cloning of this gene and the high levels of overexpression obtained will facilitate future structure-function studies. PMID:9531482

  7. The Embryonically Active Gene, Unkempt, of Drosophila Encodes a Cys(3)his Finger Protein

    PubMed Central

    Mohler, J.; Weiss, N.; Murli, S.; Mohammadi, S.; Vani, K.; Vasilakis, G.; Song, C. H.; Epstein, A.; Kuang, T.; English, J.; Cherdak, D.

    1992-01-01

    The unkempt gene of Drosophila encodes a set of embryonic RNAs, which are abundant during early stages of embryogenesis and are present ubiquitously in most somatic tissues from the syncytial embryo through stage 15 of embryogenesis. Expression of unkempt RNAs becomes restricted predominantly to the central nervous system in stages 16 and early 17. Analysis of cDNAs from this locus reveals the presence of five Cys(3)His fingers in the protein product. Isolation and analysis of mutations affecting the unkempt gene, including complete deletions of this gene, indicate that there is no zygotic requirement for unkempt during embryogenesis, presumably due to the contribution of maternally supplied RNA, although the gene is essential during post-embryonic development. PMID:1339381

  8. Somatic instability of the DNA sequences encoding the polymorphic polyglutamine tract of the AIB1 gene

    PubMed Central

    Dai, P; Wong, L

    2003-01-01

    Background: AIB1 contains a polymorphic polyglutamine tract (poly Q) that is encoded by a trinucleotide CAG repeat. Previously there have been conflicting results regarding the effect of the poly Q tract length on breast cancer. Since poly Q is not encoded by a perfect CAG repeat, the heterozygous polymorphic alleles need to be resolved, to understand the exact DNA sequences encoding poly Q. Methods: Poly Q encoding sequences of AIB1 from 107 DNA samples, including breast cancer cell lines, sporadic primary breast tumours, and blood samples from BRCA1/BRCA2 mutation carriers and the general population, were resolved by PCR/cloning followed by sequencing of each individual clone. Results: 25 distinct poly Q encoding sequence patterns were found. More than two distinct sequence patterns were found in a significantly higher proportion of tumours and cell lines than that of the general population, suggesting somatic instability. A significantly higher proportion of cancer cell lines or primary breast tumours than that of the general population contained rare sequence patterns. The proportion of sporadic breast tumours having at least one allele ⩽27 repeats is significantly higher than that in the blood of BRCA1/BRCA2 mutation carrier breast cancer patients or the general population. Conclusion: The poly Q encoding DNA sequences are somatically unstable in tumour tissues and cell lines. A missense mutation and a very short glutamine repeat in primary tumours suggests that AIB1 activity may be modulated through poly Q, which in turn plays a role in the cotransactivation of gene expressions in breast cancers. PMID:14684685

  9. Current Bacterial Gene Encoding Capsule Biosynthesis Protein CapI Contains Nucleotides Derived from Exonization

    PubMed Central

    Wang, Yong; Tao, Xia-Fang; Su, Zhi-Xi; Liu, A-Ke; Liu, Tian-Lei; Sun, Ling; Yao, Qin; Chen, Ke-Ping; Gu, Xun

    2016-01-01

    Since the proposition of introns-early hypothesis, although many studies have shown that most eukaryotic ancestors possessed intron-rich genomes, evidence of intron existence in genomes of ancestral bacteria has still been absent. While not a single intron has been found in all protein-coding genes of current bacteria, analyses on bacterial genes horizontally transferred into eukaryotes at ancient time may provide evidence of intron existence in bacterial ancestors. In this study, a bacterial gene encoding capsule biosynthesis protein CapI was found in the genome of sea anemone, Nematostella vectensis. This horizontally transferred gene contains a phase 1 intron of 40 base pairs. The nucleotides of this intron have high sequence identity with those encoding amino acids in current bacterial CapI gene, indicating that the intron and the amino acid-coding nucleotides are originated from the same ancestor sequence. Moreover, 5′-splice site of this intron is located in a GT-poor region associated with a closely following AG-rich region, suggesting that deletion mutation at 5′-splice site has been employed to remove this intron and the intron-like amino acid-coding nucleotides in current bacterial CapI gene are derived from exonization. These data suggest that bacterial CapI gene contained intron(s) at ancient time. This is the first report providing the result of sequence analysis to suggest possible existence of spliceosomal introns in ancestral bacterial genes. The methodology employed in this study may be used to identify more such evidence that would aid in settlement of the dispute between introns-early and introns-late theories. PMID:27980385

  10. Transcription Factors Encoded on Core and Accessory Chromosomes of Fusarium oxysporum Induce Expression of Effector Genes

    PubMed Central

    van der Does, H. Charlotte; Schmidt, Sarah M.; Langereis, Léon; Hughes, Timothy R.

    2016-01-01

    Proteins secreted by pathogens during host colonization largely determine the outcome of pathogen-host interactions and are commonly called ‘effectors’. In fungal plant pathogens, coordinated transcriptional up-regulation of effector genes is a key feature of pathogenesis and effectors are often encoded in genomic regions with distinct repeat content, histone code and rate of evolution. In the tomato pathogen Fusarium oxysporum f. sp. lycopersici (Fol), effector genes reside on one of four accessory chromosomes, known as the ‘pathogenicity’ chromosome, which can be exchanged between strains through horizontal transfer. The three other accessory chromosomes in the Fol reference strain may also be important for virulence towards tomato. Expression of effector genes in Fol is highly up-regulated upon infection and requires Sge1, a transcription factor encoded on the core genome. Interestingly, the pathogenicity chromosome itself contains 13 predicted transcription factor genes and for all except one, there is a homolog on the core genome. We determined DNA binding specificity for nine transcription factors using oligonucleotide arrays. The binding sites for homologous transcription factors were highly similar, suggesting that extensive neofunctionalization of DNA binding specificity has not occurred. Several DNA binding sites are enriched on accessory chromosomes, and expression of FTF1, its core homolog FTF2 and SGE1 from a constitutive promoter can induce expression of effector genes. The DNA binding sites of only these three transcription factors are enriched among genes up-regulated during infection. We further show that Ftf1, Ftf2 and Sge1 can activate transcription from their binding sites in yeast. RNAseq analysis revealed that in strains with constitutive expression of FTF1, FTF2 or SGE1, expression of a similar set of plant-responsive genes on the pathogenicity chromosome is induced, including most effector genes. We conclude that the Fol

  11. funRNA: a fungi-centered genomics platform for genes encoding key components of RNAi

    PubMed Central

    2014-01-01

    Background RNA interference (RNAi) is involved in genome defense as well as diverse cellular, developmental, and physiological processes. Key components of RNAi are Argonaute, Dicer, and RNA-dependent RNA polymerase (RdRP), which have been functionally characterized mainly in model organisms. The key components are believed to exist throughout eukaryotes; however, there is no systematic platform for archiving and dissecting these important gene families. In addition, few fungi have been studied to date, limiting our understanding of RNAi in fungi. Here we present funRNA http://funrna.riceblast.snu.ac.kr/, a fungal kingdom-wide comparative genomics platform for putative genes encoding Argonaute, Dicer, and RdRP. Description To identify and archive genes encoding the abovementioned key components, protein domain profiles were determined from reference sequences obtained from UniProtKB/SwissProt. The domain profiles were searched using fungal, metazoan, and plant genomes, as well as bacterial and archaeal genomes. 1,163, 442, and 678 genes encoding Argonaute, Dicer, and RdRP, respectively, were predicted. Based on the identification results, active site variation of Argonaute, diversification of Dicer, and sequence analysis of RdRP were discussed in a fungus-oriented manner. funRNA provides results from diverse bioinformatics programs and job submission forms for BLAST, BLASTMatrix, and ClustalW. Furthermore, sequence collections created in funRNA are synced with several gene family analysis portals and databases, offering further analysis opportunities. Conclusions funRNA provides identification results from a broad taxonomic range and diverse analysis functions, and could be used in diverse comparative and evolutionary studies. It could serve as a versatile genomics workbench for key components of RNAi. PMID:25522231

  12. Entamoeba histolytica: a unicellular organism containing two active genes encoding for members of the TBP family.

    PubMed

    Castañon-Sanchez, Carlos Alberto; Luna-Arias, Juan Pedro; de Dios-Bravo, Ma Guadalupe; Herrera-Aguirre, Maria Esther; Olivares-Trejo, Jose J; Orozco, Esther; Hernandez, Jose Manuel

    2010-03-01

    Entamoeba histolytica is the protozoan parasite which causes human amoebiasis. In this parasite, few encoding genes for transcription factors have been cloned and characterized. The E. histolytica TATA-box binding protein (EhTBP) is the first basal transcription factor that has been studied. To continue with the identification of other members of the basal transcription machinery, we performed an in silico analysis of the E. histolytica genome and found three loci encoding for polypeptides with similarity to EhTBP. One locus has a 100% identity to the previously Ehtbp gene reported by our group. The second locus encodes for a 212 aa polypeptide that is 100% identical to residues 23-234 from EhTBP. The third one encodes for a 216 aa polypeptide of 24kDa that showed 42.6% identity and 73.7% similarity to EhTBP. This protein was named E. histolytica TBP-related factor 1 (EhTRF1). Ehtrf1 gene was expressed in bacteria and the purified 28kDa recombinant polypeptide showed the capacity to bind to TATTTAAA-box by electrophoretic mobility shift assays. K(D) values for rEhTBP and rEhTRF1 were (1.71+/-2.90)x10(-12)M and (1.12+/-0.160)x10(-11)M, respectively. Homology modeling of EhTRF1 and EhTBP revealed that, although they were very similar, they showed some differences on their surfaces. Thus, E. histolytica is a unicellular organism having two members of the TBP family.

  13. Molecular characterization of genes encoding inward rectifier potassium (Kir) channels in the bed bug (Cimex lectularius).

    PubMed

    Mamidala, Praveen; Mittapelly, Priyanka; Jones, Susan C; Piermarini, Peter M; Mittapalli, Omprakash

    2013-04-01

    The molecular genetics of inward-rectifier potassium (Kir) channels in insects is poorly understood. To date, Kir channel genes have been characterized only from a few representative dipterans (i.e., fruit flies and mosquitoes). The goal of the present study was to characterize Kir channel cDNAs in a hemipteran, the bed bug (Cimex lectularius). Using our previously reported bed bug transcriptome (RNA-seq), we identified two cDNAs that encode putative Kir channels. One was a full-length cDNA that encodes a protein belonging to the insect 'Kir3' clade, which we designate as 'ClKir3'. The other was a partial cDNA that encodes a protein with similarity to both the insect 'Kir1' and 'Kir2' clades, which we designate as 'ClKir1/2'. Quantitative real-time PCR analysis revealed that ClKir1/2 and ClKir3 exhibited peak expression levels in late-instar nymphs and early-instar nymphs, respectively. Furthermore, ClKir3, but not ClKir1/2, showed tissue-specific expression in Malpighian tubules of adult bed bugs. Lastly, using an improved procedure for delivering double-stranded RNA (dsRNA) to male and female bed bugs (via the cervical membrane) we demonstrate rapid and systemic knockdown of ClKir3 transcripts. In conclusion, we demonstrate that the bed bug possesses at least two genes encoding Kir channels, and that RNAi is possible for at least Kir3, thereby offering a potential approach for elucidating the roles of Kir channel genes in bed bug physiology.

  14. Genetic analysis of the variable region genes encoding a monospecific human natural anti-DNA antibody.

    PubMed Central

    Daley, M D; Misener, V; Olee, T; Chen, P P; Siminovitch, K A

    1993-01-01

    Recent evidence suggests that natural autoantibodies may play an integral role in the development of the normal immune repertoire. To explore the genetic origins of these antibodies, we have isolated and sequenced the variable (V) region genes encoding both the heavy (H) and light (L) chains of a natural anti-DNA antibody, Kim11.4. The genes appear to be derived from the VH4.18 (subgroup VHIV), JH5, Hum1L1 (subgroup V lambda I) and J lambda 3 germline genes. The origin of the H chain diversity gene is more obscure, being potentially derived from one or more of several germline genes, arranged in either the forward or reverse orientations. Both the Kim11.4 VH and VL genes share significant degrees of similarity with those utilized in other autoantibodies, indicating that at least some degree of V restriction may exist in human autoreactive B cells. The pattern of nucleotide differences between the Kim11.4 VH and VL genes and their putative germline counterparts suggests that the Kim11.4 genes may have undergone somatic mutation and arisen as a result of antigen selection. PMID:8324896

  15. Heterogenic expression of genes encoding secreted proteins at the periphery of Aspergillus niger colonies.

    PubMed

    Vinck, Arman; de Bekker, Charissa; Ossin, Adam; Ohm, Robin A; de Vries, Ronald P; Wösten, Han A B

    2011-01-01

    Colonization of a substrate by fungi starts with the invasion of exploring hyphae. These hyphae secrete enzymes that degrade the organic material into small molecules that can be taken up by the fungus to serve as nutrients. We previously showed that only part of the exploring hyphae of Aspergillus niger highly express the glucoamylase gene glaA. This was an unexpected finding since all exploring hyphae are exposed to the same environmental conditions. Using GFP as a reporter, we here demonstrate that the acid amylase gene aamA, the α-glucuronidase gene aguA, and the feruloyl esterase gene faeA of A. niger are also subject to heterogenic expression within the exploring mycelium. Coexpression studies using GFP and dTomato as reporters showed that hyphae that highly express one of these genes also highly express the other genes encoding secreted proteins. Moreover, these hyphae also highly express the amylolytic regulatory gene amyR, and the glyceraldehyde-3-phosphate dehydrogenase gene gpdA. In situ hybridization demonstrated that the high expressers are characterized by a high 18S rRNA content. Taken together, it is concluded that two subpopulations of hyphae can be distinguished within the exploring mycelium of A. niger. The experimental data indicate that these subpopulations differ in their transcriptional and translational activity.

  16. Identification and characterization of multiple Spidroin 1 genes encoding major ampullate silk proteins in Nephila clavipes.

    PubMed

    Gaines, W A; Marcotte, W R

    2008-09-01

    Spider dragline silk is primarily composed of proteins called major ampullate spidroins (MaSps) that consist of a large repeat array flanked by nonrepetitive N- and C-terminal domains. Until recently, there has been little evidence for more than one gene encoding each of the two major spidroin silk proteins, MaSp1 and MaSp2. Here, we report the deduced N-terminal domain sequences for two distinct MaSp1 genes from Nephila clavipes (MaSp1A and MaSp1B) and for MaSp2. All three MaSp genes are co-expressed in the major ampullate gland. A search of the GenBank database also revealed two distinct MaSp1 C-terminal domain sequences. Sequencing confirmed that both MaSp1 genes are present in all seven Nephila clavipes spiders examined. The presence of nucleotide polymorphisms in these genes confirmed that MaSp1A and MaSp1B are distinct genetic loci and not merely alleles of the same gene. We experimentally determined the transcription start sites for all three MaSp genes and established preliminary pairing between the two MaSp1 N- and C-terminal domains. Phylogenetic analysis of these new sequences and other published MaSp N- and C-terminal domain sequences illustrated that duplications of MaSp genes may be widespread among spider species.

  17. Duplicated gelsolin family genes in zebrafish: a novel scinderin-like gene (scinla) encodes the major corneal crystallin.

    PubMed

    Jia, Sujuan; Omelchenko, Marina; Garland, Donita; Vasiliou, Vasilis; Kanungo, Jyotshnabala; Spencer, Michael; Wolf, Yuri; Koonin, Eugene; Piatigorsky, Joram

    2007-10-01

    We have previously identified a gelsolin-like protein (C/L-gelsolin) as a corneal crystallin in zebrafish. Here we show by phylogenetic analysis that there are at least six genes encoding gelsolin-like proteins based on their gelsolin domains in zebrafish: gsna and gsnb group with the vertebrate gelsolin gene, scina and scinb group with the scinderin (adseverin) gene, and scinla (C/L-gelsolin) and scinlb are novel scinderin-like genes. RT-PCR showed that scinla, scinlb, and gsnb are preferentially expressed in the adult cornea whereas gsna is expressed to a similar extent in cornea, lens, brain, and heart; scina and scinb expression were detectable only in whole zebrafish and not in these adult tissues. Quantitative RT-PCR and 2-dimensional polyacrylamide gel electrophoresis followed by MALDI/TOF mass spectroscopy confirmed high expression of beta-actin and scinla, moderate expression of scinlb, and very low expression of gsna and gsnb in the cornea. Finally, transgenic zebrafish carrying a green fluorescent protein reporter transgene driven by a 4 kb scinla promoter fragment showed expression in the cornea, snout, dorsal fin, and tail fin of 3-day-old zebrafish larvae. Our data suggest that scinla and scinlb are diverged paralogs of the vertebrate scinderin gene and show that scinla encodes the zebrafish corneal crystallin previously called C/L-gelsolin.

  18. Hypoxia: adapting to high altitude by mutating EPAS-1, the gene encoding HIF-2α.

    PubMed

    van Patot, Martha C Tissot; Gassmann, Max

    2011-01-01

    Living at high altitude is demanding and thus drives adaptational mechanisms. The Tibetan population has had a longer evolutionary period to adapt to high altitude than other mountain populations such as Andeans. As a result, some Tibetans living at high altitudes do not show markedly elevated red blood cell production as compared to South American high altitude natives such as Quechuas or Aymaras, thereby avoiding high blood viscosity creating cardiovascular risk. Unexpectedly, the responsible mutation(s) reducing red blood cell production do not involve either the gene encoding the blood hormone erythropoietin (Epo), or the corresponding regulatory sequences flanking the Epo gene. Similarly, functional mutations in the hypoxia-inducible transcription factor 1α (HIF-1α) gene that represents the oxygen-dependent subunit of the HIF-1 heterodimer, the latter being the main regulator of over 100 hypoxia-inducible genes, have not been described so far. It was not until very recently that three independent groups showed that the gene encoding HIF-2α, EPAS-1 (Wenger et al. 1997), represents a key gene mutated in Tibetan populations adapted to living at high altitudes (Beall et al. 2010 , Yi et al. 2010 , Simonson et al. 2010). Hypoxia-inducible transcription factors were first identified by the description of HIF-1 (Semenza et al. 1991 , 1992), which was subsequently found to enhance transcription of multiple genes that encode proteins necessary for rescuing from hypoxic exposure, including erythropoietic, angiogenic and glycolytic proteins. Then HIF-2 was identified (Ema et al. 1997 ; Flamme et al. 1997 ; Hogenesch et al. 1997 ; and Tian et al. 1997) and although it is highly similar to HIF-1 and has the potential to bind (Camenisch et al. 2001) and mediate (Mole et al. 2009) many of the same genes as HIF-1, its biological actions in response to hypoxia are distinct from those of HIF-1 (reviewed by Loboda et al. 2010). By now, several of these HIF-2 mediated

  19. Identification and characterization of a gene encoding for a nucleotidase from Phaseolus vulgaris.

    PubMed

    Cabello-Díaz, Juan Miguel; Gálvez-Valdivieso, Gregorio; Caballo, Cristina; Lambert, Rocío; Quiles, Francisco Antonio; Pineda, Manuel; Piedras, Pedro

    2015-08-01

    Nucleotidases are phosphatases that catalyze the removal of phosphate from nucleotides, compounds with an important role in plant metabolism. A phosphatase enzyme, with high affinity for nucleotides monophosphate previously identified and purified in embryonic axes from French bean, has been analyzed by MALDI TOF/TOF and two internal peptides have been obtained. The information of these peptide sequences has been used to search in the genome database and only a candidate gene that encodes for the phosphatase was identified (PvNTD1). The putative protein contains the conserved domains (motif I-IV) for haloacid dehalogenase-like hydrolases superfamily. The residues involved in the catalytic activity are also conserved. A recombinant protein overexpressed in Escherichia coli has shown molybdate resistant phosphatase activity with nucleosides monophosphate as substrate, confirming that the identified gene encodes for the phosphatase with high affinity for nucleotides purified in French bean embryonic axes. The activity of the purified protein was inhibited by adenosine. The expression of PvNTD1 gene was induced at the specific moment of radicle protrusion in embryonic axes. The gene was also highly expressed in young leaves whereas the level of expression in mature tissues was minimal.

  20. Identification of Cellular Genes Targeted by KSHV-Encoded MicroRNAs

    PubMed Central

    Samols, Mark A; Skalsky, Rebecca L; Maldonado, Ann M; Riva, Alberto; Lopez, M. Cecilia; Baker, Henry V; Renne, Rolf

    2007-01-01

    MicroRNAs (miRNAs) are 19 to 23 nucleotide–long RNAs that post-transcriptionally regulate gene expression. Human cells express several hundred miRNAs which regulate important biological pathways such as development, proliferation, and apoptosis. Recently, 12 miRNA genes have been identified within the genome of Kaposi sarcoma–associated herpesvirus; however, their functions are still unknown. To identify host cellular genes that may be targeted by these novel viral regulators, we performed gene expression profiling in cells stably expressing KSHV-encoded miRNAs. Data analysis revealed a set of 81 genes whose expression was significantly changed in the presence of miRNAs. While the majority of changes were below 2-fold, eight genes were down-regulated between 4- and 20-fold. We confirmed miRNA-dependent regulation for three of these genes and found that protein levels of thrombospondin 1 (THBS1) were decreased >10-fold. THBS1 has previously been reported to be down-regulated in Kaposi sarcoma lesions and has known activity as a strong tumor suppressor and anti-angiogenic factor, exerting its anti-angiogenic effect in part by activating the latent form of TGF-β. We show that reduced THBS1 expression in the presence of viral miRNAs translates into decreased TGF-β activity. These data suggest that KSHV-encoded miRNAs may contribute directly to pathogenesis by down-regulation of THBS1, a major regulator of cell adhesion, migration, and angiogenesis. PMID:17500590

  1. Developmental Regulation of Genes Encoding Universal Stress Proteins in Schistosoma mansoni

    PubMed Central

    Isokpehi, Raphael D.; Mahmud, Ousman; Mbah, Andreas N.; Simmons, Shaneka S.; Avelar, Lívia; Rajnarayanan, Rajendram V.; Udensi, Udensi K.; Ayensu, Wellington K.; Cohly, Hari H.; Brown, Shyretha D.; Dates, Centdrika R.; Hentz, Sonya D.; Hughes, Shawntae J.; Smith-McInnis, Dominique R.; Patterson, Carvey O.; Sims, Jennifer N.; Turner, Kelisha T.; Williams, Baraka S.; Johnson, Matilda O.; Adubi, Taiwo; Mbuh, Judith V.; Anumudu, Chiaka I.; Adeoye, Grace O.; Thomas, Bolaji N.; Nashiru, Oyekanmi; Oliveira, Guilherme

    2011-01-01

    The draft nuclear genome sequence of the snail-transmitted, dimorphic, parasitic, platyhelminth Schistosoma mansoni revealed eight genes encoding proteins that contain the Universal Stress Protein (USP) domain. Schistosoma mansoni is a causative agent of human schistosomiasis, a severe and debilitating Neglected Tropical Disease (NTD) of poverty, which is endemic in at least 76 countries. The availability of the genome sequences of Schistosoma species presents opportunities for bioinformatics and genomics analyses of associated gene families that could be targets for understanding schistosomiasis ecology, intervention, prevention and control. Proteins with the USP domain are known to provide bacteria, archaea, fungi, protists and plants with the ability to respond to diverse environmental stresses. In this research investigation, the functional annotations of the USP genes and predicted nucleotide and protein sequences were initially verified. Subsequently, sequence clusters and distinctive features of the sequences were determined. A total of twelve ligand binding sites were predicted based on alignment to the ATP-binding universal stress protein from Methanocaldococcus jannaschii. In addition, six USP sequences showed the presence of ATP-binding motif residues indicating that they may be regulated by ATP. Public domain gene expression data and RT-PCR assays confirmed that all the S. mansoni USP genes were transcribed in at least one of the developmental life cycle stages of the helminth. Six of these genes were up-regulated in the miracidium, a free-swimming stage that is critical for transmission to the snail intermediate host. It is possible that during the intra-snail stages, S. mansoni gene transcripts for universal stress proteins are low abundant and are induced to perform specialized functions triggered by environmental stressors such as oxidative stress due to hydrogen peroxide that is present in the snail hemocytes. This report serves to catalyze the

  2. [Cloning and structure of gene encoded alpha-latrocrustoxin from the Black widow spider venom].

    PubMed

    Danilevich, V N; Luk'ianov, S A; Grishin, E V

    1999-07-01

    The primary structure of the crusta gene encoding alpha-latrocrustoxin (alpha-LCT), a high molecular mass neurotoxin specific to crustaceans, was determined in the black widow spider Latrodectus mactans tredicimguttatus genome. The total length of the sequenced DNA was 4693 bp. The structural part of the black widow spider chromosome gene encoding alpha-LCT does not contain introns. The sequenced DNA contains a single extended open reading frame (4185 bp) and encodes a protein precursor of alpha-LCT, comprising 1395 aa. We assume the Met residue at position -10 relative to the N-terminal residue of Glu1 of the mature toxin to be the first one in the protein precursor. The calculated molecular mass of the precursor (156147 Da) exceeds that of the mature toxin by approximately 30 kDa. These data are in agreement with the notion that over the course of maturation the protein precursor undergoes double processing--cleavage of a decapeptide from the N-terminal part and of a approximately 200-aa fragment from the C-terminal part. alpha-LCT displayed a number of imperfect ankyrin-like repeats and areas of structural homology with earlier studied latrotoxins; the highest homology degree (62%) was revealed with alpha-latroinsectotoxin (alpha-LIT).

  3. Genome-Wide Identification and Mapping of NBS-Encoding Resistance Genes in Solanum tuberosum Group Phureja

    PubMed Central

    Lozano, Roberto; Ponce, Olga; Ramirez, Manuel; Mostajo, Nelly; Orjeda, Gisella

    2012-01-01

    The majority of disease resistance (R) genes identified to date in plants encode a nucleotide-binding site (NBS) and leucine-rich repeat (LRR) domain containing protein. Additional domains such as coiled-coil (CC) and TOLL/interleukin-1 receptor (TIR) domains can also be present. In the recently sequenced Solanum tuberosum group phureja genome we used HMM models and manual curation to annotate 435 NBS-encoding R gene homologs and 142 NBS-derived genes that lack the NBS domain. Highly similar homologs for most previously documented Solanaceae R genes were identified. A surprising ∼41% (179) of the 435 NBS-encoding genes are pseudogenes primarily caused by premature stop codons or frameshift mutations. Alignment of 81.80% of the 577 homologs to S. tuberosum group phureja pseudomolecules revealed non-random distribution of the R-genes; 362 of 470 genes were found in high density clusters on 11 chromosomes. PMID:22493716

  4. Two histone H1-encoding genes of the green alga Volvox carteri with features intermediate between plant and animal genes.

    PubMed

    Lindauer, A; Müller, K; Schmitt, R

    1993-07-15

    Southern hybridization indicated the presence of at least two and possibly four histone H1-encoding genes occurring as singlets in the Volvox carteri genome. Two of these genes, H1-I and H1-II, have been cloned and characterized. Their coding sequences are each interrupted by three introns, but only the position of the second intron is identically conserved in both H1-I and H1-II. The encoded 260-amino-acid (aa) (H1-I) and 240-aa (H1-II) polypeptides possess the typical tripartite organization of animal H1 histones, with variable N- and C-terminal domains flanking a conserved 'globular' DNA-binding domain. Extensive differences in their variable regions suggest that H1-I and H1-II (62% identity) represent two isotypes with different functions. A prominent KAPKAP-KAA motif in the H1-I N-terminal region, similarly seen in single H1 variants of a mosquito and a nematode, has a putative function in packing condensed subtypes of chromatin. Different from higher plants, but like animals, the H1 genes of V. carteri possess a typical 3' palindrome for mRNA processing, resulting in non-polyadenylated mRNAs. Transcription initiates 33 nucleotides (nt) (H1-I) and 26 nt (H1-II) downstream of typical TATA boxes. A putative 20-bp conserved enhancer element upstream of each TATA box closely resembles the consensus sequence associated with the nucleosomal histone-encoding genes in V. carteri [Müller et al., Gene 93 (1990) 167-175] and suggests stringent regulation. Accordingly, transcription of H1 was shown to be restricted to late embryogenesis, when new flagella are produced. We discuss the inferred accessory role of histone H1 proteins in stabilizing axonemal microtubules, as has been recently observed in sea urchin flagella [Multigner et al., Nature 360 (1992) 33-39].

  5. The twisted Gene Encodes Drosophila Protein O-Mannosyltransferase 2 and Genetically Interacts With the rotated abdomen Gene Encoding Drosophila Protein O-Mannosyltransferase 1

    PubMed Central

    Lyalin, Dmitry; Koles, Kate; Roosendaal, Sigrid D.; Repnikova, Elena; Van Wechel, Laura; Panin, Vladislav M.

    2006-01-01

    The family of mammalian O-mannosyltransferases includes two enzymes, POMT1 and POMT2, which are thought to be essential for muscle and neural development. Similar to mammalian organisms, Drosophila has two O-mannosyltransferase genes, rotated abdomen (rt) and DmPOMT2, encoding proteins with high homology to their mammalian counterparts. The previously reported mutant phenotype of the rt gene includes a clockwise rotation of the abdomen and defects in embryonic muscle development. No mutants have been described so far for the DmPOMT2 locus. In this study, we determined that the mutation in the twisted (tw) locus, tw1, corresponds to a DmPOMT2 mutant. The twisted alleles represent a complementation group of recessive mutations that, similar to the rt mutants, exhibit a clockwise abdomen rotation phenotype. Several tw alleles were isolated in the past; however, none of them was molecularly characterized. We used an expression rescue approach to confirm that tw locus represents DmPOMT2 gene. We found that the tw1 allele represents an amino acid substitution within the conserved PMT domain of DmPOMT2 (TW) protein. Immunostaining experiments revealed that the protein products of both rt and tw genes colocalize within Drosophila cells where they reside in the ER subcellular compartment. In situ hybridization analysis showed that both genes have essentially overlapping patterns of expression throughout most of embryogenesis (stages 8–17), while only the rt transcript is present at early embryonic stages (5 and 6), suggesting its maternal origin. Finally, we analyzed the genetic interactions between rt and tw using several mutant alleles, RNAi, and ectopic expression approaches. Our data suggest that the two Drosophila O-mannosyltransferase genes, rt and tw, have nonredundant functions within the same developmental cascade and that their activities are required simultaneously for possibly the same biochemical process. Our results establish the possibility of using

  6. 21 CFR 172.315 - Nicotinamide-ascorbic acid complex.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Nicotinamide-ascorbic acid complex. 172.315... Nutritional Additives § 172.315 Nicotinamide-ascorbic acid complex. Nicotinamide-ascorbic acid complex may be... the controlled reaction between ascorbic acid and nicotinamide, melting in the range 141 °C to 145...

  7. Cellular toxicity of nicotinamide metabolites.

    PubMed

    Rutkowski, Bolesław; Rutkowski, Przemysław; Słomińska, Ewa; Smolenski, Ryszard T; Swierczyński, Julian

    2012-01-01

    There are almost 100 different substances called uremic toxins. Nicotinamide derivatives are known as new family of uremic toxins. These uremic compounds play a role in an increased oxidative stress and disturbances in cellular repair processes by inhibiting poly (ADP-ribose) polymerase activity. New members of this family were discovered and described. Their toxic properties were a subject of recent studies. This study evaluated the concentration of 4-pyridone-3-carboxamid-1-β-ribonucleoside-triphosphate (4PYTP) and 4-pyridone-3-carboxamid-1-β-ribonucleoside-monophosphate (4PYMP) in erythrocytes of patients with chronic renal failure. Serum and red blood cells were collected from chronic renal failure patients on conservative treatment, those treated with hemodialysis, and at different times from those who underwent kidney transplantation. Healthy volunteers served as a control group. Nicotinamide metabolites were determined using liquid chromatography with mass spectrometry based on originally discovered and described method. Three novel compounds were described: 4-pyridone-3-carboxamid-1-β-ribonucleoside (4PYR), 4PYMP, and 4PYTP. 4PYR concentration was elevated in the serum, whereas 4PYMP and 4PYTP concentrations were augmented in erythrocytes of dialysis patients. Interestingly, concentrations of these compounds were less elevated during the treatment with erythropoietin-stimulating agents (ESAs). After successful kidney transplantation, concentrations of 4PYR and 4PYMP normalized according to the graft function, whereas that of 4PYTP was still elevated. During the incubation of erythrocytes in the presence of 4PYR, concentration of 4PYMP rose very rapidly while that of 4PYTP increased slowly. Therefore, we hypothesized that 4PYR, as a toxic compound, was actively absorbed by erythrocytes and metabolized to the 4PYMP and 4PYTP, which may interfere with function and life span of these cells.

  8. Phylogenetic and evolutionary analysis of NBS-encoding genes in Rosaceae fruit crops.

    PubMed

    Xu, Qiang; Wen, Xiaopeng; Deng, Xiuxin

    2007-07-01

    Phylogenetic relationships of the nucleotide binding site (NBS)-encoding resistance gene homologues (RGHs) among 12 species in five genera of Rosaceae fruit crops were evaluated. A total of 228 Rosaceous RGHs were deeply separated into two distinct clades, designated as TIR (sequences within this clade containing a Toll Interleukin-1 Receptor domain) and NonTIR (sequences lacking a TIR domain). Most Rosaceous RGH genes were phylogenetically distinct from Arabidopsis, Rice or Pine genes, except for a few Rosaceous members which grouped closely with Arabidopsis genes. Within Rosaceae, sequences from multiple species were often phylogenetically clustered together, forming heterogenous groups, however, apple- and chestnut rose-specific groups really exist. Gene duplication followed by sequence divergence were proposed as the mode for the evolution of a large number of distantly or closely related RGH genes in Rosaceae, and this mode may play a role in the generation of new resistance specificity. Positively selected sites within NBS-coding region were detected and thus nucleotide variation within NBS domain may function in determining disease resistance specificity. This study also discusses the synteny of a genomic region that encompass powdery mildew resistance locus among Malus, Prunus and Rosa, which may have potential use for fruit tree disease breeding and important gene cloning.

  9. Developmental expression of tobacco pistil-specific genes encoding novel extensin-like proteins.

    PubMed Central

    Goldman, M H; Pezzotti, M; Seurinck, J; Mariani, C

    1992-01-01

    We have sought to identify pistil-specific genes that can be used as molecular markers to study pistil development. For this purpose, a cDNA library was constructed from poly(A)+ RNA extracted from tobacco stigmas and styles at different developmental stages. Differential screening of this library led to the isolation of cDNA clones that correspond to genes preferentially or specifically expressed in the pistil. Seven of these cDNA clones encode proteins containing repetitions of the pentapeptide Ser-Pro4, which is a typical motif found in extensins. Unlike extensin genes, the extensin-like genes described here are not induced under stress conditions. RNA gel blot hybridizations demonstrated the organ-specific expression of the extensin-like genes and their temporal regulation during pistil development. After pollination, the transcript levels of the pistil-specific extensin-like genes change relative to levels in unpollinated pistils. In situ hybridization experiments showed that at least one of these pistil-specific genes is specifically expressed in cells of the transmitting tissue. The possible roles of the extensin-like proteins in pistils are discussed. PMID:1392607

  10. Nicotinamide riboside and nicotinic acid riboside salvage in fungi and mammals. Quantitative basis for Urh1 and purine nucleoside phosphorylase function in NAD+ metabolism.

    PubMed

    Belenky, Peter; Christensen, Kathryn C; Gazzaniga, Francesca; Pletnev, Alexandre A; Brenner, Charles

    2009-01-02

    NAD+ is a co-enzyme for hydride transfer enzymes and an essential substrate of ADP-ribose transfer enzymes and sirtuins, the type III protein lysine deacetylases related to yeast Sir2. Supplementation of yeast cells with nicotinamide riboside extends replicative lifespan and increases Sir2-dependent gene silencing by virtue of increasing net NAD+ synthesis. Nicotinamide riboside elevates NAD+ levels via the nicotinamide riboside kinase pathway and by a pathway initiated by splitting the nucleoside into a nicotinamide base followed by nicotinamide salvage. Genetic evidence has established that uridine hydrolase, purine nucleoside phosphorylase, and methylthioadenosine phosphorylase are required for Nrk-independent utilization of nicotinamide riboside in yeast. Here we show that mammalian purine nucleoside phosphorylase but not methylthioadenosine phosphorylase is responsible for mammalian nicotinamide riboside kinase-independent nicotinamide riboside utilization. We demonstrate that so-called uridine hydrolase is 100-fold more active as a nicotinamide riboside hydrolase than as a uridine hydrolase and that uridine hydrolase and mammalian purine nucleoside phosphorylase cleave nicotinic acid riboside, whereas the yeast phosphorylase has little activity on nicotinic acid riboside. Finally, we show that yeast nicotinic acid riboside utilization largely depends on uridine hydrolase and nicotinamide riboside kinase and that nicotinic acid riboside bioavailability is increased by ester modification.

  11. The Hd0053 gene of Haemophilus ducreyi encodes an alpha2,3-sialyltransferase.

    PubMed

    Li, Yanhong; Sun, Mingchi; Huang, Shengshu; Yu, Hai; Chokhawala, Harshal A; Thon, Vireak; Chen, Xi

    2007-09-21

    Haemophilus ducreyi is a Gram-negative bacterium that causes chancroid, a sexually transmitted genital ulcer disease. Different lipooligosaccharide (LOS) structures have been identified from H. ducreyi strain 35000, including those sialylated glycoforms. Surface LOS of H. ducreyi is considered an important virulence factor that is involved in ulcer formation, cell adhesion, and invasion of host tissue. Gene Hd0686 of H. ducreyi, designated lst (for lipooligosaccharide sialyltransferase), was identified to encode an alpha2,3-sialyltransferase that is important for the formation of sialylated LOS. Here, we show that Hd0053 of H. ducreyi genomic strain 35000HP, the third member of the glycosyltransferase family 80 (GT80), also encodes an alpha2,3-sialyltransferase that may be important for LOS sialylation.

  12. Isolation and sequence analysis of the gene encoding triose phosphate isomerase from Zygosaccharomyces bailii.

    PubMed

    Merico, A; Rodrigues, F; Côrte-Real, M; Porro, D; Ranzi, B M; Compagno, C

    2001-06-30

    The ZbTPI1 gene encoding triose phosphate isomerase (TIM) was cloned from a Zygosaccharomyces bailii genomic library by complementation of the Saccharomyces cerevisiae tpi1 mutant strain. The nucleotide sequence of a 1.5 kb fragment showed an open reading frame (ORF) of 746 bp, encoding a protein of 248 amino acid residues. The deduced amino acid sequence shares a high degree of homology with TIMs from other yeast species, including some highly conserved regions. The analysis of the promoter sequence of the ZbTPI1 revealed the presence of putative motifs known to have regulatory functions in S. cerevisiae. The GenBank Accession No. of ZbTPI1 is AF325852.

  13. The Chlamydomonas reinhardtii Nar1 Gene Encodes a Chloroplast Membrane Protein Involved in Nitrite Transport

    PubMed Central

    Rexach, Jesus; Fernández, Emilio; Galván, Aurora

    2000-01-01

    A key step for nitrate assimilation in photosynthetic eukaryotes occurs within chloroplasts, where nitrite is reduced to ammonium, which is incorporated into carbon skeletons. The Nar1 gene from Chlamydomonas reinhardtii is clustered with five other genes for nitrate assimilation, all of them regulated by nitrate. Sequence analysis of genomic DNA and cDNA of Nar1 and comparative studies of strains having or lacking Nar1 have been performed. The deduced amino acid sequence indicates that Nar1 encodes a chloroplast membrane protein with substantial identity to putative formate and nitrite transporters in bacteria. Use of antibodies against NAR1 has corroborated its location in the plastidic membrane. Characterization of strains having or lacking this gene suggests that NAR1 is involved in nitrite transport in plastids, which is critical for cell survival under limiting nitrate conditions, and controls the amount of nitrate incorporated by the cells under limiting CO2 conditions. PMID:10948261

  14. Cloning and characterization of the gene encoding inorganic pyrophosphatase of Escherichia coli K-12.

    PubMed

    Lahti, R; Pitkäranta, T; Valve, E; Ilta, I; Kukko-Kalske, E; Heinonen, J

    1988-12-01

    Escherichia coli K-12 gene ppa encoding inorganic pyrophosphatase (PPase) was cloned and sequenced. The 5' end of the ppa mRNA was identified by primer extension mapping. A typical E. coli sigma 70 promoter was identified immediately upstream of the mRNA 5' end. The structural gene of ppa contains 528 base pairs, from which a 175-amino-acid translation product, Mr 19,572, was deduced. The deduced amino acid composition perfectly fitted with that of PPase as previously determined (P. Burton, D. C. Hall, and J. Josse, J. Biol. Chem. 245:4346-4351, 1970). Furthermore, the partial amino acid sequence (residues 1 to 108) of E. coli PPase determined by S. A. Cohen (Ph.D. thesis, University of Chicago, 1978) was the same as that deduced from the nucleotide sequence. This is the first report of the cloning of a PPase gene.

  15. Identification and characterization of the Vibrio anguillarum prtV gene encoding a new metalloprotease

    NASA Astrophysics Data System (ADS)

    Mo, Zhaolan; Guo, Dongsheng; Mao, Yunxiang; Ye, Xuhong; Zou, Yuxia; Xiao, Peng; Hao, Bin

    2010-01-01

    We cloned and sequenced a prtV-like gene from Vibrio anguillarum M3 strain. This prtV gene encodes a putative protein of 918 amino acids, and is highly homologous to the V. cholerae prtV gene. We found that a prtV insertion mutant strain displayed lower gelatinase activity on gelatin agar, lower protease activity against azocasein, and lower activity for four glycosidases. This prtV mutant strain also had increased activity for two esterases in its extracellular products, as analyzed by the API ZYM system. In addition, the prtV mutant strain exhibited decreased growth in turbot intestinal mucus and reduced hemolytic activity on turbot erythrocytes. Infection experiments showed that the LD50 of the prtV mutant strain increased by at least 1 log compared to the wild-type in turbot fish. We propose that prtV plays an important role in the pathogenesis of V. anguillarum.

  16. Molecular cloning and sequencing of the gene encoding the fimbrial subunit protein of Bacteroides gingivalis.

    PubMed Central

    Dickinson, D P; Kubiniec, M A; Yoshimura, F; Genco, R J

    1988-01-01

    The gene encoding the fimbrial subunit protein of Bacteroides gingivalis 381, fimbrilin, has been cloned and sequenced. The gene was present as a single copy on the bacterial chromosome, and the codon usage in the gene conformed closely to that expected for an abundant protein. The predicted size of the mature protein was 35,924 daltons, and the secretory form may have had a 10-amino-acid, hydrophilic leader sequence similar to the leader sequences of the MePhe fimbriae family. The protein sequence had no marked similarity to known fimbrial sequences, and no homologous sequences could be found in other black-pigmented Bacteroides species, suggesting that fimbrillin represents a class of fimbrial subunit protein of limited distribution. Images PMID:2895100

  17. Function-Based Metagenomic Library Screening and Heterologous Expression Strategy for Genes Encoding Phosphatase Activity.

    PubMed

    Villamizar, Genis A Castillo; Nacke, Heiko; Daniel, Rolf

    2017-01-01

    The release of phosphate from inorganic and organic phosphorus compounds can be mediated enzymatically. Phosphate-releasing enzymes, comprising acid and alkaline phosphatases, are recognized as useful biocatalysts in applications such as plant and animal nutrition, bioremediation and diagnostic analysis. Metagenomic approaches provide access to novel phosphatase-encoding genes. Here, we describe a function-based screening approach for rapid identification of genes conferring phosphatase activity from small-insert and large-insert metagenomic libraries derived from various environments. This approach bears the potential for discovery of entirely novel phosphatase families or subfamilies and members of known enzyme classes hydrolyzing phosphomonoester bonds such as phytases. In addition, we provide a strategy for efficient heterologous phosphatase gene expression.

  18. Identification and Characterization of Multi-gene Family Encoding Germin-like Proteins in Cultivated Peanut (Arachis hypogaea L.)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Germins and germin-like proteins (GLPs) play diversified roles in plant development and basic defense. In this study, 36 EST-clones encoding GLPs were identified. Sequence similarity analysis demonstrated that the peanut genome possessed multi-gene family encoding at least 8 GLPs, named AhGLP1 to Ah...

  19. Nicotinic acid, nicotinamide, and nicotinamide riboside: a molecular evaluation of NAD+ precursor vitamins in human nutrition.

    PubMed

    Bogan, Katrina L; Brenner, Charles

    2008-01-01

    Although baseline requirements for nicotinamide adenine dinucleotide (NAD+) synthesis can be met either with dietary tryptophan or with less than 20 mg of daily niacin, which consists of nicotinic acid and/or nicotinamide, there is growing evidence that substantially greater rates of NAD+ synthesis may be beneficial to protect against neurological degeneration, Candida glabrata infection, and possibly to enhance reverse cholesterol transport. The distinct and tissue-specific biosynthetic and/or ligand activities of tryptophan, nicotinic acid, nicotinamide, and the newly identified NAD+ precursor, nicotinamide riboside, reviewed herein, are responsible for vitamin-specific effects and side effects. Because current data suggest that nicotinamide riboside may be the only vitamin precursor that supports neuronal NAD+ synthesis, we present prospects for human nicotinamide riboside supplementation and propose areas for future research.

  20. Cloning of the genes encoding two murine and human cochlear unconventional type I myosins

    SciTech Connect

    Crozet, F.; El Amraoui, Z.; Blanchard, S.

    1997-03-01

    Several lines of evidence indicate a crucial role for unconventional myosins in the function of the sensory hair cells of the inner ear. We report here the characterization of the cDNAs encoding two unconventional type I myosins from a mouse cochlear cDNA library. The first cDNA encodes a putative protein named Myo1c, which is likely to be the murine orthologue of the bullfrog myosin I{beta} and which may be involved in the gating of the mechanotransduction channel of the sensory hair cells. This myosin belongs to the group of short-tailed myosins I, with its tail ending shortly after a polybasic, TH-1-like domain. The second cDNA encodes a novel type I myosin Myo1f which displays three regions: a head domain with the conserved ATP- and actin-binding sites, a neck domain with a single IQ motif, and a tail domain with the tripartite structure initially described in protozoan myosins I. The tail of Myo1f includes (1) a TH-1 region rich in basic residues, which may interact with anionic membrane phospholipids; (2) a TH-2 proline-rich region, expected to contain an ATP-insensitive actin-binding site; and (3) an SH-3 domain found in a variety of cytoskeletal and signaling proteins. Northern blot analysis indicated that the genes encoding Myo1c and Myo1f display a widespread tissue expression in the adult mouse. Myo1c and Myo1f were mapped by in situ hybridization to the chromosomal regions 11D-11E and 17B-17C, respectively. The human orthologuous genes MYO1C and MYO1F were also characterized, and mapped to the human chromosomal regions 17p13 and 19p13.2- 19p1.3.3, respectively. 45 refs., 5 figs., 2 tabs.

  1. A bacterial gene codA encoding cytosine deaminase is an effective conditional negative selectable marker in Glycine max

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background Conditional negative selection is a powerful technique whereby the absence of a gene product allows survival in otherwise lethal conditions. In plants, the Escherichia coli gene codA has been employed as a negative selection marker. CodA is a conditionally lethal dominant gene encoding cy...

  2. PCR detection of enzyme-encoding genes in Leuconostoc mesenteroides strains of wine origin.

    PubMed

    Mtshali, Phillip Senzo; Divol, Benoit; du Toit, Maret

    2012-04-01

    Fifteen isolates of lactic acid bacteria originating from South African grape and wine samples were identified as Leuconostoc mesenteroides subsp. mesenteroides through the taxonomic analysis of their 16S rDNA gene sequences. These isolates were further tested for the presence of genes coding for enzymes of oenological relevance using PCR detection technique. A type strain of Leuc. mesenteroides (NCDO 529(T)) was also incorporated for comparative analysis. From the PCR detection results, the estA, prtP, alsD, alsS, metK, metC and metB genes were present in all the strains tested. The bgl and gshR genes encoding β-glucosidase and glutathione reductase, respectively, were not detected in some strains. On the other hand, none of the tested strains possessed the genes encoding phenolic acid decarboxylase (padA), citrate permease (citP), citrate lyase (citD, citE and citF) and arginine deiminase pathway enzymes (arcA, arcB and arcC). The verification of PCR-generated fragments was performed by sequencing. GenBank database was used to search for homologous DNA sequences. Neighbour-joining trees based on nucleotide sequences of alsS, estA, metK and mleA genes were also constructed in order to study the phylogenetic relationship between Leuc. mesenteroides strains and closely related species. The phylogenetic analyses revealed that there are genetic heterogeneities between strains of Leuc. mesenteroides species. In conclusion, this study has improved our knowledge on the genetics of oenological strains of Leuc. mesenteroides and their genetic potential to contribute to certain wine aroma compounds.

  3. Nucleotide sequence and characterization of a Bacillus subtilis gene encoding a flagellar switch protein.

    PubMed Central

    Zuberi, A R; Bischoff, D S; Ordal, G W

    1991-01-01

    The nucleotide sequence of the Bacillus subtilis fliM gene has been determined. This gene encodes a 38-kDa protein that is homologous to the FliM flagellar switch proteins of Escherichia coli and Salmonella typhimurium. Expression of this gene in Che+ cells of E. coli and B. subtilis interferes with normal chemotaxis. The nature of the chemotaxis defect is dependent upon the host used. In B. subtilis, overproduction of FliM generates mostly nonmotile cells. Those cells that are motile switch less frequently. Expression of B. subtilis FliM in E. coli also generates nonmotile cells. However, those cells that are motile have a tumble bias. The B. subtilis fliM gene cannot complement an E. coli fliM mutant. A frameshift mutation was constructed in the fliM gene, and the mutation was transferred onto the B. subtilis chromosome. The mutant has a Fla- phenotype. This phenotype is consistent with the hypothesis that the FliM protein encodes a component of the flagellar switch in B. subtilis. Additional characterization of the fliM mutant suggests that the hag and mot loci are not expressed. These loci are regulated by the SigD form of RNA polymerase. We also did not observe any methyl-accepting chemotaxis proteins in an in vivo methylation experiment. The expression of these proteins is also dependent upon SigD. It is possible that a functional basal body-hook complex may be required for the expression of SigD-regulated chemotaxis and motility genes. Images PMID:1898932

  4. Evolutionary Characteristics of Missing Proteins: Insights into the Evolution of Human Chromosomes Related to Missing-Protein-Encoding Genes.

    PubMed

    Xu, Aishi; Li, Guang; Yang, Dong; Wu, Songfeng; Ouyang, Hongsheng; Xu, Ping; He, Fuchu

    2015-12-04

    Although the "missing protein" is a temporary concept in C-HPP, the biological information for their "missing" could be an important clue in evolutionary studies. Here we classified missing-protein-encoding genes into two groups, the genes encoding PE2 proteins (with transcript evidence) and the genes encoding PE3/4 proteins (with no transcript evidence). These missing-protein-encoding genes distribute unevenly among different chromosomes, chromosomal regions, or gene clusters. In the view of evolutionary features, PE3/4 genes tend to be young, spreading at the nonhomology chromosomal regions and evolving at higher rates. Interestingly, there is a higher proportion of singletons in PE3/4 genes than the proportion of singletons in all genes (background) and OTCSGs (organ, tissue, cell type-specific genes). More importantly, most of the paralogous PE3/4 genes belong to the newly duplicated members of the paralogous gene groups, which mainly contribute to special biological functions, such as "smell perception". These functions are heavily restricted into specific type of cells, tissues, or specific developmental stages, acting as the new functional requirements that facilitated the emergence of the missing-protein-encoding genes during evolution. In addition, the criteria for the extremely special physical-chemical proteins were first set up based on the properties of PE2 proteins, and the evolutionary characteristics of those proteins were explored. Overall, the evolutionary analyses of missing-protein-encoding genes are expected to be highly instructive for proteomics and functional studies in the future.

  5. Cloning of the rice seed alpha-globulin-encoding gene: sequence similarity of the 5'-flanking region to those of the genes encoding wheat high-molecular-weight glutenin and barley D hordein.

    PubMed

    Nakase, M; Hotta, H; Adachi, T; Aoki, N; Nakamura, R; Masumura, T; Tanaka, K; Matsuda, T

    1996-05-08

    A genomic clone encoding the rice endosperm major globulin (alpha-globulin) with an apparent molecular mass of 26 kDa was isolated, and its nucleotide (nt) sequence and transcription start point (tsp) were determined. The tsp was identical to that of the gene encoding the wheat high-molecular-weight (HMW) glutenin subunit. The consensus '-300 element' and an A + T-rich sequence exist upstream from the TATA box in the 5'-flanking region. A nt sequence of about 130 bp in the 5'-flanking region was found to be markedly homologous to those of the genes encoding the wheat HMW glutenin subunit and barley D hordein.

  6. The lethal toxin from Australian funnel-web spiders is encoded by an intronless gene.

    PubMed

    Pineda, Sandy Steffany; Wilson, David; Mattick, John S; King, Glenn F

    2012-01-01

    Australian funnel-web spiders are generally considered the most dangerous spiders in the world, with envenomations from the Sydney funnel-web spider Atrax robustus resulting in at least 14 human fatalities prior to the introduction of an effective anti-venom in 1980. The clinical envenomation syndrome resulting from bites by Australian funnel-web spiders is due to a single 42-residue peptide known as δ-hexatoxin. This peptide delays the inactivation of voltage-gated sodium channels, which results in spontaneous repetitive firing and prolongation of action potentials, thereby causing massive neurotransmitter release from both somatic and autonomic nerve endings. Here we show that δ-hexatoxin from the Australian funnel-web spider Hadronyche versuta is produced from an intronless gene that encodes a prepropeptide that is post-translationally processed to yield the mature toxin. A limited sampling of genes encoding unrelated venom peptides from this spider indicated that they are all intronless. Thus, in distinct contrast to cone snails and scorpions, whose toxin genes contain introns, spiders may have developed a quite different genetic strategy for evolving their venom peptidome.

  7. Cloning and characterization of glyceraldehyde-3-phosphate dehydrogenase encoding gene in Gracilaria/Gracilariopsis lemaneiformis

    NASA Astrophysics Data System (ADS)

    Xueying, Ren; Zhenghong, Sui; Xuecheng, Zhang

    2006-04-01

    Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) plays important roles in various cellular processes. A cytosolic GAPDH encoding gene ( gpd) of Gracilaria/Gracilariopsis lemaneiformis was cloned and characterized. Deduced amino acid sequence of the enzyme of G. lemaneiformis had high homology with those of seven red algae. The 5'-untranslated regions of the GAPDHs encoding genes of these red algae varied greatly. GAPDHs of these red algae shared the highly conserved glyceraldehyde 3-phosphate dehydrogenase active site ASCTTNCL. However, such active site of Cyanidium caldarium was different from those of the other six algae at the last two residues (CL to LF), thus the spatial structure of its GAPDH active center may be different from those of the other six. Phylogenetic analysis indicated that GAPDH of G. lemaneiformis might have undergone an evolution similar to those of Porphyra yezoensis, Chondrus crispus, and Gracilaria verrucosa. C. caldarium had a closer evolutionary relationship with Cyanidioschyzon merolae than with Cyanidium sp. Virtual Northern blot analysis revealed that gpd of G. lemaneiformis expressed constitutively, which suggested that it might be house-keeping and could be adapted as an inner control in gene expression analysis of G. lemaneiformis.

  8. The Lethal Toxin from Australian Funnel-Web Spiders Is Encoded by an Intronless Gene

    PubMed Central

    Pineda, Sandy Steffany; Wilson, David; Mattick, John S.; King, Glenn F.

    2012-01-01

    Australian funnel-web spiders are generally considered the most dangerous spiders in the world, with envenomations from the Sydney funnel-web spider Atrax robustus resulting in at least 14 human fatalities prior to the introduction of an effective anti-venom in 1980. The clinical envenomation syndrome resulting from bites by Australian funnel-web spiders is due to a single 42-residue peptide known as δ-hexatoxin. This peptide delays the inactivation of voltage-gated sodium channels, which results in spontaneous repetitive firing and prolongation of action potentials, thereby causing massive neurotransmitter release from both somatic and autonomic nerve endings. Here we show that δ-hexatoxin from the Australian funnel-web spider Hadronyche versuta is produced from an intronless gene that encodes a prepropeptide that is post-translationally processed to yield the mature toxin. A limited sampling of genes encoding unrelated venom peptides from this spider indicated that they are all intronless. Thus, in distinct contrast to cone snails and scorpions, whose toxin genes contain introns, spiders may have developed a quite different genetic strategy for evolving their venom peptidome. PMID:22928020

  9. The bean. alpha. -amylase inhibitor is encoded by a lectin gene

    SciTech Connect

    Moreno, J.; Altabella, T.; Chrispeels, M.J. )

    1989-04-01

    The common bean, Phaseolus vulgaris, contains an inhibitor of insect and mammalian {alpha}-amylases that does not inhibit plant {alpha}-amylase. This inhibitor functions as an anti-feedant or seed-defense protein. We purified this inhibitor by affinity chromatography and found that it consists of a series of glycoforms of two polypeptides (Mr 14,000-19,000). Partial amino acid sequencing was carried out, and the sequences obtained are identical with portions of the derived amino acid sequence of a lectin-like gene. This lectin gene encodes a polypeptide of MW 28,000, and the primary in vitro translation product identified by antibodies to the {alpha}-amylase inhibitor has the same size. Co- and posttranslational processing of this polypeptide results in glycosylated polypeptides of 14-19 kDa. Our interpretation of these results is that the bean lectins constitute a gene family that encodes diverse plant defense proteins, including phytohemagglutinin, arcelin and {alpha}-amylase inhibitor.

  10. Characterization of the nuclear gene encoding mitochondrial aconitase in the marine red alga Gracilaria verrucosa.

    PubMed

    Zhou, Y H; Ragan, M A

    1995-07-01

    We have cloned a nuclear gene from the marine red alga Gracilaria verrucosa that encodes the complete 779 amino-acid mitochondrial aconitase (m-ACN), the first characterized from a photosynthetic organism. The N-terminal 28 deduced amino acids are predicted to constitute the mitochondrial transit peptide, the first described from a red alga. Putative transcriptional cis-acting elements were identified in the upstream untranslated region. The G. verrucosa m-ACN gene (m-ACN) is present in a single copy and is located ca. 1.5 kb upstream from the single-copy polyubiquitin gene. The single spliceosomal intron is located near the 5' end of the region encoding the mature m-ACN in precisely the same location and phase as intron 2 in Caenorhabditis elegans m-ACN; sequences at its 3' and 5' splice junctions and at the predicted lariat branch point conform well to the eukaryote consensus sequences. Multiple protein-sequence alignment of m-ACN, bacterial aconitase (b-ACN) and iron-responsive element-binding protein (IRE-BP), and phylogenetic analyses, revealed that m-ACN does not share a recent common ancestry with either b-ACN or IRE-BP.

  11. A Single Arabidopsis Gene Encodes Two Differentially Targeted Geranylgeranyl Diphosphate Synthase Isoforms1[OPEN

    PubMed Central

    Schipper, Bert; Beekwilder, Jules

    2016-01-01

    A wide diversity of isoprenoids is produced in different plant compartments. Most groups of isoprenoids synthesized in plastids, and some produced elsewhere in the plant cell derive from geranylgeranyl diphosphate (GGPP) synthesized by GGPP synthase (GGPPS) enzymes. In Arabidopsis (Arabidopsis thaliana), five genes appear to encode GGPPS isoforms localized in plastids (two), the endoplasmic reticulum (two), and mitochondria (one). However, the loss of function of the plastid-targeted GGPPS11 isoform (referred to as G11) is sufficient to cause lethality. Here, we show that the absence of a strong transcription initiation site in the G11 gene results in the production of transcripts of different lengths. The longer transcripts encode an isoform with a functional plastid import sequence that produces GGPP for the major groups of photosynthesis-related plastidial isoprenoids. However, shorter transcripts are also produced that lack the first translation initiation codon and rely on a second in-frame ATG codon to produce an enzymatically active isoform lacking this N-terminal domain. This short enzyme localizes in the cytosol and is essential for embryo development. Our results confirm that the production of differentially targeted enzyme isoforms from the same gene is a central mechanism to control the biosynthesis of isoprenoid precursors in different plant cell compartments. PMID:27707890

  12. The porcine lymphotropic herpesvirus 1 encodes functional regulators of gene expression

    SciTech Connect

    Lindner, I.; Ehlers, B. . E-mail: ehlersb@rki.de; Noack, S.; Dural, G.; Yasmum, N.; Bauer, C.; Goltz, M.

    2007-01-20

    The porcine lymphotropic herpesviruses (PLHV) are discussed as possible risk factors in xenotransplantation because of the high prevalence of PLHV-1, PLHV-2 and PLHV-3 in pig populations world-wide and the fact that PLHV-1 has been found to be associated with porcine post-transplant lymphoproliferative disease. To provide structural and functional knowledge on the PLHV immediate-early (IE) transactivator genes, the central regions of the PLHV genomes were characterized by genome walking, sequence and splicing analysis. Three spliced genes were identified (ORF50, ORFA6/BZLF1{sub h}, ORF57) encoding putative IE transactivators, homologous to (i) ORF50 and BRLF1/Rta (ii) K8/K-bZIP and BZLF1/Zta and (iii) ORF57 and BMLF1 of HHV-8 and EBV, respectively. Expressed as myc-tag or HA-tag fusion proteins, they were located to the cellular nucleus. In reporter gene assays, several PLHV-promoters were mainly activated by PLHV-1 ORF50, to a lower level by PLHV-1 ORFA6/BZLF1{sub h} and not by PLHV-1 ORF57. However, the ORF57-encoded protein acted synergistically on ORF50-mediated activation.

  13. Myxococcus xanthus sasS encodes a sensor histidine kinase required for early developmental gene expression.

    PubMed Central

    Yang, C; Kaplan, H B

    1997-01-01

    Initiation of Myxococcus xanthus multicellular development requires integration of information concerning the cells' nutrient status and density. A gain-of-function mutation, sasB7, that bypasses both the starvation and high cell density requirements for developmental expression of the 4521 reporter gene, maps to the sasS gene. The wild-type sasS gene was cloned and sequenced. This gene is predicted to encode a sensor histidine protein kinase that appears to be a key element in the transduction of starvation and cell density inputs. The sasS null mutants express 4521 at a basal level, form defective fruiting bodies, and exhibit reduced sporulation efficiencies. These data indicate that the wild-type sasS gene product functions as a positive regulator of 4521 expression and participates in M. xanthus development. The N terminus of SasS is predicted to contain two transmembrane domains that would locate the protein to the cytoplasmic membrane. The sasB7 mutation, an E139K missense mutation, maps to the predicted N-terminal periplasmic region. The C terminus of SasS contains all of the conserved residues typical of the sensor histidine protein kinases. SasS is predicted to be the sensor protein in a two-component system that integrates information required for M. xanthus developmental gene expression. PMID:9401035

  14. Divergence among genes encoding the elongation factor Tu of Yersinia Species.

    PubMed

    Isabel, Sandra; Leblanc, Eric; Boissinot, Maurice; Boudreau, Dominique K; Grondin, Myrian; Picard, François J; Martel, Eric A; Parham, Nicholas J; Chain, Patrick S G; Bader, Douglas E; Mulvey, Michael R; Bryden, Louis; Roy, Paul H; Ouellette, Marc; Bergeron, Michel G

    2008-11-01

    Elongation factor Tu (EF-Tu), encoded by tuf genes, carries aminoacyl-tRNA to the ribosome during protein synthesis. Duplicated tuf genes (tufA and tufB), which are commonly found in enterobacterial species, usually coevolve via gene conversion and are very similar to one another. However, sequence analysis of tuf genes in our laboratory has revealed highly divergent copies in 72 strains spanning the genus Yersinia (representing 12 Yersinia species). The levels of intragenomic divergence between tufA and tufB sequences ranged from 8.3 to 16.2% for the genus Yersinia, which is significantly greater than the 0.0 to 3.6% divergence observed for other enterobacterial genera. We further explored tuf gene evolution in Yersinia and other Enterobacteriaceae by performing directed sequencing and phylogenetic analyses. Phylogenetic trees constructed using concatenated tufA and tufB sequences revealed a monophyletic genus Yersinia in the family Enterobacteriaceae. Moreover, Yersinia strains form clades within the genus that mostly correlate with their phenotypic and genetic classifications. These genetic analyses revealed an unusual divergence between Yersinia tufA and tufB sequences, a feature unique among sequenced Enterobacteriaceae and indicative of a genus-wide loss of gene conversion. Furthermore, they provided valuable phylogenetic information for possible reclassification and identification of Yersinia species.

  15. The Novel Gene CRNDE Encodes a Nuclear Peptide (CRNDEP) Which Is Overexpressed in Highly Proliferating Tissues

    PubMed Central

    Szafron, Lukasz Michal; Balcerak, Anna; Grzybowska, Ewa Anna; Pienkowska-Grela, Barbara; Felisiak-Golabek, Anna; Podgorska, Agnieszka; Kulesza, Magdalena; Nowak, Natalia; Pomorski, Pawel; Wysocki, Juliusz; Rubel, Tymon; Dansonka-Mieszkowska, Agnieszka; Konopka, Bozena; Lukasik, Martyna; Kupryjanczyk, Jolanta

    2015-01-01

    CRNDE, recently described as the lncRNA-coding gene, is overexpressed at RNA level in human malignancies. Its role in gametogenesis, cellular differentiation and pluripotency has been suggested as well. Herein, we aimed to verify our hypothesis that the CRNDE gene may encode a protein product, CRNDEP. By using bioinformatics methods, we identified the 84-amino acid ORF encoded by one of two CRNDE transcripts, previously described by our research team. This ORF was cloned into two expression vectors, subsequently utilized in localization studies in HeLa cells. We also developed a polyclonal antibody against CRNDEP. Its specificity was confirmed in immunohistochemical, cellular localization, Western blot and immunoprecipitation experiments, as well as by showing a statistically significant decrease of endogenous CRNDEP expression in the cells with transient shRNA-mediated knockdown of CRNDE. Endogenous CRNDEP localizes predominantly to the nucleus and its expression seems to be elevated in highly proliferating tissues, like the parabasal layer of the squamous epithelium, intestinal crypts or spermatocytes. After its artificial overexpression in HeLa cells, in a fusion with either the EGFP or DsRed Monomer fluorescent tag, CRNDEP seems to stimulate the formation of stress granules and localize to them. Although the exact role of CRNDEP is unknown, our preliminary results suggest that it may be involved in the regulation of the cell proliferation. Possibly, CRNDEP also participates in oxygen metabolism, considering our in silico results, and the correlation between its enforced overexpression and the formation of stress granules. This is the first report showing the existence of a peptide encoded by the CRNDE gene. PMID:25978564

  16. The Caenorhabditis elegans unc-93 gene encodes a putative transmembrane protein that regulates muscle contraction

    PubMed Central

    1992-01-01

    unc-93 is one of a set of five interacting genes involved in the regulation or coordination of muscle contraction in Caenorhabditis elegans. Rare altered-function alleles of unc-93 result in sluggish movement and a characteristic "rubber band" uncoordinated phenotype. By contrast, null alleles cause no visibly abnormal phenotype, presumably as a consequence of the functional redundancy of unc-93. To understand better the role of unc-93 in regulating muscle contraction, we have cloned and molecularly characterized this gene. We isolated transposon- insertion alleles and used them to identify the region of DNA encoding the unc-93 protein. Two unc-93 proteins differing at their NH2 termini are potentially encoded by transcripts that differ at their 5' ends. The putative unc-93 proteins are 700 and 705 amino acids in length and have two distinct regions: the NH2 terminal portion of 240 or 245 amino acids is extremely hydrophilic, whereas the rest of the protein has multiple potential membrane-spanning domains. The unc-93 transcripts are low in abundance and the unc-93 gene displays weak codon usage bias, suggesting that the unc-93 protein is relatively rare. The unc-93 protein has no sequence similarity to proteins listed in current data- bases. Thus, unc-93 is likely to encode a novel membrane-associated muscle protein. We discuss possible roles for the unc-93 protein either as a component of an ion transport system involved in excitation- contraction coupling in muscle or in coordinating muscle contraction between muscle cells by affecting the functioning of gap junctions. PMID:1313436

  17. Cloning and characterization of the gene encoding 1-cyclohexenylcarbonyl coenzyme A reductase from Streptomyces collinus.

    PubMed Central

    Wang, P; Denoya, C D; Morgenstern, M R; Skinner, D D; Wallace, K K; Digate, R; Patton, S; Banavali, N; Schuler, G; Speedie, M K; Reynolds, K A

    1996-01-01

    We report the cloning of the gene encoding the 1-cyclohexenylcarbonyl coenzyme A reductase (ChcA) of Streptomyces collinus, an enzyme putatively involved in the final reduction step in the formation of the cyclohexyl moiety of ansatrienin from shikimic acid. The cloned gene, with a proposed designation of chcA, encodes an 843-bp open reading frame which predicts a primary translation product of 280 amino acids and a calculated molecular mass of 29.7 kDa. Highly significant sequence similiarity extending along almost the entire length of the protein was observed with members of the short-chain alcohol dehydrogenase superfamily. The S. collinus chcA gene was overexpressed in Escherichia coli by using a bacteriophage T7 transient expression system, and a protein with a specific ChcA activity was detected. The E. coli-produced ChcA protein was purified and shown to have similar steady-state kinetics and electrophoretic mobility on sodium dodecyl sulfate-polyacrylamide gels as the enoyl-coenzyme A reductase protein prepared from S. collinus. The enzyme demonstrated the ability to catalyze, in vitro, three of the reductive steps involved in the formation of cyclohexanecarboxylic acid. An S. collinus chcA mutant, constructed by deletion of a genomic region comprising the 5' end of chcA, lost the ChcA activity and the ability to synthesize either cyclohexanecarboxylic acid or ansatrienin. These results suggest that chcA encodes the ChcA that is involved in catalyzing multiple reductive steps in the pathway that provides the cyclohexanecarboxylic acid from shikimic acid. PMID:8955309

  18. Identification and characterization of the Arabidopsis gene encoding the tetrapyrrole biosynthesis enzyme uroporphyrinogen III synthase.

    PubMed

    Tan, Fui-Ching; Cheng, Qi; Saha, Kaushik; Heinemann, Ilka U; Jahn, Martina; Jahn, Dieter; Smith, Alison G

    2008-03-01

    UROS (uroporphyrinogen III synthase; EC 4.2.1.75) is the enzyme responsible for the formation of uroporphyrinogen III, the precursor of all cellular tetrapyrroles including haem, chlorophyll and bilins. Although UROS genes have been cloned from many organisms, the level of sequence conservation between them is low, making sequence similarity searches difficult. As an alternative approach to identify the UROS gene from plants, we used functional complementation, since this does not require conservation of primary sequence. A mutant of Saccharomyces cerevisiae was constructed in which the HEM4 gene encoding UROS was deleted. This mutant was transformed with an Arabidopsis thaliana cDNA library in a yeast expression vector and two colonies were obtained that could grow in the absence of haem. The rescuing plasmids encoded an ORF (open reading frame) of 321 amino acids which, when subcloned into an Escherichia coli expression vector, was able to complement an E. coli hemD mutant defective in UROS. Final proof that the ORF encoded UROS came from the fact that the recombinant protein expressed with an N-terminal histidine-tag was found to have UROS activity. Comparison of the sequence of AtUROS (A. thaliana UROS) with the human enzyme found that the seven invariant residues previously identified were conserved, including three shown to be important for enzyme activity. Furthermore, a structure-based homology search of the protein database with AtUROS identified the human crystal structure. AtUROS has an N-terminal extension compared with orthologues from other organisms, suggesting that this might act as a targeting sequence. The precursor protein of 34 kDa translated in vitro was imported into isolated chloroplasts and processed to the mature size of 29 kDa. Confocal microscopy of plant cells transiently expressing a fusion protein of AtUROS with GFP (green fluorescent protein) confirmed that AtUROS was targeted exclusively to chloroplasts in vivo.

  19. Diversity of Beetle Genes Encoding Novel Plant Cell Wall Degrading Enzymes

    PubMed Central

    Pauchet, Yannick; Wilkinson, Paul; Chauhan, Ritika; ffrench-Constant, Richard H.

    2010-01-01

    Plant cell walls are a heterogeneous mixture of polysaccharides and proteins that require a range of different enzymes to degrade them. Plant cell walls are also the primary source of cellulose, the most abundant and useful biopolymer on the planet. Plant cell wall degrading enzymes (PCWDEs) are therefore important in a wide range of biotechnological processes from the production of biofuels and food to waste processing. However, despite the fact that the last common ancestor of all deuterostomes was inferred to be able to digest, or even synthesize, cellulose using endogenous genes, all model insects whose complete genomes have been sequenced lack genes encoding such enzymes. To establish if the apparent “disappearance” of PCWDEs from insects is simply a sampling problem, we used 454 mediated pyrosequencing to scan the gut transcriptomes of beetles that feed on a variety of plant derived diets. By sequencing the transcriptome of five beetles, and surveying publicly available ESTs, we describe 167 new beetle PCWDEs belonging to eight different enzyme families. This survey proves that these enzymes are not only present in non-model insects but that the multigene families that encode them are apparently undergoing complex birth-death dynamics. This reinforces the observation that insects themselves, and not just their microbial symbionts, are a rich source of PCWDEs. Further it emphasises that the apparent absence of genes encoding PCWDEs from model organisms is indeed simply a sampling artefact. Given the huge diversity of beetles alive today, and the diversity of their lifestyles and diets, we predict that beetle guts will emerge as an important new source of enzymes for use in biotechnology. PMID:21179425

  20. Core-Gene-Encoded Peptide Regulating Virulence-Associated Traits in Streptococcus mutans

    PubMed Central

    Kim, Jeong Nam; Stanhope, Michael J.

    2013-01-01

    Recently, high-coverage genome sequence of 57 isolates of Streptococcus mutans, the primary etiological agent of human dental caries, was completed. The SMU.1147 gene, encoding a 61-amino-acid (61-aa) peptide, was present in all sequenced strains of S. mutans but absent in all bacteria in current databases. Reverse transcription-PCR revealed that SMU.1147 is cotranscribed with scnK and scnR, which encode the histidine kinase and response regulator, respectively, of a two-component system (TCS). The C terminus of the SMU.1147 gene product was tagged with a FLAG epitope and shown to be expressed in S. mutans by Western blotting with an anti-FLAG antibody. A nonpolar mutant of SMU.1147 formed less biofilm in glucose-containing medium and grew slower than did the wild-type strain under aerobic and anaerobic conditions, at low pH, or in the presence of H2O2. Mutation of SMU.1147 dramatically reduced genetic competence and expression of comX and comY, compared to S. mutans UA159. The competence defect of the SMU.1147 mutant could not be overcome by addition of sigX-inducing peptide (XIP) in defined medium or by competence-stimulating peptide (CSP) in complex medium. Complementation with SMU.1147 on a plasmid restored all phenotypes. Interestingly, mutants lacking either one of the TCS components and a mutant lacking all three genes behaved like the wild-type strain for all phenotypes mentioned above, but all mutant strains grew slower than UA159 in medium supplemented with 0.3 M NaCl. Thus, the SMU.1147-encoded peptide affects virulence-related traits and dominantly controls quorum-sensing pathways for development of genetic competence in S. mutans. PMID:23603743

  1. Regulation of Aspergillus genes encoding plant cell wall polysaccharide-degrading enzymes; relevance for industrial production.

    PubMed

    de Vries, R P

    2003-03-01

    The genus Aspergillus is widely used for the production of plant cell wall polysaccharide-degrading enzymes. The range of enzymes purified from these fungi covers nearly every function required for the complete degradation of cellulose, xyloglucan, xylan, galacto(gluco)mannan and pectin. This paper describes the Aspergillus enzymes involved in the degradation of these polysaccharides and discusses the regulatory systems involved in the expression of the genes encoding these proteins. The latter is of major importance in the large-scale production of these enzymes for industrial applications.

  2. Mutations in the Drosophila gene encoding ribosomal protein S6 cause tissue overgrowth.

    PubMed Central

    Stewart, M J; Denell, R

    1993-01-01

    We have characterized two P-element-induced, lethal mutations in Drosophila melanogaster which affect the larval hemocytes, mediators of the insect immune response. Each mutant displays larval melanotic tumors characteristic of mutations affecting the insect cellular immune system, and the moribund animals develop grossly hypertrophied hematopoietic organs because of increased cell proliferation and extra rounds of endoreduplication in some hematopoietic cells. Surprisingly, these mutations are due to P element insertions in the 5' regulatory region of the Drosophila gene encoding ribosomal protein S6 and cause a reduction of S6 transcript abundance in mutant larvae. Images PMID:8384310

  3. Nucleotide sequencing and characterization of the genes encoding benzene oxidation enzymes of Pseudomonas putida.

    PubMed Central

    Irie, S; Doi, S; Yorifuji, T; Takagi, M; Yano, K

    1987-01-01

    The nucleotide sequence of the genes from Pseudomonas putida encoding oxidation of benzene to catechol was determined. Five open reading frames were found in the sequence. Four corresponding protein molecules were detected by a DNA-directed in vitro translation system. Escherichia coli cells containing the fragment with the four open reading frames transformed benzene to cis-benzene glycol, which is an intermediate of the oxidation of benzene to catechol. The relation between the product of each cistron and the components of the benzene oxidation enzyme system is discussed. Images PMID:3667527

  4. Cloning and sequence analysis of the gene encoding isocitrate lyase from Rhodococcus fascians.

    PubMed

    Vereecke, D; Villarroel, R; Van Montagu, M; Desomer, J

    1994-07-22

    An isocitrate lyase (Icl)-encoding gene (icl) from the Gram+ plant pathogen Rhodococcus fascians was identified serendipitously as part of a scrambled fragment after shotgun cloning in the promoter probe vector, pDP1. The Icl protein is 429 amino acids long (47.11 kDa) and has a predicted pI of 4.84; it is 54% similar to the Escherichia coli Icl and 24-27% to eukaryotic homologues. Comparison of the prokaryotic and eukaryotic Icl confirms the earlier proposal of Matsuoka and McFadden [J. Bacteriol. 143 (1988) 4528-4536] that the enzyme has enlarged during evolution.

  5. Identification and characterization of an oleate hydratase-encoding gene from Bifidobacterium breve

    PubMed Central

    O'Connell, Kerry Joan; Motherway, Mary O'Connell; Hennessey, Alan A; Brodhun, Florian; Ross, R Paul; Feussner, Ivo; Stanton, Catherine; Fitzgerald, Gerald F; van Sinderen, Douwe

    2013-01-01

    Bifidobacteria are common commensals of the mammalian gastrointestinal tract. Previous studies have suggested that a bifidobacterial myosin cross reactive antigen (MCRA) protein plays a role in bacterial stress tolerance, while this protein has also been linked to the biosynthesis of conjugated linoleic acid (CLA) in bifidobacteria. In order to increase our understanding on the role of MCRA in bifidobacteria we created and analyzed an insertion mutant of the MCRA-encoding gene of B. breve NCFB 2258. Our results demonstrate that the MCRA protein of B. breve NCFB 2258 does not appear to play a role in CLA production, yet is an oleate hydratase, which contributes to bifidobacterial solvent stress protection. PMID:23851389

  6. Metabolic regulation of the gene encoding glutamine-dependent asparagine synthetase in Arabidopsis thaliana.

    PubMed Central

    Lam, H M; Peng, S S; Coruzzi, G M

    1994-01-01

    Here, we characterize a cDNA encoding a glutamine-dependent asparagine synthetase (ASN1) from Arabidopsis thaliana and assess the effects of metabolic regulation on ASN1 mRNA levels. Sequence analysis shows that the predicted ASN1 peptide contains a purF-type glutamine-binding domain. Southern blot experiments and cDNA clone analysis suggest that ASN1 is the only gene encoding glutamine-dependent asparagine synthetase in A. thaliana. The ASN1 gene is expressed predominantly in shoot tissues, where light has a negative effect on its mRNA accumulation. This negative effect of light on ASN1 mRNA levels was shown to be mediated, at least in part, via the photoreceptor phytochrome. We also investigated whether light-induced changes in nitrogen to carbon ratios might exert a metabolic regulation of the ASN1 mRNA accumulation. These experiments demonstrated that the accumulation of ASN1 mRNA in dark-grown plants is strongly repressed by the presence of exogenous sucrose. Moreover, this sucrose repression of ASN1 expression can be partially rescued by supplementation with exogenous amino acids such as asparagine, glutamine, and glutamate. These findings suggest that the expression of the ASN1 gene is under the metabolic control of the nitrogen to carbon ratio in cells. This is consistent with the fact that asparagine, synthesized by the ASN1 gene product, is a favored compound for nitrogen storage and nitrogen transport in dark-grown plants. We have put forth a working model suggesting that when nitrogen to carbon ratios are high, the gene product of ASN1 functions to re-direct the flow of nitrogen into asparagine, which acts as a shunt for storage and/or long-distance transport of nitrogen. PMID:7846154

  7. Contrasted patterns of selection since maize domestication on duplicated genes encoding a starch pathway enzyme.

    PubMed

    Corbi, J; Debieu, M; Rousselet, A; Montalent, P; Le Guilloux, M; Manicacci, D; Tenaillon, M I

    2011-03-01

    Maize domestication from teosinte (Zea mays ssp. parviglumis) was accompanied by an increase of kernel size in landraces. Subsequent breeding has led to a diversification of kernel size and starch content among major groups of inbred lines. We aim at investigating the effect of domestication on duplicated genes encoding a key enzyme of the starch pathway, the ADP-glucose pyrophosphorylase (AGPase). Three pairs of paralogs encode the AGPase small (SSU) and large (LSU) subunits mainly expressed in the endosperm, the embryo and the leaf. We first validated the putative sequence of LSU(leaf) through a comparative expression assay of the six genes. Second, we investigated the patterns of molecular evolution on a 2 kb coding region homologous among the six genes in three panels: teosintes, landraces, and inbred lines. We corrected for demographic effects by relying on empirical distributions built from 580 previously sequenced ESTs. We found contrasted patterns of selection among duplicates: three genes exhibit patterns of directional selection during domestication (SSU(end), LSU(emb)) or breeding (LSU(leaf)), two exhibit patterns consistent with diversifying (SSU(leaf)) and balancing selection (SSU(emb)) accompanying maize breeding. While patterns of linkage disequilibrium did not reveal sign of coevolution between genes expressed in the same organ, we detected an excess of non-synonymous substitutions in the small subunit functional domains highlighting their role in AGPase evolution. Our results offer a different picture on AGPase evolution than the one depicted at the Angiosperm level and reveal how genetic redundancy can provide flexibility in the response to selection.

  8. Cloning, sequencing and characterization of a gene encoding dihydroxyacetone kinase from Zygosaccharomyces rouxii NRRL2547.

    PubMed

    Wang, Zheng-Xiang; Kayingo, Gerald; Blomberg, Anders; Prior, Bernard A

    2002-12-01

    The dihydroxyacetone pathway, an alternative pathway for the dissimilation of glycerol via reduction by glycerol dehydrogenase and subsequent phosphorylation by dihydroxyacetone (DHA) kinase, is activated in the yeasts Saccharomyces cerevisiae and Zygosaccharomyces rouxii during osmotic stress. In experiments aimed at investigating the physiological function of the DHA pathway in Z. rouxii, a typical osmotolerant yeast, we cloned and characterized a DAK gene encoding dihydroxyacetone kinase from Z. rouxii NRRL 2547. Sequence analysis revealed a 1761 bp open reading frame, encoding a peptide composed of 587 deduced amino acids with the predicted molecular weight of 61 664 Da. As the amino acid sequence was most closely homologous (68% identity) to the S. cerevisiae Dak1p, we named the gene and protein ZrDAK1 and ZrDak1p, respectively. A putative ATP binding site was also found but no consensus element associated with osmoregulation was found in the upstream region of the ZrDAK1 gene. The ZrDAK1 gene complemented a S. cerevisiae W303-1A dak1delta dak2 delta strain by improving the growth of the mutant on 50 mmol/l dihydroxyacetone and by increasing the tolerance to dihydroxyacetone in a medium containing 5% sodium chloride, suggesting that it is a functional homologue of the S. cerevisiae DAK1. However, expression of the ZrDAK1 gene in the S. cerevisiae dak1delta dak2 delta strain had no significant effect on glycerol levels during osmotic stress. The ZrDAK1 sequence has been deposited in the public data bases under Accession No. AJ294719; regions upstream and downstream of ZrDAK1are deposited as Accession Nos AJ294739 and AJ294720, respectively.

  9. Effects of TCDD on the expression of nuclear encoded mitochondrial genes

    SciTech Connect

    Forgacs, Agnes L.; Burgoon, Lyle D.; Lynn, Scott G.; LaPres, John J.; Zacharewski, Timothy

    2010-07-15

    Generation of mitochondrial reactive oxygen species (ROS) can be perturbed following exposure to environmental chemicals such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Reports indicate that the aryl hydrocarbon receptor (AhR) mediates TCDD-induced sustained hepatic oxidative stress by decreasing hepatic ATP levels and through hyperpolarization of the inner mitochondrial membrane. To further elucidate the effects of TCDD on the mitochondria, high-throughput quantitative real-time PCR (HTP-QRTPCR) was used to evaluate the expression of 90 nuclear genes encoding mitochondrial proteins involved in electron transport, oxidative phosphorylation, uncoupling, and associated chaperones. HTP-QRTPCR analysis of time course (30 {mu}g/kg TCDD at 2, 4, 8, 12, 18, 24, 72, and 168 h) liver samples obtained from orally gavaged immature, ovariectomized C57BL/6 mice identified 54 differentially expressed genes (|fold change| > 1.5 and P-value < 0.1). Of these, 8 exhibited a sigmoidal or exponential dose-response profile (0.03 to 300 {mu}g/kg TCDD) at 4, 24 or 72 h. Dose-responsive genes encoded proteins associated with electron transport chain (ETC) complexes I (NADH dehydrogenase), III (cytochrome c reductase), IV (cytochrome c oxidase), and V (ATP synthase) and could be generally categorized as having proton gradient, ATP synthesis, and chaperone activities. In contrast, transcript levels of ETC complex II, succinate dehydrogenase, remained unchanged. Putative dioxin response elements were computationally found in the promoter regions of all 8 dose-responsive genes. This high-throughput approach suggests that TCDD alters the expression of genes associated with mitochondrial function which may contribute to TCDD-elicited mitochondrial toxicity.

  10. Enzymatic synthesis of polymers containing nicotinamide mononucleotide

    NASA Technical Reports Server (NTRS)

    Liu, Rihe

    1995-01-01

    Nicotinamide mononucleoside 5'-diphosphate in its reduced form is an excellent substrate for polynucleotide phosphorylase from Micrococcus luteus both in de novo polymerization reactions and in primer extension reactions. The oxidized form of the diphosphate is a much less efficient substrate; it can be used to extend primers but does not oligomerize in the absence of a primer. The cyanide adduct of the oxidized substrate, like the reduced substrate, polymerizes efficiently. Loss of cyanide yields high molecular weight polymers of the oxidized form. Terminal transferase from calf thymus accepts nicotinamide mononucleoside 5'-triphosphate as a substrate and efficiently adds one residue to the 3'-end of an oligodeoxynucleotide. T4 polynucleotide kinase accepts oligomers of nicotinamide mononucleotide as substrates. However, RNA polymerases do not incorporate nicotinamide mononucleoside 5'-triphosphate into products on any of the templates that we used.

  11. Enzymatic Synthesis of Polymers Containing Nicotinamide Mononucleotide

    NASA Technical Reports Server (NTRS)

    Liu, Rihe; Orgel, Leslie E.

    1995-01-01

    Nicotinamide mononucleoside 5'-diphosphate in its reduced form is an excellent substrate for polynucleotide phosphorylase from Micrococcus luteus both in de novo polymerization reactions and in primer extension reactions. The oxidized form of the diphosphate is a much less efficient substrate; it can be used to extend primers but does not oligomerize in the absence of a primer. The cyanide adduct of the oxidized substrate, like the reduced substrate, polymerizes efficiently. Loss of cyanide yields high molecular weight polymers of the oxidized form. Terminal transferase from calf thymus accepts nicotinamide mononucleoside 5'-triphosphate as a substrate and efficiently adds one residue to the 3'-end of an oligodeoxynucleotide. T4 polynucleotide kinase accepts oligomers of nicotinamide mononucleotide as substrates. However, RNA polymerases do not incorporate nicotinamide mononucleoside 5'-triphosphate into products on any of the templates that we used.

  12. The diageotropica gene of tomato encodes a cyclophilin: a novel player in auxin signaling.

    PubMed

    Oh, Kwangchul; Ivanchenko, Maria G; White, T J; Lomax, Terri L

    2006-06-01

    The single gene, auxin-resistant diageotropica (dgt) mutant of tomato displays a pleiotropic auxin-related phenotype that includes a slow gravitropic response, lack of lateral roots, reduced apical dominance, altered vascular development, and reduced fruit growth. Some auxin responses are unaltered in dgt plants, however, and the levels, metabolism, and transport of auxin appear normal, indicating that the Dgt gene encodes a component of a specific auxin signaling pathway. By combining map-based cloning with comparative microsynteny, we determined that the Dgt gene encodes a cyclophilin (CYP) (LeCYP1; gi:170439) that has not previously been identified as a component of auxin signaling and plant development. Each of the three known dgt alleles contains a unique mutation in the coding sequence of LeCyp1. Alleles dgt(1-1)and dgt(1-2) contain single nucleotide point mutations that generate an amino acid change (G137R) and a stop codon (W128stop), respectively, while dgt(dp) has an amino acid change (W128CDelta129-133) preceding a 15 bp deletion. Complementation of dgt plants with the wild-type LeCyp1 gene restored the wild-type phenotype. Each dgt mutation reduced or nullified the peptidyl-prolyl isomerase activity of the GST-LeCYP1 fusion proteins in vitro. RT-PCR and immunoblot analyses indicated that the dgt mutations do not affect the expression of LeCyp1 mRNA, but the accumulation of LeCYP1 protein is greatly reduced for all three mutant alleles. The CYP inhibitor, cyclosporin A, partially mimics the effects of the dgt mutation in inhibiting auxin-induced adventitious root initiation in tomato hypocotyl sections and reducing the auxin-induced expression of the early auxin response genes, LeIAA10 and 11. These observations confirm that the PPIase activity of the tomato CYP, LeCYP1, encoded by the Dgt gene is important for specific aspects of auxin regulation of plant growth, development, and environmental responses.

  13. Nicotinamide mononucleotide adenylyltransferase displays alternate binding modes for nicotinamide nucleotides

    PubMed Central

    Pfoh, Roland; Pai, Emil F.; Saridakis, Vivian

    2015-01-01

    Nicotinamide mononucleotide adenylyltransferase (NMNAT) catalyzes the biosynthesis of NAD+ and NaAD+. The crystal structure of NMNAT from Methanobacterium thermoautotrophicum complexed with NAD+ and SO4 2− revealed the active-site residues involved in binding and catalysis. Site-directed mutagenesis was used to further characterize the roles played by several of these residues. Arg11 and Arg136 were implicated in binding the phosphate groups of the ATP substrate. Both of these residues were mutated to lysine individually. Arg47 does not interact with either NMN or ATP substrates directly, but was deemed to play a role in binding as it is proximal to Arg11 and Arg136. Arg47 was mutated to lysine and glutamic acid. Surprisingly, when expressed in Escherichia coli all of these NMNAT mutants trapped a molecule of NADP+ in their active sites. This NADP+ was bound in a conformation that was quite different from that displayed by NAD+ in the native enzyme complex. When NADP+ was co-crystallized with wild-type NMNAT, the same structural arrangement was observed. These studies revealed a different conformation of NADP+ in the active site of NMNAT, indicating plasticity of the active site. PMID:26457427

  14. Molecular cloning of the gene encoding the bovine brain ribonuclease and its expression in different regions of the brain.

    PubMed Central

    Sasso, M P; Carsana, A; Confalone, E; Cosi, C; Sorrentino, S; Viola, M; Palmieri, M; Russo, E; Furia, A

    1991-01-01

    In this paper we report the molecular cloning of the gene encoding the bovine brain ribonuclease. The nucleotide sequence determined in this work shows a high degree of identity to the homologous gene encoding the bovine pancreatic ribonuclease. Processing of the primary transcripts of these genes also follows a similar pathway, splicing of the unique intron in the 5' untranslated region occurs at corresponding positions. Expression of the bovine brain ribonuclease gene can be detected both at the transcriptional and translational levels in all the regions of the brain examined. Images PMID:1754384

  15. Construction, cloning, and expression of synthetic genes encoding spider dragline silk.

    PubMed

    Prince, J T; McGrath, K P; DiGirolamo, C M; Kaplan, D L

    1995-08-29

    Synthetic genes encoding recombinant spider silk proteins have been constructed, cloned, and expressed. Protein sequences were derived from Nephila clavipes dragline silk proteins and reverse-translated to the corresponding DNA sequences. Codon selection was chosen to maximize expression levels in Escherichia coli. DNA "monomer" sequences were multimerized to encode high molecular weight synthetic spider silks using a "head-to-tail" construction strategy. Multimers were cloned into a prokaryotic expression vector and the encoded silk proteins were expressed in E. coli upon induction with IPTG. Four multimer, ranging in size from 14.7 to 41.3 kDa, were chosen for detailed analysis. These proteins were isolated by immobilized metal affinity chromatography and purified using reverse-phase HPLC. The composition and identity of the purified proteins were confirmed by amino acid composition analysis, N-terminal sequencing, laser desorption mass spectroscopy, and Western analysis using antibodies reactive to native spider dragline silk. Circular dichroism measurements indicate that the synthetic spider silks have substantial beta-sheet structure.

  16. Nicotinamide mononucleotide adenylyltransferase promotes hypoxic survival by activating the mitochondrial unfolded protein response

    PubMed Central

    Mao, X R; Kaufman, D M; Crowder, C M

    2016-01-01

    Gain-of-function mutations in the mouse nicotinamide mononucleotide adenylyltransferase type 1 (Nmnat1) produce two remarkable phenotypes: protection against traumatic axonal degeneration and reduced hypoxic brain injury. Despite intensive efforts, the mechanism of Nmnat1 cytoprotection remains elusive. To develop a new model to define this mechanism, we heterologously expressed a mouse Nmnat1 non-nuclear-localized gain-of-function mutant gene (m-nonN-Nmnat1) in the nematode Caenorhabditis elegans and show that it provides protection from both hypoxia-induced animal death and taxol-induced axonal pathology. Additionally, we find that m-nonN-Nmnat1 significantly lengthens C. elegans lifespan. Using the hypoxia-protective phenotype in C. elegans, we performed a candidate screen for genetic suppressors of m-nonN-Nmnat1 cytoprotection. Loss of function in two genes, haf-1 and dve-1, encoding mitochondrial unfolded protein response (mitoUPR) factors were identified as suppressors. M-nonN-Nmnat1 induced a transcriptional reporter of the mitoUPR gene hsp-6 and provided protection from the mitochondrial proteostasis toxin ethidium bromide. M-nonN-Nmnat1 was also protective against axonal degeneration in C. elegans induced by the chemotherapy drug taxol. Taxol markedly reduced basal expression of a mitoUPR reporter; the expression was restored by m-nonN-Nmnat1. Taken together, these data implicate the mitoUPR as a mechanism whereby Nmnat1 protects from hypoxic and axonal injury. PMID:26913604

  17. Nicotinamide extends replicative lifespan of human cells.

    PubMed

    Kang, Hyun Tae; Lee, Hyung Il; Hwang, Eun Seong

    2006-10-01

    We found that an ongoing application of nicotinamide to normal human fibroblasts not only attenuated expression of the aging phenotype but also increased their replicative lifespan, causing a greater than 1.6-fold increase in the number of population doublings. Although nicotinamide by itself does not act as an antioxidant, the cells cultured in the presence of nicotinamide exhibited reduced levels of reactive oxygen species (ROS) and oxidative damage products associated with cellular senescence, and a decelerated telomere shortening rate without a detectable increase in telomerase activity. Furthermore, in the treated cells growing beyond the original Hayflick limit, the levels of p53, p21WAF1, and phospho-Rb proteins were similar to those in actively proliferating cells. The nicotinamide treatment caused a decrease in ATP levels, which was stably maintained until the delayed senescence point. Nicotinamide-treated cells also maintained high mitochondrial membrane potential but a lower respiration rate and superoxide anion level. Taken together, in contrast to its demonstrated pro-aging effect in yeast, nicotinamide extends the lifespan of human fibroblasts, possibly through reduction in mitochondrial activity and ROS production.

  18. The Maltase Involved in Starch Metabolism in Barley Endosperm Is Encoded by a Single Gene

    PubMed Central

    Andriotis, Vasilios M. E.; Saalbach, Gerhard; Waugh, Robbie; Field, Robert A.; Smith, Alison M.

    2016-01-01

    During germination and early seedling growth of barley (Hordeum vulgare), maltase is responsible for the conversion of maltose produced by starch degradation in the endosperm to glucose for seedling growth. Despite the potential relevance of this enzyme for malting and the production of alcoholic beverages, neither the nature nor the role of maltase is fully understood. Although only one gene encoding maltase has been identified with certainty, there is evidence for the existence of other genes and for multiple forms of the enzyme. It has been proposed that maltase may be involved directly in starch granule degradation as well as in maltose hydrolysis. The aim of our work was to discover the nature of maltase in barley endosperm. We used ion exchange chromatography to fractionate maltase activity from endosperm of young seedlings, and we partially purified activity for protein identification. We compared maltase activity in wild-type barley and transgenic lines with reduced expression of the previously-characterised maltase gene Agl97, and we used genomic and transcriptomic information to search for further maltase genes. We show that all of the maltase activity in the barley endosperm can be accounted for by a single gene, Agl97. Multiple forms of the enzyme most likely arise from proteolysis and other post-translational modifications. PMID:27011041

  19. Genome mining demonstrates the widespread occurrence of gene clusters encoding bacteriocins in cyanobacteria.

    PubMed

    Wang, Hao; Fewer, David P; Sivonen, Kaarina

    2011-01-01

    Cyanobacteria are a rich source of natural products with interesting biological activities. Many of these are peptides and the end products of a non-ribosomal pathway. However, several cyanobacterial peptide classes were recently shown to be produced through the proteolytic cleavage and post-translational modification of short precursor peptides. A new class of bacteriocins produced through the proteolytic cleavage and heterocyclization of precursor proteins was recently identified from marine cyanobacteria. Here we show the widespread occurrence of bacteriocin gene clusters in cyanobacteria through comparative analysis of 58 cyanobacterial genomes. A total of 145 bacteriocin gene clusters were discovered through genome mining. These clusters encoded 290 putative bacteriocin precursors. They ranged in length from 28 to 164 amino acids with very little sequence conservation of the core peptide. The gene clusters could be classified into seven groups according to their gene organization and domain composition. This classification is supported by phylogenetic analysis, which further indicated independent evolutionary trajectories of gene clusters in different groups. Our data suggests that cyanobacteria are a prolific source of low-molecular weight post-translationally modified peptides.

  20. Nucleotide sequence of the Agrobacterium tumefaciens octopine Ti plasmid-encoded tmr gene.

    PubMed Central

    Heidekamp, F; Dirkse, W G; Hille, J; van Ormondt, H

    1983-01-01

    The nucleotide sequence of the tmr gene, encoded by the octopine Ti plasmid from Agrobacterium tumefaciens (pTiAch5), was determined. The T-DNA, which encompasses this gene, is involved in tumor formation and maintenance, and probably mediates the cytokinin-independent growth of transformed plant cells. The nucleotide sequence of the tmr gene displays a continuous open reading frame specifying a polypeptide chain of 240 amino acids. The 5'- terminus of the polyadenylated tmr mRNA isolated from octopine tobacco tumor cell lines was determined by nuclease S1 mapping. The nucleotide sequence 5'-TATAAAA-3', which sequence is identical to the canonical "TATA" box, was found 29 nucleotides upstream from the major initiation site for RNA synthesis. Two potential polyadenylation signals 5'-AATAAA-3' were found at 207 and 275 nucleotides downstream from the TAG stopcodon of the tmr gene. A comparison was made of nucleotide stretches, involved in transcription control of T-DNA genes. Images PMID:6312414

  1. Orf virus encodes a homolog of the vaccinia virus interferon-resistance gene E3L.

    PubMed

    McInnes, C J; Wood, A R; Mercer, A A

    1998-01-01

    A homolog of the vaccinia virus (VAC) interferon resistance gene E3L has been discovered in orf virus strain NZ-2, a parapoxvirus that infects sheep, goats and humans. The gene is located 20 kb from the left terminus of the orf virus genome and is transcribed towards this terminus. RNase protection studies have been used to define the limits of the gene and Northern analysis revealed that it is expressed early in infection. The predicted amino acid sequence of the orf virus protein shares 31% identity (57% similarity) with the VAC E3L protein. Four of the six residues identified as being essential to dsRNA binding in the vaccinia virus protein are conserved in the orf virus protein whilst the other two amino acid changes are conservative substitutions. The orf virus gene has been sequenced in two other orf virus strains which vary markedly in their ability to produce experimental lesions in vivo. Their predicted protein sequences vary by less than 3% from the NZ-2 protein. The recombinant orf virus protein, expressed as a fusion protein in E. coli, bound double-stranded (ds)RNA but not dsDNA, single-stranded (ss)DNA or ssRNA . This is the first demonstration of a VAC E3L-like gene encoded by a parapoxvirus.

  2. Bacterial Biosynthetic Gene Clusters Encoding the Anti-cancer Haterumalide Class of Molecules

    PubMed Central

    Matilla, Miguel A.; Stöckmann, Henning; Leeper, Finian J.; Salmond, George P. C.

    2012-01-01

    Haterumalides are halogenated macrolides with strong antitumor properties, making them attractive targets for chemical synthesis. Unfortunately, current synthetic routes to these molecules are inefficient. The potent haterumalide, oocydin A, was previously identified from two plant-associated bacteria through its high bioactivity against plant pathogenic fungi and oomycetes. In this study, we describe oocydin A (ooc) biosynthetic gene clusters identified by genome sequencing, comparative genomics, and chemical analysis in four plant-associated enterobacteria of the Serratia and Dickeya genera. Disruption of the ooc gene cluster abolished oocydin A production and bioactivity against fungi and oomycetes. The ooc gene clusters span between 77 and 80 kb and encode five multimodular polyketide synthase (PKS) proteins, a hydroxymethylglutaryl-CoA synthase cassette and three flavin-dependent tailoring enzymes. The presence of two free-standing acyltransferase proteins classifies the oocydin A gene cluster within the growing family of trans-AT PKSs. The amino acid sequences and organization of the PKS domains are consistent with the chemical predictions and functional peculiarities associated with trans-acyltransferase PKS. Based on extensive in silico analysis of the gene cluster, we propose a biosynthetic model for the production of oocydin A and, by extension, for other members of the haterumalide family of halogenated macrolides exhibiting anti-cancer, anti-fungal, and other interesting biological properties. PMID:23012376

  3. Localization of genes encoding three distinct flavin-containing monooxygenases to human chromosome 1q

    SciTech Connect

    Shephard, E.A.; Fox, M.F.; Povey, S. ); Dolphin, C.T.; Phillips, I.R.; Smith, R. )

    1993-04-01

    The authors have used the polymerase chain reaction to map the gene encoding human flavin-containing monooxygenase (FMO) form II (N. Lomri, Q. Gu, and J. R. Cashman, 1992, Proc. Natl. Acad. Sci. USA 89: 1685--1689) to chromosome 1. They propose the designation FMO3 for this gene as it is the third FMO gene to be mapped. The two other human FMO genes identified to date, FMO1 and FMO2, are also located on chromosome 1 (C. Dolphin, E. A. Shephard, S. Povey, C. N. A. Palmer, D. M. Ziegler, R. Ayesh, R. L. Smith, and 1. R. Phillips, 1991, J. Biol. Chem. 266: 12379--12385; C. Dolphin, E. A. Shephard, S. F. Povey, R. L. Smith, and I. R. Phillips, 1992, Biochem. J. 286: 261--267). The localization of FMO1, FMO2, and FMO3 has been refined to the long arm of chromosome 1. Analysis of human metaphase chromosomes by in situ hybridization confirmed the mapping of FMO1 and localized this gene more precisely to 1 q23-q25. 28 refs., 3 figs., 2 tabs.

  4. The KUP gene, located on human chromosome 14, encodes a protein with two distant zinc fingers.

    PubMed

    Chardin, P; Courtois, G; Mattei, M G; Gisselbrecht, S

    1991-04-11

    We have isolated a human cDNA (kup), encoding a new protein with two distantly spaced zinc fingers of the C2H2 type. This gene is highly conserved in mammals and is expressed mainly in hematopoietic cells and testis. Its expression was not higher in the various transformed cells tested than in the normal corresponding tissues. The kup gene is located in region q23-q24 of the long arm of human chromosome 14. The kup protein is 433 a.a. long, has a M.W. close to 50 kD and binds to DNA. Although the structure of the kup protein is unusual, the isolated fingers resemble closely those of the Krüppel family, suggesting that this protein is also a transcription factor. The precise function and DNA motif recognized by the kup protein remain to be determined.

  5. Cloning and characterization of a delta-6 desaturase encoding gene from Nannochloropsis oculata

    NASA Astrophysics Data System (ADS)

    Ma, Xiaolei; Yu, Jianzhong; Zhu, Baohua; Pan, Kehou; Pan, Jin; Yang, Guanpin

    2011-03-01

    A gene ( NANOC-D6D) encoding a desaturase that removes two hydrogen atoms from fatty acids at delta 6 position was isolated from a cDNA library of Nannochloropsis oculata (Droop) D. J. Hibberd (Eustigmatophyceae). The unicellular marine microalga N. oculata synthesizes rich long chain polyunsaturated fatty acids (LCPUFAs), including eicosapentaenoic acid (20:5n-3, EPA). The deduced protein contains 474 amino acids that fold into 4 trans-membrane domains. The neighbor-joining phylogenetic tree indicates that NANOC-D6D is phylogenetically close to the delta-6 fatty acid desaturase of marine microalgae such as Glossomastix chrysoplasta, Thalassiosira pseudonana, and Phaeodactylum tricornutum. The gene was expressed in Saccharomyces cerevisiae INVScl to verify the substrate specificity of NANOC-D6D. Our results suggest that the recombinant NANOC-D6D simultaneously desaturates linoleic acid (LA) and α-linolenic acid (ALA).

  6. The Arabidopsis ERECTA gene encodes a putative receptor protein kinase with extracellular leucine-rich repeats.

    PubMed Central

    Torii, K U; Mitsukawa, N; Oosumi, T; Matsuura, Y; Yokoyama, R; Whittier, R F; Komeda, Y

    1996-01-01

    Arabidopsis Landsberg erecta is one of the most popular ecotypes and is used widely for both molecular and genetic studies. It harbors the erecta (er) mutation, which confers a compact inflorescence, blunt fruits, and short petioles. We have identified five er mutant alleles from ecotypes Columbia and Wassilewskija. Phenotypic characterization of the mutant alleles suggests a role for the ER gene in regulating the shape of organs originating from the shoot apical meristem. We cloned the ER gene, and here, we report that it encodes a putative receptor protein kinases. The deduced ER protein contains a cytoplasmic protein kinase catalytic domain, a transmembrane region, and an extracellular domain consisting of leucine-rich repeats, which are thought to interact with other macromolecules. Our results suggest that cell-cell communication mediated by a receptor kinase has an important role in plant morphogenesis. PMID:8624444

  7. Parkinson's disease in relation to pesticide exposure and nuclear encoded mitochondrial complex I gene variants.

    PubMed

    Corder, Elizabeth H; Mellick, George D

    2006-01-01

    Parkinson's disease (PD) is a common age-related neurodegenerative disorder thought to result from the integrated effects of genetic background and exposure to neuronal toxins. Certain individual nuclear-encoded mitochondrial complex I gene polymorphisms were found to be associated with approximately 2-fold risk variation in an Australian case-control sample. We further characterized this sample of 306 cases and 321 controls to determine the mutual information contained in the 22 SNPs and, additionally, level of pesticide exposure: five distinct risk sets were identified using grade-of-membership analysis. Of these, one was robust to pesticide exposure (I), three were vulnerable (II, III, IV), and another (V) denoted low risk for unexposed persons. Risk for individual subjects varied > 16-fold according to level of membership in the vulnerable groups. We conclude that inherited variation in mitochondrial complex I genes and pesticide exposure together modulate risk for PD.

  8. NGF induction of the gene encoding the protease transin accompanies neuronal differentiation in PC12 cells.

    PubMed

    Machida, C M; Rodland, K D; Matrisian, L; Magun, B E; Ciment, G

    1989-06-01

    Various proteases have been found to be released by the growth cones of developing neurons in culture and have been hypothesized to play a role in the process of axon elongation. We report here that nerve growth factor (NGF) induced the gene encoding the metalloprotease transin in PC12 cells with a time course coincident with the initial appearance of neurites by these cells. Acidic and basic fibroblast growth factors also stimulated transin mRNA expression and neurite outgrowth, whereas various other agents had no effects on either of these phenomena. In contrast, dexamethasone was found to inhibit the induction of transin mRNA when added with, or following, NGF treatment. Finally, we show that sequences contained within 750 bp of the 5' untranscribed region of the transin gene confer responsiveness to NGF and dexamethasone.

  9. Identification and functional analysis of the gene encoding methionine-gamma-lyase in Brevibacterium linens.

    PubMed

    Amarita, Felix; Yvon, Mireille; Nardi, Michele; Chambellon, Emilie; Delettre, Jerôme; Bonnarme, Pascal

    2004-12-01

    The enzymatic degradation of L-methionine and subsequent formation of volatile sulfur compounds (VSCs) is believed to be essential for flavor development in cheese. L-methionine-gamma-lyase (MGL) can convert L-methionine to methanethiol (MTL), alpha-ketobutyrate, and ammonia. The mgl gene encoding MGL was cloned from the type strain Brevibacterium linens ATCC 9175 known to produce copious amounts of MTL and related VSCs. The disruption of the mgl gene, achieved in strain ATCC 9175, resulted in a 62% decrease in thiol-producing activity and a 97% decrease in total VSC production in the knockout strain. Our work shows that L-methionine degradation via gamma-elimination is a key step in the formation of VSCs in B. linens.

  10. Expression of the gene encoding growth hormone in the human mammary gland

    SciTech Connect

    Mol, J.A.; Misdorp, W.; Rijnberk, A.

    1995-10-01

    Progestins cause a syndrome of growth hormone (GH) excess and enhanced mammary tumorigenesis in the dog. This has been regarded as being specific for the dog. Recently we reported that progestin-induced GH excess originates from foci of hyperplastic ductular epithelium of the mammary gland in the dog. In the present report we demonstrate by reverse-transcriptase PCR and immunohistochemistry that a main factor involved in tissue growth, i.e. GH, is also expressed in normal and neoplastic human mammary glands. The gene expressed in the human mammary gland proved to be identical to the gene encoding GH in the pituitary gland. The role of progesterone in the GH expression of the human mammary gland needs, however, to be proven. It is hypothesized that this locally produced hGH may play a pathogenetic role in breast cancer. 21 refs., 2 figs., 1 tab.

  11. Cloning, overexpression and nucleotide sequence of a thermostable DNA ligase-encoding gene.

    PubMed

    Barany, F; Gelfand, D H

    1991-12-20

    Thermostable DNA ligase has been harnessed for the detection of single-base genetic diseases using the ligase chain reaction [Barany, Proc. Natl. Acad. Sci. USA 88 (1991) 189-193]. The Thermus thermophilus (Tth) DNA ligase-encoding gene (ligT) was cloned in Escherichia coli by genetic complementation of a ligts 7 defect in an E. coli host. Nucleotide sequence analysis of the gene revealed a single chain of 676 amino acid residues with 47% identity to the E. coli ligase. Under phoA promoter control, Tth ligase was overproduced to greater than 10% of E. coli cellular proteins. Adenylated and deadenylated forms of the purified enzyme were distinguished by apparent molecular weights of 81 kDa and 78 kDa, respectively, after separation via sodium dodecyl sulfate-polyacrylamide-gel electrophoresis.

  12. Conserved serine/threonine kinase encoded by CBK1 regulates expression of several hypha-associated transcripts and genes encoding cell wall proteins in Candida albicans.

    PubMed

    McNemar, Mark D; Fonzi, William A

    2002-04-01

    The opportunistic fungal pathogen, Candida albicans, is reported to have several potential virulence factors. A potentially significant factor is the ability to undergo morphological transition from yeast to hypha. This alteration of form is accompanied by many changes within the cell, including alterations in gene expression and cell wall composition. We have isolated a gene that encodes a highly conserved serine/threonine kinase that appears to be involved in the regulation of proteins associated with the cell wall. We have assigned the designation CBK1 (cell wall biosynthesis kinase 1) to this gene. Mutants lacking CBK1 form large aggregates of round cells under all growth conditions and lack the ability to undergo morphological differentiation. Additionally, these mutants show an altered pattern of expression of several transcripts encoding proteins associated with the cell wall. The results suggest that the kinase encoded by CBK1 plays a general role in the maintenance and alteration of the cell wall of C. albicans in all morphologies.

  13. Characterization of three genomic loci encoding Rhizobium sp. strain ORS571 N2 fixation genes.

    PubMed

    Donald, R G; Nees, D W; Raymond, C K; Loroch, A I; Ludwig, R A

    1986-01-01

    Sixty-five independent, N2 fixation-defective (Nif-) vector insertion (Vi) mutants were selected, cloned, and mapped to the ORS571 genome. The recombinant Nif::Vi plasmids obtained in this way were used as DNA hybridization probes to isolate homologous phages from a genomic library of ORS571 constructed in lambda EMBL3. Genomic maps were drawn for three ORS571 Nif gene loci. Forty-five Nif::Vi mutants in genomic Nif locus 1 defined two gene clusters separated by 8 kilobase pairs (kb) of DNA. In the first cluster, 36 Nif::Vi mutants mapped to a 7-kb DNA segment that showed DNA homology with Klebsiella pneumoniae nifHDKE and encoded at least two Nif operons. In the other cluster, nine Nif::Vi mutants mapped to a 1.5-kb DNA segment that showed homology with K. pneumoniae and Rhizobium meliloti nifA; this DNA segment encoded a separate Nif operon. Fifteen Nif::Vi mutants mapped to a 3.5-kb DNA segment defined as Nif locus 2 and showed DNA homology with the R. meliloti P2 fixABC operon. Nif locus 2 carries a second nifH (nifH2) gene. Four Nif::Vi mutants mapped to a 2-kb DNA segment defined as Nif locus 3 and showed DNA homology with K. pneumoniae nifB. DNA from lambda Nif phages comprising all three genomic Nif loci was subcloned in plasmid vectors able to stably replicate in ORS571. These plasmid subclones were introduced into ORS571 strains carrying physically mapped Nif::Vi insertions, and genetic complementations were conducted. With the exception of certain mutants mapping to the nifDK genes, all mutants could be complemented to Nif+ when they carried plasmid subclones of defined genomic DNA regions. Conversely, most nifDK mutants behaved as pseudodominant alleles.

  14. TAFII40 Protein Is Encoded by the e(y)1 Gene: Biological Consequences of Mutations

    PubMed Central

    Soldatov, Aleksei; Nabirochkina, Elena; Georgieva, Sofia; Belenkaja, Tatiana; Georgiev, Pavel

    1999-01-01

    The enhancer of yellow 1 gene, e(y)1, of Drosophila melanogaster has been cloned and demonstrated to encode the TAFII40 protein. The e(y)1 gene is expressed in females much more strongly than in males due to the accumulation of e(y)1 mRNA in the ovaries. Two different e(y)1 mutations have been obtained. The e(y)1ul mutation, induced by the insertion of Stalker into the coding region, leads to the replacement of 25 carboxy-terminal amino acids by 17 amino acids encoded by the Stalker sequences and to a decrease of the e(y)1 transcription level. The latter is the main cause of dramatic underdevelopment of the ovaries and sterility of females bearing the e(y)1 mutation. This follows from the restoration of female fertility upon transformation of e(y)1u1 flies with a construction synthesizing the mutant protein. The e(y)1P1 mutation induced by P element insertion into the transcribed nontranslated region of the gene has almost no influence on the phenotype of flies. However, in combination with the phP1 mutation, which leads to a strong P element-mediated suppression of e(y)1 transcription, this mutation is lethal. Genetic studies of the e(y)1u1 mutation revealed a sensitivity of the yellow and white expression to the TAFII40/e(y)1 level. The su(Hw)-binding region, Drosophila insulator, stabilizes the expression of the white gene and makes it independent of the e(y)1u1 mutation. PMID:10207100

  15. Multidrug resistance in fungi: regulation of transporter-encoding gene expression

    PubMed Central

    Paul, Sanjoy; Moye-Rowley, W. Scott

    2014-01-01

    A critical risk to the continued success of antifungal chemotherapy is the acquisition of resistance; a risk exacerbated by the few classes of effective antifungal drugs. Predictably, as the use of these drugs increases in the clinic, more resistant organisms can be isolated from patients. A particularly problematic form of drug resistance that routinely emerges in the major fungal pathogens is known as multidrug resistance. Multidrug resistance refers to the simultaneous acquisition of tolerance to a range of drugs via a limited or even single genetic change. This review will focus on recent progress in understanding pathways of multidrug resistance in fungi including those of most medical relevance. Analyses of multidrug resistance in Saccharomyces cerevisiae have provided the most detailed outline of multidrug resistance in a eukaryotic microorganism. Multidrug resistant isolates of S. cerevisiae typically result from changes in the activity of a pair of related transcription factors that in turn elicit overproduction of several target genes. Chief among these is the ATP-binding cassette (ABC)-encoding gene PDR5. Interestingly, in the medically important Candida species, very similar pathways are involved in acquisition of multidrug resistance. In both C. albicans and C. glabrata, changes in the activity of transcriptional activator proteins elicits overproduction of a protein closely related to S. cerevisiae Pdr5 called Cdr1. The major filamentous fungal pathogen, Aspergillus fumigatus, was previously thought to acquire resistance to azole compounds (the principal antifungal drug class) via alterations in the azole drug target-encoding gene cyp51A. More recent data indicate that pathways in addition to changes in the cyp51A gene are important determinants in A. fumigatus azole resistance. We will discuss findings that suggest azole resistance in A. fumigatus and Candida species may share more mechanistic similarities than previously thought. PMID:24795641

  16. Expression of a gene encoding mitochondrial aldehyde dehydrogenase in rice increases under submerged conditions.

    PubMed

    Nakazono, M; Tsuji, H; Li, Y; Saisho, D; Arimura, S; Tsutsumi, N; Hirai, A

    2000-10-01

    It is known that alcoholic fermentation is important for survival of plants under anaerobic conditions. Acetaldehyde, one of the intermediates of alcoholic fermentation, is not only reduced by alcohol dehydrogenase but also can be oxidized by aldehyde dehydrogenase (ALDH). To determine whether ALDH plays a role in anaerobic metabolism in rice (Oryza sativa L. cv Nipponbare), we characterized a cDNA clone encoding mitochondrial ALDH from rice (Aldh2a). Analysis of sub-cellular localization of ALDH2a protein using green fluorescent protein and an in vitro ALDH assay using protein extracts from Escherichia coli cells that overexpressed ALDH2a indicated that ALDH2a functions in the oxidation of acetaldehyde in mitochondria. A Southern-blot analysis indicated that mitochondrial ALDH is encoded by at least two genes in rice. We found that the Aldh2a mRNA was present at high levels in leaves of dark-grown seedlings, mature leaf sheaths, and panicles. It is interesting that expression of the rice Aldh2a gene, unlike the expression of the tobacco (Nicotiana tabacum) Aldh2a gene, was induced in rice seedlings by submergence. Experiments with ruthenium red, which is a blocker of Ca(2+) fluxes in rice as well as maize (Zea mays), suggest that the induction of expression of Adh1 and Pdc1 by low oxygen stress is regulated by elevation of the cytosolic Ca(2+) level. However, the induction of Aldh2a gene expression may not be controlled by the cytosolic Ca(2+) level elevation. A possible involvement of ALDH2a in the submergence tolerance of rice is discussed.

  17. A turquoise mutant genetically separates expression of genes encoding phycoerythrin and its associated linker peptides.

    PubMed

    Seib, Laura Ort; Kehoe, David M

    2002-02-01

    During complementary chromatic adaptation (CCA), cyanobacterial light harvesting structures called phycobilisomes are restructured in response to ambient light quality shifts. Transcription of genes encoding components of the phycobilisome is differentially regulated during this process: red light activates cpcB2A2, whereas green light coordinately activates the cpeCDE and cpeBA operons. Three signal transduction components that regulate CCA have been isolated to date: a sensor-photoreceptor (RcaE) and two response regulators (RcaF and RcaC). Mutations in the genes encoding these components affect the accumulation of both cpcB2A2 and cpeBA gene products. We have isolated and characterized a new pigmentation mutant called Turquoise 1. We demonstrate that this mutant phenotype is due to a dramatic decrease in cpeBA transcript abundance and results from a lesion in the cpeR gene. However, in this mutant cpeCDE RNA levels remain near those found in wild-type cells. Our results show that the coordinate regulation of cpeBA and cpeCDE by green light can be uncoupled by the loss of CpeR, and we furnish the first genetic evidence that different regulatory mechanisms control these two operons. Sequence analysis of CpeR reveals that it shares limited sequence similarity to members of the PP2C class of protein serine/threonine phosphatases. We also demonstrate that cpeBA and cpeCDE retain light quality responsiveness in a mutant lacking the RcaE photoreceptor. This provides compelling evidence for the partial control of CCA through an as-yet-uncharacterized second light quality sensing system.

  18. Gene structure and spatiotemporal expression profile of tomato genes encoding YUCCA-like flavin monooxygenases: the ToFZY gene family.

    PubMed

    Expósito-Rodríguez, Marino; Borges, Andrés A; Borges-Pérez, Andrés; Pérez, José A

    2011-07-01

    The flavin monooxygenases (FMO) encoded by plant YUCCA genes are thought to catalyze a rate-limiting step in the tryptamine pathway for indole-3-acetic acid biosynthesis. Recent experiments with different plant models have indicate that YUCCA genes play essential roles in growth and development through their contribution to the local pool of free auxin. In this study we have characterized five new genes that encode YUCCA-like FMOs in the tomato genome (ToFZY2 to ToFZY6), including gene structure, conserved motifs and phylogenetic analyses. As a first step towards clarifying the individual functions of ToFZY genes, we have used quantitative real-time RT-PCR to conduct a systematic comparison of the steady-state mRNA levels of 6 ToFZY genes, in 33 samples representing major organs and the entire tomato life cycle. We followed an absolute quantification strategy which allowed us to cross-compare transcript levels among different ToFZY genes in a given spatiotemporal coordinate. Our results indicate that expression of ToFZY genes is temporally and spatially regulated, and that the distinctive expression pattern of each ToFZY gene partially overlaps with other members of the multigenic family. We compare our data with previous results in other plant species and make some predictions about the role of tryptamine pathway in tomato growth and development.

  19. Molecular cloning of ADIR, a novel interferon responsive gene encoding a protein related to the torsins.

    PubMed

    Dron, Michel; Meritet, Jean François; Dandoy-Dron, Françoise; Meyniel, Jean-Philippe; Maury, Chantal; Tovey, Michael G

    2002-03-01

    The expression of the previously uncharacterized gene Adir (for ATP dependent interferon responsive gene) was increased by 5- to 15-fold in tissue of the oral cavity or in spleen and liver of mice treated orally or intraperitoneally with IFN-alpha, and in mouse cells treated in vitro with IFN-alpha or IFN-gamma. The level of Adir mRNA was also increased 20- to 40-fold in the brains of animals infected with encephalomyocarditis virus. Adir is expressed ubiquitously in mouse tissues as 1.9-, 2.4-, and 3.5-kb mRNA transcripts encoding a 385-amino-acid protein with a conserved ATP binding domain containing typical nucleotide and Mg(2+) binding sites. We also characterized the human ortholog, ADIR, which is located on chromosome 1q25-q31 and contains six exons encoding a 397-amino-acid protein with 80% homology to the mouse protein. A single 2.3-kb mRNA was detected in all human tissues examined, except for placenta, which also contained a 1.25-kb tissue-specific transcript generated by alternative splicing and encoding a putative 336-amino-acid protein. Although ADIR exhibits low homology to DYT1 and TOR1B, the deduced ADIR protein sequences are highly homologous to torsin A and torsin B and more distantly related to members of the Clp/HSP100 family of proteins, suggesting that ADIR, like torsins, is related to the AAA chaperone-like family of ATPases. An ADIR-EGFP fusion protein expressed in HeLa cells was shown to be associated with the endoplasmic reticulum.

  20. A Gene Encoding Antigenic Peptides of Human Squamous Cell Carcinoma Recognized by Cytotoxic T Lymphocytes

    PubMed Central

    Shichijo, Shigeki; Nakao, Masanobu; Imai, Yasuhisa; Takasu, Hideo; Kawamoto, Mayumi; Niiya, Fumihiko; Yang, Damu; Toh, Yuji; Yamana, Hideaki; Itoh, Kyogo

    1998-01-01

    Except for melanomas, tumor antigens recognized by cytotoxic T lymphocytes (CTLs) are yet unidentified. We have identified a gene encoding antigenic peptides of human squamous cell carcinomas (SCCs) recognized by human histocompatibility leukocyte antigens (HLA)- A2601–restricted CTLs. This gene showed no similarity to known sequences, and encoded two (125- and 43-kilodalton [kD]) proteins. The 125-kD protein with the leucine zipper motif was expressed in the nucleus of the majority of proliferating cells tested, including normal and malignant cells. The 43-kD protein was expressed in the cytosol of most SCCs from various organs and half of lung adenocarcinomas, but was not expressed in other cancers nor in a panel of normal tissues. The three nonapeptides shared by the two proteins were recognized by the KE4 CTLs, and one of the peptides induced in vitro from peripheral blood mononuclear cells (PBMCs) the CTLs restricted to the autologous tumor cells. The 43-kD protein and this nonapeptide (KGSGKMKTE) may be useful for the specific immunotherapy of HLA-A2601+ epithelial cancer patients. PMID:9449708

  1. Cloning, sequencing and expression of the gene encoding the extracellular metalloprotease of Aeromonas caviae.

    PubMed

    Kawakami, K; Toma, C; Honma, Y

    2000-01-01

    A gene (apk) encoding the extracellular protease of Aeromonas caviae Ae6 has been cloned and sequenced. For cloning the gene, the DNA genomic library was screened using skim milk LB agar. One clone harboring plasmid pKK3 was selected for sequencing. Nucleotide sequencing of the 3.5 kb region of pKK3 revealed a single open reading frame (ORF) of 1,785 bp encoding 595 amino acids. The deduced polypeptide contained a putative 16-amino acid signal peptide followed by a large propeptide. The N-terminal amino acid sequence of purified recombinant protein (APK) was consistent with the DNA sequence. This result suggested a mature protein of 412 amino acids with a molecular mass of 44 kDa. However, the molecular mass of purified recombinant APK revealed 34 kDa by SDS-PAGE, suggesting that further processing at the C-terminal region took place. The 2 motifs of zinc binding sites deduced are highly conserved in the APK as well as in other zinc metalloproteases including Vibrio proteolyticus neutral protease, Emp V from Vibrio vulnificus, HA/P from Vibrio cholerae, and Pseudomonas aeruginosa elastase. Proteolytic activity was inhibited by EDTA, Zincov, 1,10-phenanthroline and tetraethylenepentamine while unaffected by the other inhibitors tested. The protease showed maximum activity at pH 7.0 and was inactivated by heating at 80 C for 15 min. These results together suggest that APK belongs to the thermolysin family of metalloendopeptidases.

  2. The European Eel NCCβ Gene Encodes a Thiazide-resistant Na-Cl Cotransporter.

    PubMed

    Moreno, Erika; Plata, Consuelo; Rodríguez-Gama, Alejandro; Argaiz, Eduardo R; Vázquez, Norma; Leyva-Ríos, Karla; Islas, León; Cutler, Christopher; Pacheco-Alvarez, Diana; Mercado, Adriana; Cariño-Cortés, Raquel; Castañeda-Bueno, María; Gamba, Gerardo

    2016-10-21

    The thiazide-sensitive Na-Cl cotransporter (NCC) is the major pathway for salt reabsorption in the mammalian distal convoluted tubule. NCC plays a key role in the regulation of blood pressure. Its inhibition with thiazides constitutes the primary baseline therapy for arterial hypertension. However, the thiazide-binding site in NCC is unknown. Mammals have only one gene encoding for NCC. The eel, however, contains a duplicate gene. NCCα is an ortholog of mammalian NCC and is expressed in the kidney. NCCβ is present in the apical membrane of the rectum. Here we cloned and functionally characterized NCCβ from the European eel. The cRNA encodes a 1043-amino acid membrane protein that, when expressed in Xenopus oocytes, functions as an Na-Cl cotransporter with two major characteristics, making it different from other known NCCs. First, eel NCCβ is resistant to thiazides. Single-point mutagenesis supports that the absence of thiazide inhibition is, at least in part, due to the substitution of a conserved serine for a cysteine at position 379. Second, NCCβ is not activated by low-chloride hypotonic stress, although the unique Ste20-related proline alanine-rich kinase (SPAK) binding site in the amino-terminal domain is conserved. Thus, NCCβ exhibits significant functional differences from NCCs that could be helpful in defining several aspects of the structure-function relationship of this important cotransporter.

  3. Plasmid-encoded genes influence exosporium assembly and morphology in Bacillus megaterium QM B1551 spores.

    PubMed

    Manetsberger, Julia; Hall, Elizabeth A H; Christie, Graham

    2015-09-01

    Spores of Bacillus megaterium QM B1551 are encased in a morphologically distinctive exosporium. We demonstrate here that genes encoded on the indigenous pBM500 and pBM600 plasmids are required for exosporium assembly and or stability in spores of this strain. Bioinformatic analyses identified genes encoding orthologues of the B. cereus-family exosporium nap and basal layer proteins within the B. megaterium genome. Transcriptional analyses, supported by electron and fluorescent microscopy, indicate that the pole-localized nap, identified here for the first time in B. megaterium QM B1551 spores, is comprised of the BclA1 protein. The role of the BxpB protein, which forms the basal layer of the exosporium in B. cereus spores, is less clear since spores of a null mutant strain display an apparently normal morphology. Retention of the localized nap in bxpB null spores suggests that B. megaterium employs an alternative mechanism to that used by B. cereus spores in anchoring the nap to the spore surface.

  4. Plasmid-encoded genes influence exosporium assembly and morphology in Bacillus megaterium QM B1551 spores

    PubMed Central

    Manetsberger, Julia; Hall, Elizabeth A. H.; Christie, Graham

    2015-01-01

    Spores of Bacillus megaterium QM B1551 are encased in a morphologically distinctive exosporium. We demonstrate here that genes encoded on the indigenous pBM500 and pBM600 plasmids are required for exosporium assembly and or stability in spores of this strain. Bioinformatic analyses identified genes encoding orthologues of the B. cereus-family exosporium nap and basal layer proteins within the B. megaterium genome. Transcriptional analyses, supported by electron and fluorescent microscopy, indicate that the pole-localized nap, identified here for the first time in B. megaterium QM B1551 spores, is comprised of the BclA1 protein. The role of the BxpB protein, which forms the basal layer of the exosporium in B. cereus spores, is less clear since spores of a null mutant strain display an apparently normal morphology. Retention of the localized nap in bxpB null spores suggests that B. megaterium employs an alternative mechanism to that used by B. cereus spores in anchoring the nap to the spore surface. PMID:26316548

  5. The Dh gene of Drosophila melanogaster encodes a diuretic peptide that acts through cyclic AMP.

    PubMed

    Cabrero, Pablo; Radford, Jonathan C; Broderick, Kate E; Costes, Laurence; Veenstra, Jan A; Spana, Eric P; Davies, Shireen A; Dow, Julian A T

    2002-12-01

    Dh, the gene that encodes a CRF-like peptide in Drosophila melanogaster, is described. The product of this gene is a 44-amino-acid peptide (Drome-DH(44)) with a sequence almost identical to the Musca domestica and Stomoxys calcitrans diuretic hormones. There are no other similar peptides encoded within the known Drosophila genomic sequence. Functional studies showed that the deduced peptide stimulated fluid production, and that this effect was mediated by cyclic AMP in principal cells only: there was no effect on the levels of either cyclic GMP or intracellular calcium. Stimulation also elevated levels of cyclic AMP (but not cyclic GMP) phosphodiesterase, a new mode of action for this class of hormone. The transcript was localised by in situ hybridisation, and the peptide by immunocytochemistry, to two groups of three neurones in the pars intercerebralis within the brain. These cells also express receptors for leucokinin, another major diuretic peptide, implying that the cells may be important in homeostatic regulation.

  6. Life without putrescine: disruption of the gene-encoding polyamine oxidase in Ustilago maydis odc mutants.

    PubMed

    Valdés-Santiago, Laura; Guzmán-de-Peña, Doralinda; Ruiz-Herrera, José

    2010-11-01

    In previous communications the essential role of spermidine in Ustilago maydis was demonstrated by means of the disruption of the genes encoding ornithine decarboxylase (ODC) and spermidine synthase (SPE). However, the assignation of specific roles to each polyamine in different cellular functions was not possible because the spermidine added to satisfy the auxotrophic requirement of odc/spe double mutants is partly back converted into putrescine. In this study, we have approached this problem through the disruption of the gene-encoding polyamine oxidase (PAO), required for the conversion of spermidine into putrescine, and the construction of odc/pao double mutants that were unable to synthesize putrescine by either ornithine decarboxylation or retroconversion from spermidine. Phenotypic analysis of the mutants provided evidence that putrescine is only an intermediary in spermidine biosynthesis, and has no direct role in cell growth, dimorphic transition, or any other vital function of U. maydis. Nevertheless, our results show that putrescine may play a role in the protection of U. maydis against salt and osmotic stress, and possibly virulence. Evidence was also obtained that the retroconversion of spermidine into putrescine is not essential for U. maydis growth but may be important for its survival under natural conditions.

  7. Characterization of a gene which encodes a mannosyltransferase homolog of Paracoccidioides brasiliensis.

    PubMed

    Costa, Alessandra A; Gómez, Francisco J; Pereira, Maristela; Felipe, M Sueli S; Jesuino, Rosália S A; Deepe, George S; de Almeida Soares, Célia M

    2002-08-01

    We screened an expression library of the yeast form of Paracoccidioides brasiliensis with a pool of human sera that was pre-adsorbed with mycelium, from patients with paracoccidioidomycosis (PCM). A sequence (PbYmnt) was obtained and characterized. A genomic clone was obtained by PCR of P. brasiliensis total DNA. The sequence contained a single open reading frame (ORF) encoding a protein of 357 amino acid residues, with a molecular mass of 39.78 kDa. The deduced amino acid sequence exhibited identity to mannosyl- and glycosyltransferases from several sources. A DXD motif was present in the translated gene and this sequence is characteristic of the glycosyltransferases. Hydropathy analysis revealed a single transmembrane region near the amino terminus of the molecule that suggested a type II membrane protein. The PbYmnt was expressed preferentially in the yeast parasitic phase. The accession number of the nucleotide sequence of PbYmnt and its flanking regions is AF374353. A recombinant protein was generated in Escherichia coli. Our data suggest that PbYmnt encodes one member of a glycosyltransferase family of proteins and that our strategy was useful in the isolation of differentially expressed genes.

  8. A cotton gene encoding a polygalacturonase inhibitor-like protein is specifically expressed in petals.

    PubMed

    Shi, Haiyan; Zhu, Li; Zhou, Ying; Li, Gang; Chen, Liang; Li, Xuebao

    2009-04-01

    A cDNA encoding a polygalacturonase-inhibitor-like protein (PGIP) was isolated from cotton flower cDNA library. The cDNA, designated GhPS1 (GenBank accession No. ABO47744), encodes a protein with 370 amino acids that shares high similarity with the known plant PGIPs. Fluorescent microscopy indicated that GhPS1 protein localizes on the cell membranes as well as in cytoplasm. Real-time quantitative RT-PCR and Northern blot analyses showed that GhPS1 was specifically expressed in cotton petals. Furthermore, the GhPS1 expression was gradually up-regulated in petal development, and its transcripts were accumulated to the highest level in the petals at anthesis. However, its expression level was declined rapidly in senesced petals after flowering. At low temperature, the GhPS1 gene expression was gradually decreased to very low level in petals. Collectively, our results suggest that GhPS1 gene might be involved in cotton petal development and senescence, and in response to cold stress.

  9. Cloning and expression in Escherichia coli of Pseudomonas strain LB400 genes encoding polychlorinated biphenyl degradation.

    PubMed Central

    Mondello, F J

    1989-01-01

    Pseudomonas strain LB400 is able to degrade an unusually wide variety of polychlorinated biphenyls (PCBs). A genomic library of LB400 was constructed by using the broad-host-range cosmid pMMB34 and introduced into Escherichia coli. Approximately 1,600 recombinant clones were tested, and 5 that expressed 2,3-dihydroxybiphenyl dioxygenase activity were found. This enzyme is encoded by the bphC gene of the 2,3-dioxygenase pathway for PCB-biphenyl metabolism. Two recombinant plasmids encoding the ability to transform PCBs to chlorobenzoic acids were identified, and one of these, pGEM410, was chosen for further study. The PCB-degrading genes (bphA, -B, -C, and -D) were localized by subcloning experiments to a 12.4-kilobase region of pGEM410. The ability of recombinant strains to degrade PCBs was compared with that of the wild type. In resting-cell assays, PCB degradation by E. coli strain FM4560 (containing a pGEM410 derivative) approached that of LB400 and was significantly greater than degradation by the original recombinant strain. High levels of PCB metabolism by FM4560 did not depend on the growth of the organism on biphenyl, as it did for PCB metabolism by LB400. When cells were grown with succinate as the carbon source, PCB degradation by FM4560 was markedly superior to that by LB400. Images PMID:2493454

  10. A satellite phage-encoded antirepressor induces repressor aggregation and cholera toxin gene transfer.

    PubMed

    Davis, Brigid M; Kimsey, Harvey H; Kane, Anne V; Waldor, Matthew K

    2002-08-15

    CTXphi is a filamentous bacteriophage whose genome encodes cholera toxin, the principal virulence factor of Vibrio cholerae. We have found that the CTXphi-related element RS1 is a satellite phage whose transmission depends upon proteins produced from a CTX prophage (its helper phage). However, unlike other satellite phages and satellite animal viruses, RS1 can aid the CTX prophage as well as exploit it, due to the RS1-encoded protein RstC. RstC, whose function previously was unknown, is an antirepressor that counteracts the activity of the phage repressor RstR. RstC promotes transcription of genes required for phage production and thereby promotes transmission of both RS1 and CTXphi. Antirepression by RstC also induces expression of the cholera toxin genes, ctxAB, and thus may contribute to the virulence of V.cholerae. In vitro, RstC binds directly to RstR, producing unusual, insoluble aggregates containing both proteins. In vivo, RstC and RstR are both found at the cell pole, where they again appear to form stable complexes. The sequestration/inactivation process induced by RstC resembles those induced by mutant polyglutamine-containing proteins implicated in human neurodegenerative disorders.

  11. Proanthocyanidin synthesis and expression of genes encoding leucoanthocyanidin reductase and anthocyanidin reductase in developing grape berries and grapevine leaves.

    PubMed

    Bogs, Jochen; Downey, Mark O; Harvey, John S; Ashton, Anthony R; Tanner, Gregory J; Robinson, Simon P

    2005-10-01

    Proanthocyanidins (PAs), also called condensed tannins, can protect plants against herbivores and are important quality components of many fruits. Two enzymes, leucoanthocyanidin reductase (LAR) and anthocyanidin reductase (ANR), can produce the flavan-3-ol monomers required for formation of PA polymers. We isolated and functionally characterized genes encoding both enzymes from grapevine (Vitis vinifera L. cv Shiraz). ANR was encoded by a single gene, but we found two highly related genes encoding LAR. We measured PA content and expression of genes encoding ANR, LAR, and leucoanthocyanidin dioxygenase in grape berries during development and in grapevine leaves, which accumulated PA throughout leaf expansion. Grape flowers had high levels of PA, and accumulation continued in skin and seeds from fruit set until the onset of ripening. VvANR was expressed throughout early flower and berry development, with expression increasing after fertilization. It was expressed in berry skin and seeds until the onset of ripening, and in expanding leaves. The genes encoding LAR were expressed in developing fruit, particularly in seeds, but had low expression in leaves. The two LAR genes had different patterns of expression in skin and seeds. During grape ripening, PA levels decreased in both skin and seeds, and expression of genes encoding ANR and LAR were no longer detected. The results indicate that PA accumulation occurs early in grape development and is completed when ripening starts. Both ANR and LAR contribute to PA synthesis in fruit, and the tissue and temporal-specific regulation of the genes encoding ANR and LAR determines PA accumulation and composition during grape berry development.

  12. Small gene family encoding an eggshell (chorion) protein of the human parasite Schistosoma mansoni

    SciTech Connect

    Bobek, L.A.; Rekosh, D.M.; Lo Verde, P.T.

    1988-08-01

    The authors isolated six independent genomic clones encoding schistosome chorion or eggshell proteins from a Schistosoma mansoni genomic library. A linkage map of five of the clones spanning 35 kilobase pairs (kbp) of the S. mansoni genome was constructed. The region contained two eggshell protein genes closely linked, separated by 7.5 kbp of intergenic DNA. The two genes of the cluster were arranged in the same orientation, that is, they were transcribed from the same strand. The sixth clone probably represents a third copy of the eggshell gene that is not contained within the 35-kbp region. The 5- end of the mRNA transcribed from these genes was defined by primer extension directly off the RNA. The ATCAT cap site sequence was homologous to a silkmoth chorion PuTCATT cap site sequence, where Pu indicates any purine. DNA sequence analysis showed that there were no introns in these genes. The DNA sequences of the three genes were very homologous to each other and to a cDNA clone, pSMf61-46, differing only in three or four nucleotices. A multiple TATA box was located at positions -23 to -31, and a CAAAT sequence was located at -52 upstream of the eggshell transcription unit. Comparison of sequences in regions further upstream with silkmoth and Drosophila sequences revealed very short elements that were shared. One such element, TCACGT, recently shown to be an essential cis-regulatory element for silkmoth chorion gene promoter function, was found at a similar position in all three organisms.

  13. Genetic Organization of Plasmid ColJs, Encoding Colicin Js Activity, Immunity, and Release Genes

    PubMed Central

    Šmajs, David; Weinstock, George M.

    2001-01-01

    The 5.2-kb ColJs plasmid of a colicinogenic strain of Shigella sonnei (colicin type 7) was isolated and sequenced. pColJs was partly homologous to pColE1 and to pesticin-encoding plasmid pPCP1, mainly in the rep, mob, and cer regions. A 1.2-kb unique region of pColJs showed significantly different G+C content (34%) compared to the rest of pColJs (53%). Within the unique region, seven open reading frames (ORFs) were identified. ORF94 was shown to code for colicin Js activity (cja), a 94-amino-acid polypeptide (molecular mass, 10.4 kDa); ORF129 (cji) was shown to code for the 129-amino-acid colicin Js immunity protein (molecular mass, 14.3 kDa); and ORF65 was shown to be involved in colicin Js release by producer bacteria (cjl) coding for a 65-amino-acid polypeptide (molecular mass, 7.5 kDa). In contrast to the gene order in other colicin operons, the cjl gene was found upstream from cja. Moreover, the promoter upstream from cjl was similar to promoters described upstream from several colicin activity genes. The cji gene was found to be located downstream from cja with a transcription polarity opposite to that of the cjl and cja genes. The cja, cji, and cjl genes were not similar to other known colicin genes. Colicin Js was purified as an inactive fusion protein with an N-terminal histidine tag. Activity of the purified fusion form of colicin Js was restored after cleavage of the amino acids fused to the colicin Js N terminus. PMID:11395458

  14. The facC Gene of Aspergillus nidulans Encodes an Acetate-Inducible Carnitine Acetyltransferase

    PubMed Central

    Stemple, Christopher J.; Davis, Meryl A.; Hynes, Michael J.

    1998-01-01

    Mutations in the facC gene of Aspergillus nidulans result in an inability to use acetate as a sole carbon source. This gene has been cloned by complementation. The proposed translation product of the facC gene has significant similarity to carnitine acetyltransferases (CAT) from other organisms. Total CAT activity was found to be inducible by acetate and fatty acids and repressed by glucose. Acetate-inducible activity was found to be absent in facC mutants, while fatty acid-inducible activity was absent in an acuJ mutant. Acetate induction of facC expression was dependent on the facB regulatory gene, and an expressed FacB fusion protein was demonstrated to bind to 5′ facC sequences. Carbon catabolite repression of facC expression was affected by mutations in the creA gene and a CreA fusion protein bound to 5′ facC sequences. Mutations in the acuJ gene led to increased acetate induction of facC expression and also of an amdS-lacZ reporter gene, and it is proposed that this results from accumulation of acetate, as well as increased expression of facB. A model is presented in which facC encodes a cytosolic CAT enzyme, while a different CAT enzyme, which is acuJ dependent, is present in peroxisomes and mitochondria, and these activities are required for the movement of acetyl groups between intracellular compartments. PMID:9829933

  15. Ammonia-regulated expression of a soybean gene encoding cytosolic glutamine synthetase in transgenic Lotus corniculatus.

    PubMed

    Miao, G H; Hirel, B; Marsolier, M C; Ridge, R W; Verma, D P

    1991-01-01

    A full-length cDNA clone encoding cytosolic glutamine synthetase (GS), expressed in roots and root nodules of soybean, was isolated by direct complementation of an Escherichia coli gln A- mutant. This sequence is induced in roots by the availability of ammonia. A 3.5-kilobase promoter fragment of a genomic clone (lambda GS15) corresponding to this cDNA was isolated and fused with a reporter [beta-glucuronidase (GUS)] gene. The GS-GUS fusion was introduced into a legume (Lotus corniculatus) and a nonlegume (tobacco) plant by way of Agrobacterium-mediated transformations. This chimeric gene was found to be expressed in a root-specific manner in both tobacco and L. corniculatus, the expression being restricted to the growing root apices and the vascular bundles of the mature root. Treatment with ammonia increased the expression of this chimeric gene in the legume background (i.e., L. corniculatus); however, no induction was observed in tobacco roots. Histochemical localization of GUS activity in ammonia-treated transgenic L. corniculatus roots showed a uniform distribution across all cell types. These data suggest that the tissue specificity of the soybean cytosolic GS gene is conserved in both tobacco and L. corniculatus; however, in the latter case, this gene is ammonia inducible. Furthermore, the ammonia-enhanced GS gene expression in L. corniculatus is due to an increase in transcription. That this gene is directly regulated by externally supplied or symbiotically fixed nitrogen is also evident from the expression of GS-GUS in the infection zone, including the uninfected cells, and the inner cortex of transgenic L. corniculatus nodules, where a flux of ammonia is encountered by this tissue. The lack of expression of GS-GUS in the outer cortex of the nodules suggests that ammonia may not be able to diffuse outside the endodermis.

  16. Analysis of essential Arabidopsis nuclear genes encoding plastid-targeted proteins.

    PubMed

    Savage, Linda J; Imre, Kathleen M; Hall, David A; Last, Robert L

    2013-01-01

    The Chloroplast 2010 Project (http://www.plastid.msu.edu/) identified and phenotypically characterized homozygous mutants in over three thousand genes, the majority of which encode plastid-targeted proteins. Despite extensive screening by the community, no homozygous mutant alleles were available for several hundred genes, suggesting that these might be enriched for genes of essential function. Attempts were made to generate homozygotes in ~1200 of these lines and 521 of the homozygous viable lines obtained were deposited in the Arabidopsis Biological Resource Center (http://abrc.osu.edu/). Lines that did not yield a homozygote in soil were tested as potentially homozygous lethal due to defects either in seed or seedling development. Mutants were characterized at four stages of development: developing seed, mature seed, at germination, and developing seedlings. To distinguish seed development or seed pigment-defective mutants from seedling development mutants, development of seeds was assayed in siliques from heterozygous plants. Segregating seeds from heterozygous parents were sown on supplemented media in an attempt to rescue homozygous seedlings that could not germinate or survive in soil. Growth of segregating seeds in air and air enriched to 0.3% carbon dioxide was compared to discover mutants potentially impaired in photorespiration or otherwise responsive to CO2 supplementation. Chlorophyll fluorescence measurements identified CO2-responsive mutants with altered photosynthetic parameters. Examples of genes with a viable mutant allele and one or more putative homozygous-lethal alleles were documented. RT-PCR of homozygotes for potentially weak alleles revealed that essential genes may remain undiscovered because of the lack of a true null mutant allele. This work revealed 33 genes with two or more lethal alleles and 73 genes whose essentiality was not confirmed with an independent lethal mutation, although in some cases second leaky alleles were identified.

  17. Isolation and characterization of 17 different genes encoding putative endopolygalacturonase genes from Rhizopus oryzae

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Polygalacturonase enzymes are a valuable aid in the retting of flax for production of linens and, more recently, production of biofuels from citrus wastes. In a search of the recently sequenced Rhizopus oryzae strain 99-880 genome database, 18 putative endopolygalacturonase genes were identified, w...

  18. Hyphal tip extension in Aspergillus nidulans requires the manA gene, which encodes phosphomannose isomerase.

    PubMed Central

    Smith, D J; Payton, M A

    1994-01-01

    A strain of Aspergillus nidulans carrying a temperature-sensitive mutation in the manA gene produces cell walls depleted of D-mannose and forms hyphal tip balloons at the restrictive temperature (B.P. Valentine and B.W. Bainbridge, J. Gen. Microbiol. 109:155-168, 1978). We have isolated and characterized the manA gene and physically located it between 3.5 and 5.5 kb centromere distal of the riboB locus on chromosome VIII. The manA gene contains four introns and encodes a 50.6-kDa protein which has significant sequence identity to type I phosphomannose isomerase proteins from other eukaryotes. We have constructed by integrative transformation a null mutation in the manA gene which can only be maintained in a heterokaryotic strain with wild-type manA+ nuclei. Thus, a manA null mutation is lethal in A. nidulans. The phenotype of the mutation was analyzed in germinating conidia. Such conidia are able to commence germination but swell abnormally, sometimes producing a misshapen germ tube, before growth ceases. The reason for the lethality is probably the lack of synthesis of mannose-containing cell wall polymers that must be required for normal cell wall development in growing hyphae. Images PMID:8065336

  19. BAGEL3: automated identification of genes encoding bacteriocins and (non-)bactericidal posttranslationally modified peptides

    PubMed Central

    van Heel, Auke J.; de Jong, Anne; Montalbán-López, Manuel; Kok, Jan; Kuipers, Oscar P.

    2013-01-01

    Identifying genes encoding bacteriocins and ribosomally synthesized and posttranslationally modified peptides (RiPPs) can be a challenging task. Especially those peptides that do not have strong homology to previously identified peptides can easily be overlooked. Extensive use of BAGEL2 and user feedback has led us to develop BAGEL3. BAGEL3 features genome mining of prokaryotes, which is largely independent of open reading frame (ORF) predictions and has been extended to cover more (novel) classes of posttranslationally modified peptides. BAGEL3 uses an identification approach that combines direct mining for the gene and indirect mining via context genes. Especially for heavily modified peptides like lanthipeptides, sactipeptides, glycocins and others, this genetic context harbors valuable information that is used for mining purposes. The bacteriocin and context protein databases have been updated and it is now easy for users to submit novel bacteriocins or RiPPs. The output has been simplified to allow user-friendly analysis of the results, in particular for large (meta-genomic) datasets. The genetic context of identified candidate genes is fully annotated. As input, BAGEL3 uses FASTA DNA sequences or folders containing multiple FASTA formatted files. BAGEL3 is freely accessible at http://bagel.molgenrug.nl. PMID:23677608

  20. Genes encoding defensins of important Chagas disease vectors used for phylogenetic studies.

    PubMed

    de Araújo, Catarina Andréa Chaves; Lima, Ana Carolina Bastos; Jansen, Ana Maria; Galvão, Cleber; Jurberg, José; Costa, Jane; Azambuja, Patricia; Waniek, Peter Josef

    2015-12-01

    Insects possess both cellular and humoral immune responses. The latter makes them capable to recognize and control invading pathogens after synthesis of a variety of small proteins, also known as antimicrobial peptides. Defensins, cysteine-rich cationic peptides with major activity against Gram-positive bacteria, are one ubiquitous class of antimicrobial peptides, widely distributed in different animal and plant taxa. Regarding triatomines in each of the so far analyzed species, various defensin gene isoforms have been identified. In the present study, these genes were sequenced and used as a molecular marker for phylogenetic analysis. Considering the vectors of Chagas disease the authors are reporting for the first time the presence of these genes in Triatoma sordida (Stål, 1859), Rhodnius nasutus (Stål, 1859), and Panstrongylus megistus (Burmeister, 1835). Members of the Triatoma brasiliensis species complex were included into the study to verify the genetic variability within these taxa. Mainly in their mature peptide, the deduced defensin amino acid sequences were highly conserved. In the dendrogram based on defensin encoding nucleotide, sequences the Triatoma Def3/4 genes were separated from the rest. In the dendrogram based on deduced amino acid sequences the Triatoma Def2/3/4 together with Rhodnius DefA/B pre-propeptides were separated from the rest. In the sub-branches of both the DNA and amino acid dendrograms, the genus Triatoma was separated from the genus Rhodnius as well as from P. megistus.

  1. Regulation of the ald gene encoding alanine dehydrogenase by AldR in Mycobacterium smegmatis.

    PubMed

    Jeong, Ji-A; Baek, Eun-Young; Kim, Si Wouk; Choi, Jong-Soon; Oh, Jeong-Il

    2013-08-01

    The regulatory gene aldR was identified 95 bp upstream of the ald gene encoding L-alanine dehydrogenase in Mycobacterium smegmatis. The AldR protein shows sequence similarity to the regulatory proteins of the Lrp/AsnC family. Using an aldR deletion mutant, we demonstrated that AldR serves as both activator and repressor for the regulation of ald gene expression, depending on the presence or absence of L-alanine. The purified AldR protein exists as a homodimer in the absence of L-alanine, while it adopts the quaternary structure of a homohexamer in the presence of L-alanine. The binding affinity of AldR for the ald control region was shown to be increased significantly by L-alanine. Two AldR binding sites (O1 and O2) with the consensus sequence GA-N₂-ATC-N₂-TC and one putative AldR binding site with the sequence GA-N₂-GTT-N₂-TC were identified upstream of the ald gene. Alanine and cysteine were demonstrated to be the effector molecules directly involved in the induction of ald expression. The cellular level of L-alanine was shown to be increased in M. smegmatis cells grown under hypoxic conditions, and the hypoxic induction of ald expression appears to be mediated by AldR, which senses the intracellular level of alanine.

  2. Antibacterial Compounds-Macrolactin Alters the Soil Bacterial Community and Abundance of the Gene Encoding PKS

    PubMed Central

    Yuan, Jun; Zhao, Mengli; Li, Rong; Huang, Qiwei; Rensing, Christopher; Raza, Waseem; Shen, Qirong

    2016-01-01

    Macrolactin produced by many soil microbes has been shown to be an efficient antibacterial agent against many bacterial pathogens. However, studies examining the effect of macrolactin on both the soil bacterial community and the intrinsic bacterial species that harbor genes responsible for the production of this antibiotic have not been conducted so far. In this study, a mixture of macrolactin was isolated from the liquid culture of Bacillus amyloliquefaciens NJN-6, and applied to the soil once a week for four weeks. 16S rRNA Illumina MiSeq sequencing showed that continuous application of macrolactin reduced the α-diversity of the soil bacterial community and thereby changed the relative abundance of microbes at both the phylum and genus level. The relative abundance of Proteobacteria and Firmicutes was significantly increased along with a significant decrease in the relative abundance of Acidobacteria. However, the application of macrolactins had an insignificant effect on the total numbers of bacteria. Further, the native gene responsible for the production of macrolactin, the gene encoding polyketide synthase was reduced in copy number after the application of macrolactin. The results of this study suggested that a bactericide from a microbial source could decrease the diversity of the soil bacterial community and change the bacterial community structure. Moreover, the populations of the intrinsic bacterial species which harbor genes responsible for macrolactin production were inhibited when the external source antibiotic was applied. PMID:27965639

  3. Mutations in the Drosophila melanogaster gene encoding S-adenosylmethionine suppress position-effect variegation

    SciTech Connect

    Larsson, J.; Rasmuson-Lestander, A.; Zhang, Jingpu

    1996-06-01

    In Drosophila melanogaster, the study of trans-acting modifier mutations of position-effect variegation and Polycomb group (Pc-G) genes have been useful tools to investigate genes involved in chromatin structure. We have cloned a modifier gene, Suppressor of zeste 5 (Su(z)5), which encodes S-adenosylmethionine synthetase, and we present here molecular results and data concerning its expression in mutants and genetic interactions. The mutant alleles Su(z)5, l(2)R23 and l(2)M6 show suppression of w{sup m4} and also of two white mutants induced by roo element insertions in the regulatory region i.e., w{sup is} (in combination with z{sup 1}) and w{sup sp1}. Two of the Su(z)5 alleles, as well as a deletion of the gene, also act as enhancers of Polycomb by increasing the size of sex combes on midleg. The results suggest that Su(z)5 is connected with regulation of chromatin structure. The enzyme S-adenosylmethionine synthetase is involved in the synthesis of S-adenosylmethionine, a methyl group donor and also, after decarboxylation, a propylamino group donor in the biosynthesis of polyamines. Our results from HPLC analysis show that in ovaries from heterozygous Su(z)5 mutants the content of spermine is significantly reduced. Results presented here suggest that polyamines are an important molecule class in the regulation of chromatin structure. 50 refs., 5 figs., 3 tabs.

  4. Unexpected Diversity of pepA Genes Encoding Leucine Aminopeptidases in Sediments from a Freshwater Lake

    PubMed Central

    Tsuboi, Shun; Yamamura, Shigeki; Imai, Akio; Iwasaki, Kazuhiro

    2016-01-01

    We herein designed novel PCR primers for universal detection of the pepA gene, which encodes the representative leucine aminopeptidase gene, and investigated the genetic characteristics and diversity of pepA genes in sediments of hypereutrophic Lake Kasumigaura, Japan. Most of the amino acid sequences deduced from the obtained clones (369 out of 370) were related to PepA-like protein sequences in the M17 family of proteins. The developed primers broadly detected pepA-like clones associated with diverse bacterial phyla—Alpha-, Beta-, Gamma-, and Deltaproteobacteria, Acidobacteria, Actinobacteria, Aquificae, Chlamydiae, Chloroflexi, Cyanobacteria, Firmicutes, Nitrospirae, Planctomycetes, and Spirochetes as well as the archaeal phylum Thaumarchaeota, indicating that prokaryotes in aquatic environments possessing leucine aminopeptidase are more diverse than previously reported. Moreover, prokaryotes related to the obtained pepA-like clones appeared to be r- and K-strategists, which was in contrast to our previous findings showing that the neutral metalloprotease gene clones obtained were related to the r-strategist genus Bacillus. Our results suggest that an unprecedented diversity of prokaryotes with a combination of different proteases participate in sedimentary proteolysis. PMID:26936797

  5. Unexpected Diversity of pepA Genes Encoding Leucine Aminopeptidases in Sediments from a Freshwater Lake.

    PubMed

    Tsuboi, Shun; Yamamura, Shigeki; Imai, Akio; Iwasaki, Kazuhiro

    2016-01-01

    We herein designed novel PCR primers for universal detection of the pepA gene, which encodes the representative leucine aminopeptidase gene, and investigated the genetic characteristics and diversity of pepA genes in sediments of hypereutrophic Lake Kasumigaura, Japan. Most of the amino acid sequences deduced from the obtained clones (369 out of 370) were related to PepA-like protein sequences in the M17 family of proteins. The developed primers broadly detected pepA-like clones associated with diverse bacterial phyla-Alpha-, Beta-, Gamma-, and Deltaproteobacteria, Acidobacteria, Actinobacteria, Aquificae, Chlamydiae, Chloroflexi, Cyanobacteria, Firmicutes, Nitrospirae, Planctomycetes, and Spirochetes as well as the archaeal phylum Thaumarchaeota, indicating that prokaryotes in aquatic environments possessing leucine aminopeptidase are more diverse than previously reported. Moreover, prokaryotes related to the obtained pepA-like clones appeared to be r- and K-strategists, which was in contrast to our previous findings showing that the neutral metalloprotease gene clones obtained were related to the r-strategist genus Bacillus. Our results suggest that an unprecedented diversity of prokaryotes with a combination of different proteases participate in sedimentary proteolysis.

  6. BAGEL3: Automated identification of genes encoding bacteriocins and (non-)bactericidal posttranslationally modified peptides.

    PubMed

    van Heel, Auke J; de Jong, Anne; Montalbán-López, Manuel; Kok, Jan; Kuipers, Oscar P

    2013-07-01

    Identifying genes encoding bacteriocins and ribosomally synthesized and posttranslationally modified peptides (RiPPs) can be a challenging task. Especially those peptides that do not have strong homology to previously identified peptides can easily be overlooked. Extensive use of BAGEL2 and user feedback has led us to develop BAGEL3. BAGEL3 features genome mining of prokaryotes, which is largely independent of open reading frame (ORF) predictions and has been extended to cover more (novel) classes of posttranslationally modified peptides. BAGEL3 uses an identification approach that combines direct mining for the gene and indirect mining via context genes. Especially for heavily modified peptides like lanthipeptides, sactipeptides, glycocins and others, this genetic context harbors valuable information that is used for mining purposes. The bacteriocin and context protein databases have been updated and it is now easy for users to submit novel bacteriocins or RiPPs. The output has been simplified to allow user-friendly analysis of the results, in particular for large (meta-genomic) datasets. The genetic context of identified candidate genes is fully annotated. As input, BAGEL3 uses FASTA DNA sequences or folders containing multiple FASTA formatted files. BAGEL3 is freely accessible at http://bagel.molgenrug.nl.

  7. Cloning and sequencing of the genes from Salmonella typhimurium encoding a new bacterial ribonucleotide reductase.

    PubMed Central

    Jordan, A; Gibert, I; Barbé, J

    1994-01-01

    A plasmid library of Salmonella typhimurium was used to complement a temperature-sensitive nrdA mutant of Escherichia coli. Complementation was obtained with two different classes of plasmids, one carrying the E. coli nrdAB-like genes and the second containing an operon encoding a new bacterial ribonucleotide reductase. Plasmids harboring these new reductase genes also enable obligately anaerobic nrdB::Mud1 E. coli mutants to grow in the presence of oxygen. This operon consists of two open reading frames, which have been designated nrdE (2,145 bp) and nrdF (969 bp). The deduced amino acid sequences of the nrdE and nrdF products include the catalytically important residues conserved in ribonucleotide reductase enzymes of class I and show 25 and 28% overall identity with the R1 and R2 protein, respectively, of the aerobic ribonucleoside diphosphate reductase of E. coli. The 3' end of the sequenced 4.9-kb fragment corresponds to the upstream region of the previously published proU operon of both S. typhimurium and E. coli, indicating that the nrdEF genes are at 57 min on the chromosomal maps of these two bacterial species. Analysis of the nrdEF and proU sequences demonstrates that transcription of the nrdEF genes is in the clockwise direction on the S. typhimurium and E. coli maps. Images PMID:8195103

  8. A dynamin-like protein encoded by the yeast sporulation gene SPO15.

    PubMed

    Yeh, E; Driscoll, R; Coltrera, M; Olins, A; Bloom, K

    1991-02-21

    The tightly centromere-linked gene SPO15 is essential for meiotic cell division in the yeast Saccharomyces cerevisiae. Diploid cells without the intact SPO15 gene product are able to complete premeiotic DNA synthesis and genetic recombination, but are unable to traverse the division cycles. Electron microscopy of blocked cells reveals a duplicated but unseparated spindle-pole body. Thus cells are unable to form a bipolar spindle. Sequence analysis of SPO15 DNA reveals an open reading frame that predicts a protein of 704 amino acids. This protein is identical to VPS1, a gene involved in vacuolar protein sorting in yeast which has significant sequence homology (45% overall, 66% over 300 amino acids) to the microtubule bundling-protein, dynamin. The SPO15 gene product expressed in Escherichia coli can be affinity-purified with microtubules. SPO15 encodes a protein that is likely to be involved in a microtubule-dependent process required for the timely separation of spindle-pole bodies in meiosis.

  9. The Pun1 gene for pungency in pepper encodes a putative acyltransferase.

    PubMed

    Stewart, Charles; Kang, Byoung-Cheorl; Liu, Kede; Mazourek, Michael; Moore, Shanna L; Yoo, Eun Young; Kim, Byung-Dong; Paran, Ilan; Jahn, Molly M

    2005-06-01

    Pungency in Capsicum fruits is due to the accumulation of the alkaloid capsaicin and its analogs. The biosynthesis of capsaicin is restricted to the genus Capsicum and results from the acylation of an aromatic moiety, vanillylamine, by a branched-chain fatty acid. Many of the enzymes involved in capsaicin biosynthesis are not well characterized and the regulation of the pathway is not fully understood. Based on the current pathway model, candidate genes were identified in public databases and the literature, and genetically mapped. A published EST co-localized with the Pun1 locus which is required for the presence of capsaicinoids. This gene, AT3, has been isolated and its nucleotide sequence has been determined in an array of genotypes within the genus. AT3 showed significant similarity to acyltransferases in the BAHD superfamily. The recessive allele at this locus contains a deletion spanning the promoter and first exon of the predicted coding region in every non-pungent accession tested. Transcript and protein expression of AT3 was tissue-specific and developmentally regulated. Virus-induced gene silencing of AT3 resulted in a decrease in the accumulation of capsaicinoids, a phenotype consistent with pun1. In conclusion, gene mapping, allele sequence data, expression profile and silencing analysis collectively indicate that the Pun1 locus in pepper encodes a putative acyltransferase, and the pun1 allele, used in pepper breeding for nearly 50 000 years, results from a large deletion at this locus.

  10. Hypoxia-inducible genes encoding small EF-hand proteins in rice and tomato.

    PubMed

    Otsuka, Chie; Minami, Ikuko; Oda, Kenji

    2010-01-01

    Rice has evolved metabolic and morphological adaptations to low-oxygen stress to grow in submerged paddy fields. To characterize the molecular components that mediate the response to hypoxia in rice, we identified low-oxygen stress early response genes by microarray analysis. Among the highly responsive genes, five genes, OsHREF1 to OsHREF5, shared strong homology. They encoded small proteins harboring two EF-hands, typical Ca(2+)-binding motifs. Homologous genes were found in many land plants, including SlHREF in tomato, which is also strongly induced by hypoxia. SlHREF induction was detected in both roots and shoots of tomato plants under hypoxia. With the exception of OsHREF5, OsHREF expression was unaffected by drought, salinity, cold, or osmotic stress. Fluorescent signals of green fluorescent protein-fused OsHREFs were detected in the cytosol and nucleus. Ruthenium red, an inhibitor of intracellular Ca(2+) release, repressed induction of OsHREF1-4 under hypoxia. The HREFs may be related to the Ca(2+) response to hypoxia.

  11. Open reading frame 176 in the photosynthesis gene cluster of Rhodobacter capsulatus encodes idi, a gene for isopentenyl diphosphate isomerase.

    PubMed Central

    Hahn, F M; Baker, J A; Poulter, C D

    1996-01-01

    Isopentenyl diphosphate (IPP) isomerase catalyzes an essential activation step in the isoprenoid biosynthetic pathway. A database search based on probes from the highly conserved regions in three eukaryotic IPP isomerases revealed substantial similarity with ORF176 in the photosynthesis gene cluster in Rhodobacter capsulatus. The open reading frame was cloned into an Escherichia coli expression vector. The encoded 20-kDa protein, which was purified in two steps by ion exchange and hydrophobic interaction chromatography, catalyzed the interconversion of IPP and dimethylallyl diphosphate. Thus, the photosynthesis gene cluster encodes all of the enzymes required to incorporate IPP into the ultimate carotenoid and bacteriochlorophyll metabolites in R. capsulatus. More recent searches uncovered additional putative open reading frames for IPP isomerase in seed-bearing plants (Oryza sativa, Arabadopsis thaliana, and Clarkia breweri), a worm (Caenorhabiditis elegans), and another eubacterium (Escherichia coli). The R. capsulatus enzyme is the smallest of the IPP isomerases to be identified thus far and may consist mostly of a fundamental catalytic core for the enzyme. PMID:8550491

  12. Characterization of five subgroups of the sieve element occlusion gene family in Glycine max reveals genes encoding non-forisome P-proteins, forisomes and forisome tails.

    PubMed

    Zielonka, Sascia; Ernst, Antonia M; Hawat, Susan; Twyman, Richard M; Prüfer, Dirk; Noll, Gundula A

    2014-09-01

    P-proteins are structural phloem proteins discussed to be involved in the rapid sealing of injured sieve elements. P-proteins are found in all dicotyledonous and some monocotyledonous plants, but additional crystalloid P-proteins, known as forisomes, have evolved solely in the Fabaceae. Both types are encoded by members of the sieve element occlusion (SEO) gene family, which comprises seven phylogenetic subgroups. The Fabaceae-specific subgroup 1 contains genes encoding forisome subunits in e.g. Medicago truncatula, Vicia faba, Dipteryx panamensis and Canavalia gladiata whereas basal subgroup 5 encodes P-proteins in Nicotiana tabacum (tobacco) and Arabidopsis thaliana. The function of remaining subgroups is still unknown. We chose Glycine max (soybean) as a model to investigate SEO proteins representing different subgroups in one species. We isolated native P-proteins to determine the SEO protein composition and analyzed the expression pattern, localization and structure of the G. max SEO proteins representing five of the subgroups. We found that subgroup 1 GmSEO genes encode forisome subunits, a member of subgroup 5 encodes a non-forisome P-protein and subgroup 2 GmSEO genes encode the components of forisome tails, which are present in a restricted selection of Fabaceaen species. We therefore present the first molecular characterization of a Fabaceae non-forisome P-protein and the first evidence that forisome tails are encoded by a phylogenetically-distinct branch of the SEO gene family.

  13. Characterization of the gene encoding the polymorphic immunodominant molecule, a neutralizing antigen of Theileria parva

    SciTech Connect

    Toye, P.G.; Metzelaar, M.J.; Wijngaard, P.L.J.

    1995-08-01

    Theileria parva, a tick-transmitted protozoan parasite related to Plasmodium spp., causes the disease East Coast fever, an acute and usually fatal lymphoproliferative disorder of cattle in Africa. Previous studies using sera from cattle that have survived infection identified a polymorphic immunodominant molecule (PIM) that is expressed by both the infective sporozoite stage of the parasite and the intracellular schizont. Here we show that mAb specific for the PIM Ag can inhibit sporozoite invasion of lymphocytes in vitro. A cDNA clone encoding the PIM Ag of the T. parva (Muguga) stock was obtained by using these mAb in a novel eukaryotic expression cloning system that allows isolation of cDNA encoding cytoplasmic or surface Ags. To establish the molecular basis of the polymorphism of PIM, the cDNA of the PIM Ag from a buffalo-derived T. parva stock was isolated and its sequence was compared with that of the cattle-derived Muguga PIM. The two cDNAs showed considerable identity in both the 5{prime} and 3{prime} regions, but there was substantial sequence divergence in the central regions. Several types of repeated sequences were identified in the variant regions. In the Muguga form of the molecule, there were five tandem repeats of the tetrapeptide, QPEP, that were shown, by transfection of a deleted version of the PIM gene, not to react with several anti-PIM mAbs. By isolating and sequencing the genomic version of the gene, we identified two small introns in the 3{prime} region of the gene. Finally, we showed that polyclonal rat Abs against recombinant PIM neutralize sporozoite infectivity in vitro, suggesting that the PIM Ag should be evaluated for its capacity to immunize cattle against East Coast Fever.

  14. The gene encoding cytochrome-c oxidase subunit I from Synechocystis PCC6803.

    PubMed

    Alge, D; Schmetterer, G; Peschek, G A

    1994-01-28

    The gene (coxI or CoxA) encoding subunit I (COI) of cytochrome-c oxidase (cytochrome aa3) of Synechocystis PCC6803, Synechococcus PCC7942 (Anacystis nidulans R2) and Nostoc PCC8002 (Nostoc Mac), was identified by heterologous hybridization of chromosomal digests with a 17-bp oligodeoxyribonucleotide (probe C) derived from the coxI of Paracoccus denitrificans. A single genomic fragment was found to bind to probe C in all chromosomal digests. Due to its favorable signal-to-noise ratio, the genome of Synechocystis was chosen for the isolation and sequencing of this gene. A genomic DNA library in pUC18 was screened with probe C. The two probe C-positive plasmids, pDAUV1 and pDAUV2, contained a 1-kb overlapping region, with the conserved 17-bp sequence encoding the CuB-binding region of the COI polypeptide. These plasmids were subcloned into competent Escherichia coli DH5 alpha cells, and the nucleotide sequences were determined. The deduced amino acid (aa) sequences of Synechocystis COI and homologous proteins from a variety of prokaryotic and eukaryotic organisms showed an overall similarity of between 38.6 and 45.8%. Hydropathy plots revealed 12 potential transmembrane helices. All of the six histidines needed for the binding of heme a and the heme a3/CuB bimetallic center are present in the expected positions of the Synechocystis COI protein (533 aa, M(r) 59,390). A monospecific antibody raised against P. denitrificans COI gave an unequivocal immunological cross-reaction on Western blots of membrane preparations from Synechocystis, Anacystis and Nostoc, showing that the product of gene coxI is indeed synthesized and incorporated into cyanobacterial membranes.

  15. Constraints on intron evolution in the gene encoding the myosin alkali light chain in Drosophila

    SciTech Connect

    Leicht, B.G.; Muse, S.V.; Hanczyc, M.

    1995-01-01

    Interspecific comparisons of intron sequences reveal conserved blocks of invariant nucleotides and several other departures from the strictly neutral model of molecular evolution. To distinguish the past action of evolutionary forces in introns known to have regulatory information, we examined nucleotide sequence variation at 991 sites in a random sample of 16 Drosophila melanogaster alleles of the gene encoding the myosin alkali light chain (Mlc1). The Mlc1 gene of D. melanogaster encodes two Mlc1 isoforms via developmentally regulated alternative pre-mRNA splicing. Analyses of these data reveal that introns 4 and 5, which flank the alternatively spliced exon 5, have reduced levels of both intraspecific polymorphism and interspecific divergence relative to intron 3. No polymorphism was observed in any of the exons examined in D. melanogaster. A genealogical analysis clearly demonstrates the occurrence of intragenic recombination in the ancestral history of Mlc1. Recombination events are estimated to be 13 times more likely than mutation events over the span of the sequenced region. Although there is little evidence for pairwise linkage disequilibrium in the Mlc1 region, higher order disequilibrium. does seem to be present in the 5{prime} half of the portion of the gene that was examined. Predictions of the folding free energy of the pre-mRNA reveal that sampled alleles have a significantly higher (less stable) free energy than do randomly permuted sequences. These results are consistent with the hypothesis that introns surrounding an alternatively spliced exon are subjected to additional constraints, perhaps due to specific aspects of secondary structure required for appropriate splicing of the pre-mRNA molecule. 48 refs., 5 figs., 3 tabs.

  16. Impact of biotic and abiotic factors on the expression of fungal effector-encoding genes in axenic growth conditions.

    PubMed

    Meyer, Michel; Bourras, Salim; Gervais, Julie; Labadie, Karine; Cruaud, Corinne; Balesdent, Marie-Hélène; Rouxel, Thierry

    2017-02-01

    In phytopathogenic fungi, the expression of hundreds of small secreted protein (SSP)-encoding genes is induced upon primary infection of plants while no or a low level of expression is observed during vegetative growth. In some species such as Leptosphaeria maculans, this coordinated in-planta upregulation of SSP-encoding genes expression relies on an epigenetic control but the signals triggering gene expression in-planta are unknown. In the present study, biotic and abiotic factors that may relieve suppression of SSP-encoding gene expression during axenic growth of L. maculans were investigated. Some abiotic factors (temperature, pH) could have a limited effect on SSP gene expression. In contrast, two types of cellular stresses induced by antibiotics (cycloheximide, phleomycin) activated strongly the transcription of SSP genes. A transcriptomic analysis to cycloheximide exposure revealed that biological processes such as ribosome biosynthesis and rRNA processing were induced whereas important metabolic pathways such as glycogen and nitrogen metabolism, glycolysis and tricarboxylic acid cycle activity were down-regulated. A quantitatively different expression of SSP-encoding genes compared to plant infection was also detected. Interestingly, the same physico-chemical parameters as those identified here for L. maculans effectors were identified to regulate positively or negatively the expression of bacterial effectors. This suggests that apoplastic phytopathogens may react to similar physiological parameters for regulation of their effector genes.

  17. Modular synthase-encoding gene involved in α-olefin biosynthesis in Synechococcus sp. strain PCC 7002.

    PubMed

    Mendez-Perez, Daniel; Begemann, Matthew B; Pfleger, Brian F

    2011-06-01

    A gene involved in the production of medium-chain α-olefins was identified in the cyanobacterium Synechococcus sp. strain PCC 7002. The gene encodes a large multidomain protein with homology to type I polyketide synthases, suggesting a route for hydrocarbon biosynthesis from fatty acids via an elongation decarboxylation mechanism.

  18. Characterization of the nodulation plasmid encoded chemoreceptor gene mcpG from Rhizobium leguminosarum

    PubMed Central

    Yost, Christopher K; Clark, Kirsten T; Del Bel, Kate L; Hynes, Michael F

    2003-01-01

    Background In general, chemotaxis in Rhizobium has not been well characterized. Methyl accepting chemotaxis proteins are sensory proteins important in chemotaxis of numerous bacteria, but their involvement in Rhizobium chemotaxis is unclear and merits further investigation. Results A putative methyl accepting chemotaxis protein gene (mcpG) of Rhizobium leguminosarum VF39SM was isolated and characterized. The gene was found to reside on the nodulation plasmid, pRleVF39d. The predicted mcpG ORF displayed motifs common to known methyl-accepting chemotaxis proteins, such as two transmembrane domains and high homology to the conserved methylation and signaling domains of well-characterized MCPs. Phenotypic analysis of mcpG mutants using swarm plates did not identify ligands for this putative receptor. Additionally, gene knockouts of mcpG did not affect a mutant strain's ability to compete for nodulation with the wild type. Notably, mcpG was found to be plasmid-encoded in all strains of R. leguminosarum and R. etli examined, though it was found on the nodulation plasmid only in a minority of strains. Conclusions Based on sequence homology R. leguminosarum mcpG gene codes for a methyl accepting chemotaxis protein. The gene is plasmid localized in numerous Rhizobium spp. Although localized to the sym plasmid of VF39SM mcpG does not appear to participate in early nodulation events. A ligand for McpG remains to be found. Apparent McpG orthologs appear in a diverse range of proteobacteria. Identification and characterization of mcpG adds to the family of mcp genes already identified in this organism. PMID:12553885

  19. Recognition deficits in mice carrying mutations of genes encoding BLOC-1 subunits pallidin or dysbindin.

    PubMed

    Spiegel, S; Chiu, A; James, A S; Jentsch, J D; Karlsgodt, K H

    2015-11-01

    Numerous studies have implicated DTNBP1, the gene encoding dystrobrevin-binding protein or dysbindin, as a candidate risk gene for schizophrenia, though this relationship remains somewhat controversial. Variation in dysbindin, and its location on chromosome 6p, has been associated with cognitive processes, including those relying on a complex system of glutamatergic and dopaminergic interactions. Dysbindin is one of the seven protein subunits that comprise the biogenesis of lysosome-related organelles complex 1 (BLOC-1). Dysbindin protein levels are lower in mice with null mutations in pallidin, another gene in the BLOC-1, and pallidin levels are lower in mice with null mutations in the dysbindin gene, suggesting that multiple subunit proteins must be present to form a functional oligomeric complex. Furthermore, pallidin and dysbindin have similar distribution patterns in a mouse and human brain. Here, we investigated whether the apparent correspondence of pallid and dysbindin at the level of gene expression is also found at the level of behavior. Hypothesizing a mutation leading to underexpression of either of these proteins should show similar phenotypic effects, we studied recognition memory in both strains using the novel object recognition task (NORT) and social novelty recognition task (SNRT). We found that mice with a null mutation in either gene are impaired on SNRT and NORT when compared with wild-type controls. These results support the conclusion that deficits consistent with recognition memory impairment, a cognitive function that is impaired in schizophrenia, result from either pallidin or dysbindin mutations, possibly through degradation of BLOC-1 expression and/or function.

  20. Characterization of the Plesiomonas shigelloides Genes Encoding the Heme Iron Utilization System

    PubMed Central

    Henderson, D. P.; Wyckoff, E. E.; Rashidi, C. E.; Verlei, H.; Oldham, A. L.

    2001-01-01

    Plesiomonas shigelloides is a gram-negative pathogen which can utilize heme as an iron source. In previous work, P. shigelloides genes which permitted heme iron utilization in a laboratory strain of Escherichia coli were isolated. In the present study, the cloned P. shigelloides sequences were found to encode ten potential heme utilization proteins: HugA, the putative heme receptor; TonB and ExbBD; HugB, the putative periplasmic binding protein; HugCD, the putative inner membrane permease; and the proteins HugW, HugX, and HugZ. Three of the genes, hugA, hugZ, and tonB, contain a Fur box in their putative promoters, indicating that the genes may be iron regulated. When the P. shigelloides genes were tested in E. coli K-12 or in a heme iron utilization mutant of P. shigelloides, hugA, the TonB system genes, and hugW, hugX, or hugZ were required for heme iron utilization. When the genes were tested in a hemA entB mutant of E. coli, hugWXZ were not required for utilization of heme as a porphyrin source, but their absence resulted in heme toxicity when the strains were grown in media containing heme as an iron source. hugA could replace the Vibrio cholerae hutA in a heme iron utilization assay, and V. cholerae hutA could complement a P. shigelloides heme utilization mutant, suggesting that HugA is the heme receptor. Our analyses of the TonB system of P. shigelloides indicated that it could function in tonB mutants of both E. coli and V. cholerae and that it was similar to the V. cholerae TonB1 system in the amino acid sequence of the proteins and in the ability of the system to function in high-salt medium. PMID:11292789

  1. Praja1, a novel gene encoding a RING-H2 motif in mouse development.

    PubMed

    Mishra, L; Tully, R E; Monga, S P; Yu, P; Cai, T; Makalowski, W; Mezey, E; Pavan, W J; Mishra, B

    1997-11-06

    As part of a cloning strategy to identify genes involved in early mouse liver development we have isolated Praja1, a gene with similar sequences to the Drosophila melanogaster gene goliath (gl) which is involved in the fate of mesodermal cells ultimately forming gut musculatures, fat body, and the heart. Praja1 is a 2.1 kb gene encoding a putative 396 amino acid ORF and includes a COOH-terminal RING-H2 domain. Using the Jackson Laboratory BSS panel, we have localized Praja1 on chromosome X at 36 cM, which may be a candidate gene for mouse sla (sex linked sideroblastic anemia), near the X inactivation center gene, Xist. Northern blot analysis demonstrated three transcripts (3.1, 2.6 and 2.1 kb) in mRNA from adult mouse tissues brain, liver, and kidney as well as in mRNA from developing mouse embryos (days 7, 11, 15 and 17 post coitus, p.c.). In vitro transcription/translation yielded a product with an Mr of 59 kD. Immunohistochemical staining of in vitro liver explant cultures using a heterologous antibody against praja1 demonstrated cytoplasmic staining of cuboidal cells that have hepatocyte morphology and organization. The presence of the RING-H2 domain, a proline-rich region at the COOH-end, and regions rich in acidic amino acids, leads to the hypothesis that the Praja1 product is possibly involved in mediating protein-protein interactions, possibly as part of a protein sorting or transport pathway. This is strengthened by the similarity of Praja1 to rat Neurodap1, whose product has been shown to localize to the endoplasmic reticulum and golgi in brain.

  2. Gene therapy of experimental malignant mesothelioma using adenovirus vectors encoding the HSVtk gene.

    PubMed

    Esandi, M C; van Someren, G D; Vincent, A J; van Bekkum, D W; Valerio, D; Bout, A; Noteboom, J L

    1997-04-01

    Replication-defective adenovirus vectors were generated in which the gene of interest (lacZ, luciferase or HSV-tk) is driven by the adenovirus major late promoter (MLP) or the human cytomegalovirus immediate-early gene promoter/enhancer (CMV). In vitro experiments with rat (II-45) and human (MERO 25) mesothelioma cell lines revealed that the CMV promoter was stronger than the MLP promoter regarding levels of expression of the luciferase reporter gene and ganciclovir (GCV) killing efficiency after tk gene transfer. Following administration of IG.Ad.CMV.lacZ recombinant adenovirus (Introgene, IG) into the pleural cavity of Fischer rats with established mesothelioma, a widespread distribution of infectious virus particles through the thorax contents was demonstrated. However, a relatively small proportion of tumor cells were transduced. Nevertheless, a strong tumor growth inhibition was observed following treatment with IG.Ad.CMV.TK recombinant adenovirus and GCV. Separate groups of rats inoculated on day 0 with 10(5) II-45 cells in the pleural cavity, received 7 x 10(9) infectious particles of IG.Ad. CMV.TK on day 1, day 2, day 4 or day 8. One day after virus administration, 25 mg/kg GCV or PBS (controls) was injected i.p. (intraperitoneally) twice daily. On day 15, all animals were killed. Significant tumor regression, equivalent to 5 log cell kill, occurred in the treated rats suggesting an impressive bystander effect. In a survival study, animals were treated 9 days after inoculation of 10(5) tumor cells with IG.Ad.CMV.TK and a 14 days course of GCV. This treatment prolonged symptom-free survival time from 19 days in the controls to 33 days in the treated group. These responses can be best explained by assuming continued tk expression in or around the tumor tissue during GCV treatment. Our results confirm and extend earlier findings with the same model and demonstrate the potential of the herpes simplex virus thymidine kinase suicide gene therapy as a local

  3. Degradation of Benzene by Pseudomonas veronii 1YdBTEX2 and 1YB2 Is Catalyzed by Enzymes Encoded in Distinct Catabolism Gene Clusters.

    PubMed

    de Lima-Morales, Daiana; Chaves-Moreno, Diego; Wos-Oxley, Melissa L; Jáuregui, Ruy; Vilchez-Vargas, Ramiro; Pieper, Dietmar H

    2015-10-16

    Pseudomonas veronii 1YdBTEX2, a benzene and toluene degrader, and Pseudomonas veronii 1YB2, a benzene degrader, have previously been shown to be key players in a benzene-contaminated site. These strains harbor unique catabolic pathways for the degradation of benzene comprising a gene cluster encoding an isopropylbenzene dioxygenase where genes encoding downstream enzymes were interrupted by stop codons. Extradiol dioxygenases were recruited from gene clusters comprising genes encoding a 2-hydroxymuconic semialdehyde dehydrogenase necessary for benzene degradation but typically absent from isopropylbenzene dioxygenase-encoding gene clusters. The benzene dihydrodiol dehydrogenase-encoding gene was not clustered with any other aromatic degradation genes, and the encoded protein was only distantly related to dehydrogenases of aromatic degradation pathways. The involvement of the different gene clusters in the degradation pathways was suggested by real-time quantitative reverse transcription PCR.

  4. Evidence for negative selection on the gene encoding rhoptry-associated protein 1 (RAP-1) in Plasmodium spp.

    PubMed

    Pacheco, M Andreína; Ryan, Elizabeth M; Poe, Amanda C; Basco, Leonardo; Udhayakumar, Venkatachalam; Collins, Williams E; Escalante, Ananias A

    2010-07-01

    Assessing how natural selection, negative or positive, operates on genes with low polymorphism is challenging. We investigated the genetic diversity of orthologous genes encoding the rhoptry-associated protein 1 (RAP-1), a low polymorphic protein of malarial parasites that is involved in erythrocyte invasion. We applied evolutionary genetic methods to study the polymorphism in RAP-1 from Plasmodium falciparum (n=32) and Plasmodium vivax (n=6), the two parasites responsible for most human malaria morbidity and mortality, as well as RAP-1 orthologous in closely related malarial species found in non-human primates (NHPs). Overall, genes encoding RAP-1 are highly conserved in all Plasmodium spp. included in this investigation. We found no evidence for natural selection, positive or negative, acting on the gene encoding RAP-1 in P. falciparum or P. vivax. However, we found evidence that the orthologous genes in non-human primate parasites (Plasmodium cynomolgi, Plasmodium inui, and Plasmodium knowlesi) are under purifying (negative) selection. We discuss the importance of considering negative selection while studying genes encoding proteins with low polymorphism and how selective pressures may differ among orthologous genes in closely related malarial parasites species.

  5. The Drosophila pigmentation gene pink (p) encodes a homologue of human Hermansky-Pudlak syndrome 5 (HPS5).

    PubMed

    Falcón-Pérez, Juan M; Romero-Calderón, Rafael; Brooks, Elizabeth S; Krantz, David E; Dell'Angelica, Esteban C

    2007-02-01

    Lysosome-related organelles comprise a group of specialized intracellular compartments that include melanosomes and platelet dense granules (in mammals) and eye pigment granules (in insects). In humans, the biogenesis of these organelles is defective in genetic disorders collectively known as Hermansky-Pudlak syndrome (HPS). Patients with HPS-2, and two murine HPS models, carry mutations in genes encoding subunits of adaptor protein (AP)-3. Other genes mutated in rodent models include those encoding VPS33A and Rab38. Orthologs of all of these genes in Drosophila melanogaster belong to the 'granule group' of eye pigmentation genes. Other genes associated with HPS encode subunits of three complexes of unknown function, named biogenesis of lysosome-related organelles complex (BLOC)-1, -2 and -3, for which the Drosophila counterparts had not been characterized. Here, we report that the gene encoding the Drosophila ortholog of the HPS5 subunit of BLOC-2 is identical to the granule group gene pink (p), which was first studied in 1910 but had not been identified at the molecular level. The phenotype of pink mutants was exacerbated by mutations in AP-3 subunits or in the orthologs of VPS33A and Rab38. These results validate D. melanogaster as a genetic model to study the function of the BLOCs.

  6. Nicotinamide adenine dinucleotide biosynthesis promotes liver regeneration.

    PubMed

    Mukherjee, Sarmistha; Chellappa, Karthikeyani; Moffitt, Andrea; Ndungu, Joan; Dellinger, Ryan W; Davis, James G; Agarwal, Beamon; Baur, Joseph A

    2017-02-01

    The regenerative capacity of the liver is essential for recovery from surgical resection or injuries induced by trauma or toxins. During liver regeneration, the concentration of nicotinamide adenine dinucleotide (NAD) falls, at least in part due to metabolic competition for precursors. To test whether NAD availability restricts the rate of liver regeneration, we supplied nicotinamide riboside (NR), an NAD precursor, in the drinking water of mice subjected to partial hepatectomy. NR increased DNA synthesis, mitotic index, and mass restoration in the regenerating livers. Intriguingly, NR also ameliorated the steatosis that normally accompanies liver regeneration. To distinguish the role of hepatocyte NAD levels from any systemic effects of NR, we generated mice overexpressing nicotinamide phosphoribosyltransferase, a rate-limiting enzyme for NAD synthesis, specifically in the liver. Nicotinamide phosphoribosyltransferase overexpressing mice were mildly hyperglycemic at baseline and, similar to mice treated with NR, exhibited enhanced liver regeneration and reduced steatosis following partial hepatectomy. Conversely, mice lacking nicotinamide phosphoribosyltransferase in hepatocytes exhibited impaired regenerative capacity that was completely rescued by administering NR.

  7. Seven Genes of the Enhancer of Split Complex of Drosophila Melanogaster Encode Helix-Loop-Helix Proteins

    PubMed Central

    Knust, E.; Schrons, H.; Grawe, F.; Campos-Ortega, J. A.

    1992-01-01

    Enhancer of split [E(spl)] is one of the neurogenic loci of Drosophila and, as such, is required for normal segregation of neural and epidermal cell progenitors. Genetic observations indicate that the E(spl) locus is in fact a gene complex comprising a cluster of related genes and that other genes of the region are also required for normal early neurogenesis. Three of the genes of the complex were known to encode helix-loop-helix (HLH) proteins and to be transcribed in nearly identical patterns. Here, we show that four other genes in the vicinity also encode HLH proteins and, during neuroblast segregation, three of them are expressed in the same pattern. We show by germ-line transformation that these three genes are also necessary to allow epidermal development of the neuroectodermal cells. PMID:1427040

  8. Modulation of Gene Expression by Polymer Nanocapsule Delivery of DNA Cassettes Encoding Small RNAs.

    PubMed

    Yan, Ming; Wen, Jing; Liang, Min; Lu, Yunfeng; Kamata, Masakazu; Chen, Irvin S Y

    2015-01-01

    Small RNAs, including siRNAs, gRNAs and miRNAs, modulate gene expression and serve as potential therapies for human diseases. Delivery to target cells remains the fundamental limitation for use of these RNAs in humans. To address this challenge, we have developed a nanocapsule delivery technology that encapsulates small DNA molecules encoding RNAs into a small (30 nm) polymer nanocapsule. For proof of concept, we transduced DNA expression cassettes for three small RNAs. In one application, the DNA cassette encodes an shRNA transcriptional unit that downregulates CCR5 and protects from HIV-1 infection. The DNA cassette nanocapsules were further engineered for timed release of the DNA cargo for prolonged knockdown of CCR5. Secondly, the nanocapsules provide an efficient means for delivery of gRNAs in the CRISPR/Cas9 system to mutate integrated HIV-1. Finally, delivery of microRNA-125b to mobilized human CD34+ cells enhances survival and expansion of the CD34+ cells in culture.

  9. Generation of MANAbodies specific to HLA-restricted epitopes encoded by somatically mutated genes.

    PubMed

    Skora, Andrew D; Douglass, Jacqueline; Hwang, Michael S; Tam, Ada J; Blosser, Richard L; Gabelli, Sandra B; Cao, Jianhong; Diaz, Luis A; Papadopoulos, Nickolas; Kinzler, Kenneth W; Vogelstein, Bert; Zhou, Shibin

    2015-08-11

    Mutant epitopes encoded by cancer genes are virtually always located in the interior of cells, making them invisible to conventional antibodies. We here describe an approach to identify single-chain variable fragments (scFvs) specific for mutant peptides presented on the cell surface by HLA molecules. We demonstrate that these scFvs can be successfully converted to full-length antibodies, termed MANAbodies, targeting "Mutation-Associated Neo-Antigens" bound to HLA. A phage display library representing a highly diverse array of single-chain variable fragment sequences was first designed and constructed. A competitive selection protocol was then used to identify clones specific for mutant peptides bound to predefined HLA types. In this way, we obtained two scFvs, one specific for a peptide encoded by a common KRAS mutant and the other by a common epidermal growth factor receptor (EGFR) mutant. The scFvs bound to these peptides only when the peptides were complexed with HLA-A2 (KRAS peptide) or HLA-A3 (EGFR peptide). We converted one scFv to a full-length antibody (MANAbody) and demonstrate that the MANAbody specifically reacts with mutant peptide-HLA complex even when the peptide differs by only one amino acid from the normal, WT form.

  10. Effects of cholecystokinin and bombesin on the expression of preprosomatostatin-encoding genes in goldfish forebrain.

    PubMed

    Canosa, Luis Fabián; Peter, Richard E

    2004-09-15

    It was previously demonstrated that both cholecystokinin (CCK) and bombesin (BBS) stimulate growth hormone (GH) secretion in goldfish. Both peptides induce satiety and it was speculated that they integrate satiation and the postprandial increase in GH circulating levels. In the present paper we investigated the effects of CCK and BBS on the forebrain expression of the somatostatin gene family in goldfish to analyze if somatostatin peptides may be part of the effector mechanisms of CCK and BBS. We found that peripherally as well as centrally administered CCK decreases mRNA levels of preprosomatostatin (PSS)-I that encodes for SRIF-14, having no effects on PSS-II and PSS-III, which encode for gSRIF-28 and [Pro2] SRIF-14, respectively. In addition, a direct action on the pituitary to stimulate GH release, this inhibition of PSS-I expression provides a possible mechanism for CCK to increase postprandial GH levels. On the other hand, BBS inhibits the forebrain expression of PSS-I and PSS-II but does not affect PSS-III regardless of the route of administration. We conclude that this could be the most likely mechanism of action of BBS to increase GH secretion, since there are few BBS-immunoreactive (IR) fibers and BBS binding sites in the anterior pituitary of goldfish.

  11. Characterization of a ribonuclease gene and encoded protein from the reptile, Iguana iguana.

    PubMed

    Nitto, Takeaki; Lin, Cynthia; Dyer, Kimberly D; Wagner, Robert A; Rosenberg, Helene F

    2005-06-06

    In this work we identify an intronless open reading frame encoding an RNase A ribonuclease from genomic DNA from the Iguana iguana IgH2 cell line. The iguana RNase is expressed primarily in pancreas, and represents the majority of the specific enzymatic activity in this tissue. The encoded sequence shares many features with its better-known mammalian counterparts including the crucial His12, Lys40 and His114 catalytic residues and efficient hydrolytic activity against yeast tRNA substrate (k(cat)/K(m)=6 x 10(4) M(-1) s(-1)), albeit at a reduced pH optimum (pH 6.0). Although the catalytic activity of the iguana RNase is not diminished by human placental RI, iguana RNase is not bactericidal nor is it cytotoxic even at micromolar concentrations. Phylogenetic analysis indicates moderate (46%) amino acid sequence similarity to a pancreatic RNase isolated from Chelydra serpentina (snapping turtle) although no specific relationship could be determined between these RNases and the pancreatic ribonucleases characterized among mammalian species. Further analysis of ribonucleases from non-mammalian vertebrate species is needed in order to define relationships and lineages within the larger RNase A gene superfamily.

  12. The Drosophila prage Gene, Required for Maternal Transcript Destabilization in Embryos, Encodes a Predicted RNA Exonuclease

    PubMed Central

    Cui, Jun; Lai, Yun Wei; Sartain, Caroline V.; Zuckerman, Rebecca M.; Wolfner, Mariana F.

    2016-01-01

    Egg activation, the transition of mature oocytes into developing embryos, is critical for the initiation of embryogenesis. This process is characterized by resumption of meiosis, changes in the egg’s coverings and by alterations in the transcriptome and proteome of the egg; all of these occur in the absence of new transcription. Activation of the egg is prompted by ionic changes in the cytoplasm (usually a rise in cytosolic calcium levels) that are triggered by fertilization in some animals and by mechanosensitive cues in others. The egg’s transcriptome is dramatically altered during the process, including by the removal of many maternal mRNAs that are not needed for embryogenesis. However, the mechanisms and regulators of this selective RNA degradation are not yet fully known. Forward genetic approaches in Drosophila have identified maternal-effect genes whose mutations prevent the transcriptome changes. One of these genes, prage (prg), was identified by Tadros et al. in a screen for mutants that fail to destabilize maternal transcripts. We identified the molecular nature of the prg gene through a combination of deficiency mapping, complementation analysis, and DNA sequencing of both extant prg mutant alleles. We find that prg encodes a ubiquitously expressed predicted exonuclease, consistent with its role in maternal mRNA destabilization during egg activation. PMID:27172196

  13. Molecular cloning of a gene encoding the histamine H2 receptor

    SciTech Connect

    Gantz, I.; Schaeffer, M.; DelValle, J.; Logsdon, C.; Campbell, V.; Uhler, M.; Yamada, Tadataka )

    1991-01-15

    The H2 subclass of histamine receptors mediates gastric acid secretion, and antagonists for this receptor have proven to be effective therapy for acid peptic disorders of the gastrointestinal tract. The physiological action of histamine has been shown to be mediated via a guanine nucleotide-binding protein linked to adenylate cyclase activation and cellular cAMP generation. The authors capitalized on the technique of polymerase chain reaction, using degenerate oligonucleotide primers based on the known homology between cellular receptors linked to guanine nucleotide-binding proteins to obtain a partial-length clone from canine gastric parietal cell cDNA. This clone was used to obtain a full-length receptor gene from a canine genomic library. Histamine increased in a dose-dependent manner cellular cAMP content in L cells permanently transfected with this gene, and preincubation of the cells with the H2-selective antagonist cimetidine shifted the dose-response curve to the right. Cimetidine inhibited the binding of the radiolabeled H2 receptor-selective ligand (methyl-{sup 3}H)tiotidine to the transfected cells in a dose-dependent fashion, but the H1-selective antagonist diphenhydramine did not. These data indicate that they have cloned a gene that encodes the H2 subclass of histamine receptors.

  14. Kallmann Syndrome: Mutations in the Genes Encoding Prokineticin-2 and Prokineticin Receptor-2

    PubMed Central

    Dodé, Catherine; Teixeira, Luis; Levilliers, Jacqueline; Fouveaut, Corinne; Bouchard, Philippe; Kottler, Marie-Laure; Lespinasse, James; Lienhardt-Roussie, Anne; Mathieu, Michèle; Moerman, Alexandre; Morgan, Graeme; Murat, Arnaud; Toublanc, Jean-Edmont; Wolczynski, Slawomir; Delpech, Marc; Petit, Christine; Young, Jacques; Hardelin, Jean-Pierre

    2006-01-01

    Kallmann syndrome combines anosmia, related to defective olfactory bulb morphogenesis, and hypogonadism due to gonadotropin-releasing hormone deficiency. Loss-of-function mutations in KAL1 and FGFR1 underlie the X chromosome-linked form and an autosomal dominant form of the disease, respectively. Mutations in these genes, however, only account for approximately 20% of all Kallmann syndrome cases. In a cohort of 192 patients we took a candidate gene strategy and identified ten and four different point mutations in the genes encoding the G protein-coupled prokineticin receptor-2 (PROKR2) and one of its ligands, prokineticin-2 (PROK2), respectively. The mutations in PROK2 were detected in the heterozygous state, whereas PROKR2 mutations were found in the heterozygous, homozygous, or compound heterozygous state. In addition, one of the patients heterozygous for a PROKR2 mutation was also carrying a missense mutation in KAL1, thus indicating a possible digenic inheritance of the disease in this individual. These findings reveal that insufficient prokineticin-signaling through PROKR2 leads to abnormal development of the olfactory system and reproductive axis in man. They also shed new light on the complex genetic transmission of Kallmann syndrome. PMID:17054399

  15. (Structure and expression of nuclear genes encoding rubisco activase): Progress report

    SciTech Connect

    Not Available

    1989-01-01

    Our first year's activities include: (1) completing a survey of the basic characteristics of activase gene expression in barley; and (2) isolating and structurally characterizing cDNA and genomic DNA sequences encoding activase from barley. Our goal was to determine whether activase mRNA and protein accumulation are coordinated with those of the rubisco subunits. We utilized the first leaves of barley as an experimental system for these studies because they can be used in two ways to study the expression of leaf genes: by following the naturally occurring differentiation of leaf cells, which occurs acropetally along the barley leaf; and by following the photomorphogenesis of etiolated barley seedlings. In the acropetal gradient of leaf cell differentiation, activase mRNA and mRNA and polypeptide expression is tightly coordinated with rubisco subunit mRNA and polypeptide expression. Although we have not measured their precise stoichiometry at each stage of leaf differentiation, activase protein is expressed at the level of about one polypeptide per rubisco holoenzyme in mature regions of the leaf. Coordination of the expression of activase mRNAs and polypeptides indicates that in the barley leaf gradient, activase gene expression is largely controlled at the level of transcription. However, translational controls may play a role in regulating activase expression on a short term basis.

  16. Sudden infant death syndrome caused by cardiac arrhythmias: only a matter of genes encoding ion channels?

    PubMed

    Sarquella-Brugada, Georgia; Campuzano, Oscar; Cesar, Sergi; Iglesias, Anna; Fernandez, Anna; Brugada, Josep; Brugada, Ramon

    2016-03-01

    Sudden infant death syndrome is the unexpected demise of a child younger than 1 year of age which remains unexplained after a complete autopsy investigation. Usually, it occurs during sleep, in males, and during the first 12 weeks of life. The pathophysiological mechanism underlying the death is unknown, and the lethal episode is considered multifactorial. However, in cases without a conclusive post-mortem diagnosis, suspicious of cardiac arrhythmias may also be considered as a cause of death, especially in families suffering from any cardiac disease associated with sudden cardiac death. Here, we review current understanding of sudden infant death, focusing on genetic causes leading to lethal cardiac arrhythmias, considering both genes encoding ion channels as well as structural proteins due to recent association of channelopathies and desmosomal genes. We support a comprehensive analysis of all genes associated with sudden cardiac death in families suffering of infant death. It allows the identification of the most plausible cause of death but also of family members at risk, providing cardiologists with essential data to adopt therapeutic preventive measures in families affected with this lethal entity.

  17. Cell-Free Phospholipid Biosynthesis by Gene-Encoded Enzymes Reconstituted in Liposomes

    PubMed Central

    Scott, Andrew; Noga, Marek J.; de Graaf, Paul; Westerlaken, Ilja; Yildirim, Esengul; Danelon, Christophe

    2016-01-01

    The goal of bottom-up synthetic biology culminates in the assembly of an entire cell from separate biological building blocks. One major challenge resides in the in vitro production and implementation of complex genetic and metabolic pathways that can support essential cellular functions. Here, we show that phospholipid biosynthesis, a multiple-step process involved in cell membrane homeostasis, can be reconstituted starting from the genes encoding for all necessary proteins. A total of eight E. coli enzymes for acyl transfer and headgroup modifications were produced in a cell-free gene expression system and were co-translationally reconstituted in liposomes. Acyl-coenzyme A and glycerol-3-phosphate were used as canonical precursors to generate a variety of important bacterial lipids. Moreover, this study demonstrates that two-step acyl transfer can occur from enzymes synthesized inside vesicles. Besides clear implications for growth and potentially division of a synthetic cell, we postulate that gene-based lipid biosynthesis can become instrumental for ex vivo and protein purification-free production of natural and non-natural lipids. PMID:27711229

  18. Prevalence of genes encoding extracellular proteases in Staphylococcus aureus - important targets triggering immune response in vivo.

    PubMed

    Zdzalik, Michal; Karim, Abdulkarim Y; Wolski, Krzysztof; Buda, Pawel; Wojcik, Kinga; Brueggemann, Sarah; Wojciechowski, Piotr; Eick, Sigrun; Calander, Ann-Marie; Jonsson, Ing-Marie; Kubica, Malgorzata; Polakowska, Klaudia; Miedzobrodzki, Jacek; Wladyka, Benedykt; Potempa, Jan; Dubin, Grzegorz

    2012-11-01

    Proteases of Staphylococcus aureus have long been considered to function as important virulence factors, although direct evidence of the role of particular enzymes remains incomplete and elusive. Here, we sought to provide a collective view of the prevalence of extracellular protease genes in genomes of commensal and pathogenic strains of S. aureus and their expression in the course of human and mouse infection. Data on V8 protease, staphopains A and B, aureolysin, and the recently described and poorly characterized group of six Spl proteases are provided. A phylogenetically diverse collection of 167 clinical isolates was analyzed, resulting in the comprehensive genetic survey of the prevalence of protease-encoding genes. No correlation between identified gene patterns with specific infections was established. Humoral response against the proteases of interest was examined in the sera derived from human patients and from a model mouse infection. The analysis suggests that at least some, if not all, tested proteases are expressed and secreted during the course of infection. Overall, the results presented in this study support the hypothesis that the secretory proteases as a group may contribute to the virulence of S. aureus.

  19. Biodiversity of genes encoding anti-microbial traits within plant associated microbes

    PubMed Central

    Mousa, Walaa K.; Raizada, Manish N.

    2015-01-01

    The plant is an attractive versatile home for diverse associated microbes. A subset of these microbes produces a diversity of anti-microbial natural products including polyketides, non-ribosomal peptides, terpenoids, heterocylic nitrogenous compounds, volatile compounds, bacteriocins, and lytic enzymes. In recent years, detailed molecular analysis has led to a better understanding of the underlying genetic mechanisms. New genomic and bioinformatic tools have permitted comparisons of orthologous genes between species, leading to predictions of the associated evolutionary mechanisms responsible for diversification at the genetic and corresponding biochemical levels. The purpose of this review is to describe the biodiversity of biosynthetic genes of plant-associated bacteria and fungi that encode selected examples of antimicrobial natural products. For each compound, the target pathogen and biochemical mode of action are described, in order to draw attention to the complexity of these phenomena. We review recent information of the underlying molecular diversity and draw lessons through comparative genomic analysis of the orthologous coding sequences (CDS). We conclude by discussing emerging themes and gaps, discuss the metabolic pathways in the context of the phylogeny and ecology of their microbial hosts, and discuss potential evolutionary mechanisms that led to the diversification of biosynthetic gene clusters. PMID:25914708

  20. Identification and analysis of a Saccharomyces cerevisiae copper homeostasis gene encoding a homeodomain protein.

    PubMed Central

    Knight, S A; Tamai, K T; Kosman, D J; Thiele, D J

    1994-01-01

    Yeast metallothionein, encoded by the CUP1 gene, and its copper-dependent transcriptional activator ACE1 play a key role in mediating copper resistance in Saccharomyces cerevisiae. Using an ethyl methanesulfonate mutant of a yeast strain in which CUP1 and ACE1 were deleted, we isolated a gene, designated CUP9, which permits yeast cells to grow at high concentrations of environmental copper, most notably when lactate is the sole carbon source. Disruption of CUP9, which is located on chromosome XVI, caused a loss of copper resistance in strains which possessed CUP1 and ACE1, as well as in the cup1 ace1 deletion strain. Measurement of intracellular copper levels of the wild-type and cup9-1 mutant demonstrated that total intracellular copper concentrations were unaffected by CUP9. CUP9 mRNA levels were, however, down regulated by copper when yeast cells were grown with glucose but not with lactate or glycerol-ethanol as the sole carbon source. This down regulation was independent of the copper metalloregulatory transcription factor ACE1. The DNA sequence of CUP9 predicts an open reading frame of 306 amino acids in which a 55-amino-acid sequence showed 47% identity with the homeobox domain of the human proto-oncogene PBX1, suggesting that CUP9 is a DNA-binding protein which regulates the expression of important copper homeostatic genes. Images PMID:7969120

  1. Detection and characterisation of the genes encoding glyoxalase I and II from Neisseria meningitidis.

    PubMed

    Kizil, G; Wilks, K; Wells, D; Ala'Aldeen, D A

    2000-07-01

    Glyoxalase enzymes I and II are involved in a detoxification process consisting of conversion of reactive dicarbonyl compounds (e.g., methylglyoxal) to less reactive hydroxy acids. The structural gene for meningococcal glyoxalase I (gloA) was identified by screening an expression library with a rabbit antiserum. The meningococcal gloA gene consisted of 138 deduced amino acids, with a calculated mol. wt of 15.7 kDa. The DNA and deduced protein sequence of gloA was compared to known sequences of glyoxalase I enzymes and showed high homology with gloA of several eukaryotic and prokaryotic species. Insertion of a gloA-containing plasmid in Escherichia coli increased the host organism's tolerance to methylglyoxal from <2 mM to >4 mM, thus demonstrating its functional identity. A databank search also revealed the presence of a putative gloB gene, encoding glyoxalase II (GlxII), in the recently released genomic sequences of Neisseria meningitidis and N. gonorrhoeae.

  2. MFα1, the Gene Encoding the α Mating Pheromone of Candida albicans†

    PubMed Central

    Panwar, Sneh L.; Legrand, Melanie; Dignard, Daniel; Whiteway, Malcolm; Magee, Paul. T.

    2003-01-01

    Candida albicans, the single most frequently isolated human fungal pathogen, was thought to be asexual until the recent discovery of the mating-type-like locus (MTL). Homozygous MTL strains were constructed and shown to mate. Furthermore, it has been demonstrated that opaque-phase cells are more efficient in mating than white-phase cells. The similarity of the genes involved in the mating pathway in Saccharomyces cerevisiae and C. albicans includes at least one gene (KEX2) that is involved in the processing of the α mating pheromone in the two yeasts. Taking into account this similarity, we searched the C. albicans genome for sequences that would encode the α pheromone gene. Here we report the isolation and characterization of the gene MFα1, which codes for the precursor of the α mating pheromone in C. albicans. Two active α-peptides, 13 and 14 amino acids long, would be generated after the precursor molecule is processed in C. albicans. To examine the role of this gene in mating, we constructed an mfα1 null mutant of C. albicans. The mfα1 null mutant fails to mate as MTLα, while MTLa mfα1 cells are still mating competent. Experiments performed with the synthetic α-peptides show that they are capable of inducing growth arrest, as demonstrated by halo tests, and also induce shmooing in MTLa cells of C. albicans. These peptides are also able to complement the mating defect of an MTLα kex2 mutant strain when added exogenously, thereby confirming their roles as α mating pheromones. PMID:14665468

  3. Knockdown of Five Genes Encoding Uncharacterized Proteins Inhibits Entamoeba histolytica Phagocytosis of Dead Host Cells.

    PubMed

    Sateriale, Adam; Miller, Peter; Huston, Christopher D

    2016-04-01

    Entamoeba histolytica is the protozoan parasite that causes invasive amebiasis, which is endemic to many developing countries and characterized by dysentery and liver abscesses. The virulence of E. histolytica correlates with the degree of host cell engulfment, or phagocytosis, and E. histolytica phagocytosis alters amebic gene expression in a feed-forward manner that results in an increased phagocytic ability. Here, we used a streamlined RNA interference screen to silence the expression of 15 genes whose expression was upregulated in phagocytic E. histolytica trophozoites to determine whether these genes actually function in the phagocytic process. When five of these genes were silenced, amebic strains with significant decreases in the ability to phagocytose apoptotic host cells were produced. Phagocytosis of live host cells, however, was largely unchanged, and the defects were surprisingly specific for phagocytosis. Two of the five encoded proteins, which we named E. histolytica ILWEQ (EhILWEQ) and E. histolytica BAR (EhBAR), were chosen for localization via SNAP tag labeling and localized to the site of partially formed phagosomes. Therefore, both EhILWEQ and EhBAR appear to contribute to E. histolytica virulence through their function in phagocytosis, and the large proportion (5/15 [33%]) of gene-silenced strains with a reduced ability to phagocytose host cells validates the previously published microarray data set demonstrating feed-forward control of E. histolytica phagocytosis. Finally, although only limited conclusions can be drawn from studies using the virulence-deficient G3 Entamoeba strain, the relative specificity of the defects induced for phagocytosis of apoptotic cells but not healthy cells suggests that cell killing may play a rate-limiting role in the process of Entamoeba histolytica host cell engulfment.

  4. Overproduction of lactimidomycin by cross-overexpression of genes encoding Streptomyces antibiotic regulatory proteins.

    PubMed

    Zhang, Bo; Yang, Dong; Yan, Yijun; Pan, Guohui; Xiang, Wensheng; Shen, Ben

    2016-03-01

    The glutarimide-containing polyketides represent a fascinating class of natural products that exhibit a multitude of biological activities. We have recently cloned and sequenced the biosynthetic gene clusters for three members of the glutarimide-containing polyketides-iso-migrastatin (iso-MGS) from Streptomyces platensis NRRL 18993, lactimidomycin (LTM) from Streptomyces amphibiosporus ATCC 53964, and cycloheximide (CHX) from Streptomyces sp. YIM56141. Comparative analysis of the three clusters identified mgsA and chxA, from the mgs and chx gene clusters, respectively, that were predicted to encode the PimR-like Streptomyces antibiotic regulatory proteins (SARPs) but failed to reveal any regulatory gene from the ltm gene cluster. Overexpression of mgsA or chxA in S. platensis NRRL 18993, Streptomyces sp. YIM56141 or SB11024, and a recombinant strain of Streptomyces coelicolor M145 carrying the intact mgs gene cluster has no significant effect on iso-MGS or CHX production, suggesting that MgsA or ChxA regulation may not be rate-limiting for iso-MGS and CHX production in these producers. In contrast, overexpression of mgsA or chxA in S. amphibiosporus ATCC 53964 resulted in a significant increase in LTM production, with LTM titer reaching 106 mg/L, which is five-fold higher than that of the wild-type strain. These results support MgsA and ChxA as members of the SARP family of positive regulators for the iso-MGS and CHX biosynthetic machinery and demonstrate the feasibility to improve glutarimide-containing polyketide production in Streptomyces strains by exploiting common regulators.

  5. Isolation of Drosophila genes encoding G protein-coupled receptor kinases.

    PubMed Central

    Cassill, J A; Whitney, M; Joazeiro, C A; Becker, A; Zuker, C S

    1991-01-01

    G protein-coupled receptors are regulated via phosphorylation by a variety of protein kinases. Recently, termination of the active state of two such receptors, the beta-adrenergic receptor and rhodopsin, has been shown to be mediated by agonist- or light-dependent phosphorylation of the receptor by members of a family of protein-serine/threonine kinases (here referred to as G protein-coupled receptor kinases). We now report the isolation of a family of genes encoding a set of Drosophila protein kinases that appear to code for G protein-coupled receptor kinases. These proteins share a high degree of sequence homology with the bovine beta-adrenergic receptor kinase. The presence of a conserved family of G protein-coupled receptor kinases in vertebrates and invertebrates points to the central role of these kinases in signal transduction cascades. Images PMID:1662381

  6. Reduction of antinutritional glucosinolates in Brassica oilseeds by mutation of genes encoding transporters.

    PubMed

    Nour-Eldin, Hussam Hassan; Madsen, Svend Roesen; Engelen, Steven; Jørgensen, Morten Egevang; Olsen, Carl Erik; Andersen, Jonathan Sonne; Seynnaeve, David; Verhoye, Thalia; Fulawka, Rudy; Denolf, Peter; Halkier, Barbara Ann

    2017-03-13

    The nutritional value of Brassica seed meals is reduced by the presence of glucosinolates, which are toxic compounds involved in plant defense. Mutation of the genes encoding two glucosinolate transporters (GTRs) eliminated glucosinolates from Arabidopsis thaliana seeds, but translation of loss-of-function phenotypes into Brassica crops is challenging because Brassica is polyploid. We mutated one of seven and four of 12 GTR orthologs and reduced glucosinolate levels in seeds by 60-70% in two different Brassica species (Brassica rapa and Brassica juncea). Reduction in seed glucosinolates was stably inherited over multiple generations and maintained in field trials of two mutant populations at three locations. Successful translation of the gtr loss-of-function phenotype from model plant to two Brassica crops suggests that our transport engineering approach could be broadly applied to reduce seed glucosinolate content in other oilseed crops, such as Camelina sativa or Crambe abyssinica.

  7. The daf-4 gene encodes a bone morphogenetic protein receptor controlling C. elegans dauer larva development.

    PubMed

    Estevez, M; Attisano, L; Wrana, J L; Albert, P S; Massagué, J; Riddle, D L

    1993-10-14

    The bone morphogenetic protein (BMP) family is a conserved group of signalling molecules within the transforming growth factor-beta (TGF-beta) superfamily. This group, including the Drosophila decapentaplegic (dpp) protein and the mammalian BMPs, mediates cellular interactions and tissue differentiation during development. Here we show that a homologue of human BMPs controls a developmental switch in the life cycle of the free-living soil nematode Caenorhabditis elegans. Starvation and overcrowding induce C. elegans to form a developmentally arrested, third-stage dauer larva. The daf-4 gene, which acts to inhibit dauer larva formation and promote growth, encodes a receptor protein kinase similar to the daf-1, activin and TGF-beta receptor serine/threonine kinases. When expressed in monkey COS cells, the daf-4 receptor binds human BMP-2 and BMP-4. The daf-4 receptor is the first to be identified for any growth factor in the BMP family.

  8. The abp gene in Geobacillus stearothermophilus T-6 encodes a GH27 β-L-arabinopyranosidase.

    PubMed

    Salama, Rachel; Alalouf, Onit; Tabachnikov, Orly; Zolotnitsky, Gennady; Shoham, Gil; Shoham, Yuval

    2012-07-30

    In this study we demonstrate that the abp gene in Geobacillus stearothermophilus T-6 encodes a family 27 glycoside hydrolase β-L-arabinopyranosidase. The catalytic constants towards the chromogenic substrate pNP-β-L-arabinopyranoside were 0.8±0.1 mM, 6.6±0.3 s(-1), and 8.2±0.3 s(-1) mM(-1) for K(m), k(cat) and k(cat)/K(m), respectively. (13)C NMR spectroscopy unequivocally showed that Abp is capable of removing β-L-arabinopyranose residues from the natural arabino-polysaccharide, larch arabinogalactan. Most family 27 enzymes are active on galactose and contain a conserved Asp residue, whereas in Abp this residue is Ile67, which shifts the specificity of the enzyme towards arabinopyranoside.

  9. The fas operon of Rhodococcus fascians encodes new genes required for efficient fasciation of host plants.

    PubMed

    Crespi, M; Vereecke, D; Temmerman, W; Van Montagu, M; Desomer, J

    1994-05-01

    Three virulence loci (fas, att, and hyp) of Rhodococcus fascians D188 have been identified on a 200-kb conjugative linear plasmid (pFiD188). The fas locus was delimited to a 6.5-kb DNA fragment by insertion mutagenesis, single homologous disruptive recombination, and in trans complementation of different avirulent insertion mutants. The locus is arranged as a large operon containing six open reading frames whose expression is specifically induced during the interaction with host plants. One predicted protein is homologous to P-450 cytochromes from actinomycetes. The putative ferredoxin component is of a novel type containing additional domains homologous to transketolases from chemoautotrophic, photosynthetic, and methylotrophic microorganisms. Genetic analysis revealed that fas encodes, in addition to the previously identified ipt, at least two new genes that are involved in fasciation development, one of which is only required on older tobacco plants.

  10. A gene encoding a protein modified by the phytohormone indoleacetic acid

    PubMed Central

    Walz, Alexander; Park, Seijin; Slovin, Janet P.; Ludwig-Müller, Jutta; Momonoki, Yoshie S.; Cohen, Jerry D.

    2002-01-01

    We show that the expression of an indole-3-acetic acid (IAA)-modified protein from bean seed, IAP1, is correlated to the developmental period of rapid growth during seed development. Moreover, this protein undergoes rapid degradation during germination. The gene for IAP1, the most abundant protein covalently modified by IAA (iap1, GenBank accession no. AF293023) was isolated and cloned from bush bean (Phaseolus vulgaris) seeds. The 957-bp sequence encodes a 35-kDa polypeptide. IAA-modified proteins represent a distinct class of conjugated phytohormones and appear in bean to be the major form of auxin in seeds. IAA proteins also are found at other stages of development in bean plants. Our immunological and analytical data suggest that auxin modification of a small class of proteins may be a feature common to many plants. PMID:11830675

  11. WRKY domain-encoding genes of a crop legume chickpea (Cicer arietinum): comparative analysis with Medicago truncatula WRKY family and characterization of group-III gene(s)

    PubMed Central

    Kumar, Kamal; Srivastava, Vikas; Purayannur, Savithri; Kaladhar, V. Chandra; Cheruvu, Purnima Jaiswal; Verma, Praveen Kumar

    2016-01-01

    The WRKY genes have been identified as important transcriptional modulators predominantly during the environmental stresses, but they also play critical role at various stages of plant life cycle. We report the identification of WRKY domain (WD)-encoding genes from galegoid clade legumes chickpea (Cicer arietinum L.) and barrel medic (Medicago truncatula). In total, 78 and 98 WD-encoding genes were found in chickpea and barrel medic, respectively. Comparative analysis suggests the presence of both conserved and unique WRKYs, and expansion of WRKY family in M. truncatula primarily by tandem duplication. Exclusively found in galegoid legumes, CaWRKY16 and its orthologues encode for a novel protein having a transmembrane and partial Exo70 domains flanking a group-III WD. Genomic region of galegoids, having CaWRKY16, is more dynamic when compared with millettioids. In onion cells, fused CaWRKY16-EYFP showed punctate fluorescent signals in cytoplasm. The chickpea WRKY group-III genes were further characterized for their transcript level modulation during pathogenic stress and treatments of abscisic acid, jasmonic acid, and salicylic acid (SA) by real-time PCR. Differential regulation of genes was observed during Ascochyta rabiei infection and SA treatment. Characterization of A. rabiei and SA inducible gene CaWRKY50 showed that it localizes to plant nucleus, binds to W-box, and have a C-terminal transactivation domain. Overexpression of CaWRKY50 in tobacco plants resulted in early flowering and senescence. The in-depth comparative account presented here for two legume WRKY genes will be of great utility in hastening functional characterization of crop legume WRKYs and will also help in characterization of Exo70Js. PMID:27060167

  12. WRKY domain-encoding genes of a crop legume chickpea (Cicer arietinum): comparative analysis with Medicago truncatula WRKY family and characterization of group-III gene(s).

    PubMed

    Kumar, Kamal; Srivastava, Vikas; Purayannur, Savithri; Kaladhar, V Chandra; Cheruvu, Purnima Jaiswal; Verma, Praveen Kumar

    2016-06-01

    The WRKY genes have been identified as important transcriptional modulators predominantly during the environmental stresses, but they also play critical role at various stages of plant life cycle. We report the identification of WRKY domain (WD)-encoding genes from galegoid clade legumes chickpea (Cicer arietinum L.) and barrel medic (Medicago truncatula). In total, 78 and 98 WD-encoding genes were found in chickpea and barrel medic, respectively. Comparative analysis suggests the presence of both conserved and unique WRKYs, and expansion of WRKY family in M. truncatula primarily by tandem duplication. Exclusively found in galegoid legumes, CaWRKY16 and its orthologues encode for a novel protein having a transmembrane and partial Exo70 domains flanking a group-III WD. Genomic region of galegoids, having CaWRKY16, is more dynamic when compared with millettioids. In onion cells, fused CaWRKY16-EYFP showed punctate fluorescent signals in cytoplasm. The chickpea WRKY group-III genes were further characterized for their transcript level modulation during pathogenic stress and treatments of abscisic acid, jasmonic acid, and salicylic acid (SA) by real-time PCR. Differential regulation of genes was observed during Ascochyta rabiei infection and SA treatment. Characterization of A. rabiei and SA inducible gene CaWRKY50 showed that it localizes to plant nucleus, binds to W-box, and have a C-terminal transactivation domain. Overexpression of CaWRKY50 in tobacco plants resulted in early flowering and senescence. The in-depth comparative account presented here for two legume WRKY genes will be of great utility in hastening functional characterization of crop legume WRKYs and will also help in characterization of Exo70Js.

  13. The nitrate reductase-encoding gene of Volvox carteri: map location, sequence and induction kinetics.

    PubMed

    Gruber, H; Goetinck, S D; Kirk, D L; Schmitt, R

    1992-10-12

    The nitrate reductase (NR) structural gene (nitA) of Volvox carteri has been cloned and characterized. There is a single copy of this gene in the genome, and RFLP (restriction-fragment length polymorphism) analysis assigns it to the previously defined nitA/chlR locus on linkage group IX, 20-30 cM from the two beta-tubulin-encoding loci. Determination of the 5871-nt sequence of the coding region of genomic clones, and comparisons to a cDNA sequence, revealed ten introns and eleven exons that encode a 864-aa polypeptide. Detailed comparisons with higher-plant and fungal NRs indicate that, whereas the aa sequence is strongly conserved within functional domains for the flavin adenine dinucleotide-, heme- and molybdenum-pterin cofactor-binding sites, substantial differences in the aa sequence occur in the N-terminal end and the two inter-domain regions. Two potential transcription start points 439 and 452 nt upstream from the start codon and a polyadenylation signal 355 nt downstream from the stop codon have been identified by primer-extension analysis and cDNA sequencing, respectively. Accumulation of the nitA transcript is both induced by nitrate and repressed by ammonium and urea: after the organism is transferred from ammonium to nitrate as the nitrogen source, a 3.6-kb NR transcript is readily detectable on Northern blots by 10 min, reaches maximum abundance by 30 min, and then rapidly declines to an intermediate level that is subsequently maintained. Substantial induction by nitrate is observed at the end of the dark portion of the daily light/dark cycle, but the inductive response peaks in the first hour of the light period.(ABSTRACT TRUNCATED AT 250 WORDS)

  14. The Schizosaccharomyces pombe cdc3+ gene encodes a profilin essential for cytokinesis

    PubMed Central

    1994-01-01

    The fission yeast Schizosaccharomyces pombe divides by medial fission and, like many higher eukaryotic cells, requires the function of an F- actin contractile ring for cytokinesis. In S. pombe, a class of cdc- mutants defective for cytokinesis, but not for DNA replication, mitosis, or septum synthesis, have been identified. In this paper, we present the characterization of one of these mutants, cdc3-124. Temperature shift experiments reveal that mutants in cdc3 are incapable of forming an F-actin contractile ring. We have molecularly cloned cdc3 and used the cdc3+ genomic DNA to create a strain carrying a cdc3 null mutation by homologous recombination in vivo. Cells bearing a cdc3-null allele are inviable. They arrest the cell cycle at cytokinesis without forming a contractile ring. DNA sequence analysis of the cdc3+ gene reveals that it encodes profilin, an actin-monomer-binding protein. In light of recent studies with profilins, we propose that Cdc3-profilin plays an essential role in cytokinesis by catalyzing the formation of the F-actin contractile ring. Consistent with this proposal are our observations that Cdc3-profilin localizes to the medial region of the cell where the F-actin contractile ring forms, and that it is essential for F-actin ring formation. Cells overproducing Cdc3-profilin become elongated, dumbbell shaped, and arrest at cytokinesis without any detectable F-actin staining. This effect of Cdc3-profilin overproduction is relieved by introduction of a multicopy plasmid carrying the actin encoding gene, act1+. We attribute these effects to potential sequestration of actin monomers by profilin, when present in excess. PMID:8207058

  15. The human homolog of the JE gene encodes a monocyte secretory protein.

    PubMed Central

    Rollins, B J; Stier, P; Ernst, T; Wong, G G

    1989-01-01

    The mouse fibroblast gene, JE, was one of the first platelet-derived growth factor-inducible genes to be described as such. The protein encoded by JE (mJE) is the prototype of a large family of secreted, cytokinelike glycoproteins, all of whose members are induced by a mitogenic or activation signal in monocytes macrophages, and T lymphocytes; JE is the only member to have been identified in fibroblasts. We report the identification of a human homolog for murine JE, cloned from human fibroblasts. The protein predicted by the coding sequence of human JE (hJE) is 55 amino acids shorter than mJE, and its sequence is identical to that of a recently purified monocyte chemoattractant. When expressed in COS cells, the human JE cDNA directed the secretion of N-glycosylated proteins of Mr 16,000 to 18,000 as well as proteins of Mr 15,500, 15,000, and 13,000. Antibodies raised against mJE recognized these hJE species, all of which were secreted by human fibroblasts. hJE expression was stimulated in HL60 cells during phorbol myristate acetate-induced monocytoid differentiation. However, resting human monocytes constitutively secreted hJE; treatment with gamma interferon did not enhance hJE expression in monocytes, and treatment with phorbol myristate acetate or lipopolysaccharide inhibited its expression. Thus, human JE encodes yet another member of the large family of JE-related cytokinelike proteins, in this case a novel human monocyte and fibroblast secretory protein. Images PMID:2513477

  16. Nicotinamide downregulates gene expression of interleukin-6, interleukin-10, monocyte chemoattractant protein-1, and tumour necrosis factor-α gene expression in HaCaT keratinocytes after ultraviolet B irradiation.

    PubMed

    Monfrecola, G; Gaudiello, F; Cirillo, T; Fabbrocini, G; Balato, A; Lembo, S

    2013-03-01

    Ultraviolet (UV) radiation has profound effects on human skin, causing sunburn, inflammation, cellular-tissue injury, cell death, and skin cancer. Most of these effects are mediated by a number of cytokines produced by keratinocytes. In this study we investigated whether nicotinamide (NCT), the amide form of vitamin B3, might have a protective function in reducing the expression of interleukin (IL)-1β, IL-6, IL-8, IL-10, monocyte chemoattractant protein (MCP)-1 and tumour necrosis factor (TNF)-α in UV-irradiated keratinocytes. HaCaT cells were treated with UVB in the presence or absence of NCT, and cytokine mRNA levels were examined by quantitative real-time PCR. NCT significantly downregulated IL-6, IL-10, MCP-1 and TNF-α mRNA expression, whereas it did not exert any significant effect on IL-1β or IL-8 expression. Because of its ability to decrease these cytokine mediators after UV exposure, NCT is a possible therapy to improve or prevent conditions induced or aggravated by UV light.

  17. Identification of Antithrombin-Modulating Genes. Role of LARGE, a Gene Encoding a Bifunctional Glycosyltransferase, in the Secretion of Proteins?

    PubMed Central

    de la Morena-Barrio, María Eugenia; Buil, Alfonso; Antón, Ana Isabel; Martínez-Martínez, Irene; Miñano, Antonia; Gutiérrez-Gallego, Ricardo; Navarro-Fernández, José; Aguila, Sonia; Souto, Juan Carlos; Vicente, Vicente; Soria, José Manuel; Corral, Javier

    2013-01-01

    The haemostatic relevance of antithrombin together with the low genetic variability of SERPINC1, and the high heritability of plasma levels encourage the search for modulating genes. We used a hypothesis-free approach to identify these genes, evaluating associations between plasma antithrombin and 307,984 polymorphisms in the GAIT study (352 individuals from 21 Spanish families). Despite no SNP reaching the genome wide significance threshold, we verified milder positive associations in 307 blood donors from a different cohort. This validation study suggested LARGE, a gene encoding a protein with xylosyltransferase and glucuronyltransferase activities that forms heparin-like linear polysaccharides, as a potential modulator of antithrombin based on the significant association of one SNPs, rs762057, with anti-FXa activity, particularly after adjustment for age, sex and SERPINC1 rs2227589 genotype, all factors influencing antithrombin levels (p = 0.02). Additional results sustained this association. LARGE silencing inHepG2 and HEK-EBNA cells did not affect SERPINC1 mRNA levels but significantly reduced the secretion of antithrombin with moderate intracellular retention. Milder effects were observed on α1-antitrypsin, prothrombin and transferrin. Our study suggests LARGE as the first known modifier of plasma antithrombin, and proposes a new role for LARGE in modulating extracellular secretion of certain glycoproteins. PMID:23705025

  18. The lysP gene encodes the lysine-specific permease.

    PubMed Central

    Steffes, C; Ellis, J; Wu, J; Rosen, B P

    1992-01-01

    Escherichia coli transports lysine by two distinct systems, one of which is specific for lysine (LysP) and the other of which is inhibited by arginine ornithine. The activity of the lysine-specific system increases with growth in acidic medium, anaerobiosis, and high concentrations of lysine. It is inhibited by the lysine analog S-(beta-aminoethyl)-L-cysteine (thiosine). Thiosine-resistant (Tsr) mutants were isolated by using transpositional mutagenesis with TnphoA. A Tsr mutant expressing alkaline phosphatase activity in intact cells was found to lack lysine-specific transport. This lysP mutation was mapped to about 46.5 min on the E. coli chromosome. The lysP-phoA fusion was cloned and used as a probe to clone the wild-type lysP gene. The nucleotide sequence of the 2.7-kb BamHI fragment was determined. An open reading frame from nucleotides 522 to 1989 was observed. The translation product of this open reading frame is predicted to be a hydrophobic protein of 489 residues. The lysP gene product exhibits sequence similarity to a family of amino acid transport proteins found in both prokaryotes and eukaryotes, including the aromatic amino acid permease of E. coli (aroP) and the arginine permease of Saccharomyces cerevisiae (CAN1). Cells carrying a plasmid with the lysP gene exhibited a 10- to 20-fold increase in the rate of lysine uptake above wild-type levels. These results demonstrate that the lysP gene encodes the lysine-specific permease. Images PMID:1315732

  19. UV radiation-induced immunosuppression is greater in men and prevented by topical nicotinamide.

    PubMed

    Damian, Diona L; Patterson, Clare R S; Stapelberg, Michael; Park, Joohong; Barnetson, Ross St C; Halliday, Gary M

    2008-02-01

    UV radiation-induced immunosuppression augments cutaneous carcinogenesis. The incidence of skin cancer continues to increase despite increased use of sunscreens, which are less effective at preventing immunosuppression than sunburn. Using the Mantoux reaction as a model of skin immunity, we investigated the effects of solar-simulated (ss) UV and its component UVA and UVB wavebands and tested the ability of topical nicotinamide to protect from UV-induced immunosuppression. Healthy, Mantoux-positive volunteers were UV-irradiated on their backs, with 5% nicotinamide or vehicle applied to different sites in a randomized, double-blinded manner. Subsequent Mantoux testing at irradiated and adjacent unirradiated sites enabled measurement of UV-induced immunosuppression with and without nicotinamide. Suberythemal ssUV caused significant immunosuppression, although component UVB and UVA doses delivered independently did not. Men were immunosuppressed by ssUV doses three times lower than those required to immunosuppress women. This may be an important cause of the higher skin cancer incidence and mortality observed in men. Topical nicotinamide prevented immunosuppression, with gene chip microarrays suggesting that the mechanisms of protection may include alterations in complement, energy metabolism and apoptosis pathways. Nicotinamide is a safe and inexpensive compound that could be added to sunscreens or after-sun lotions to improve protection from immunosuppression. immunosuppression.JID JOURNAL CLUB ARTICLE: For questions, answers, and open discussion about this article, please go to http://network.nature.com/group/jidclub

  20. A single cluster of coregulated genes encodes the biosynthesis of the mycotoxins roquefortine C and meleagrin in Penicillium chrysogenum.

    PubMed

    García-Estrada, Carlos; Ullán, Ricardo V; Albillos, Silvia M; Fernández-Bodega, María Ángeles; Durek, Pawel; von Döhren, Hans; Martín, Juan F

    2011-11-23

    A single gene cluster of Penicillium chrysogenum contains genes involved in the biosynthesis and secretion of the mycotoxins roquefortine C and meleagrin. Five of these genes have been silenced by RNAi. Pc21g15480 (rds) encodes a nonribosomal cyclodipeptide synthetase for the biosynthesis of both roquefortine C and meleagrin. Pc21g15430 (rpt) encodes a prenyltransferase also required for the biosynthesis of both mycotoxins. Silencing of Pc21g15460 or Pc21g15470 led to a decrease in roquefortine C and meleagrin, whereas silencing of the methyltransferase gene (Pc21g15440; gmt) resulted in accumulation of glandicolin B, indicating that this enzyme catalyzes the conversion of glandicolin B to meleagrin. All these genes are transcriptionally coregulated. Our results prove that roquefortine C and meleagrin derive from a single pathway.

  1. Cloning and expression of a bpr gene encoding Bacillopeptidase F from Bacillus amyloliquefaciens CH86-1.

    PubMed

    Kwon, Gun-Hee; Park, Jae-Yong; Kim, Jong-Sang; Lim, Jinkyu; Park, Cheon-Seok; Kwon, Dae Young; Kim, Jeong Hwan

    2011-05-01

    A gene encoding bacillopeptidase F, bpr86-1, was cloned from B. amyloliquefaciens CH86-1 isolated from cheonggukjang. This gene could encode a preproenzyme of 1,431 amino acids. When bpr86-1 was introduced into B. subtilis WB600 via pHY300PLK, an E. coli-Bacillus shuttle vector, the transformant showed fibrinolytic activity. During growth on LB, the fibrinolytic activity of cells increased sharply when they entered the stationary phase. The highest activity (761.4 mU/mg protein) was observed at 96 h of cultivation.

  2. Sequence similarities in the genes encoding polychlorinated biphenyl degradation by pseudomonas strain LB400 and alcaligenes eutrophus H850

    SciTech Connect

    Yates, J.R.; Mondello, F.J.

    1989-01-01

    DNA-DNA hybridization was used to compare the Pseudomonas strain LB400 genes for polychlorinated biphenyl (PCB) degradation with those from seven other PCB-degrading strains. Significant hybridization was detected to the genome of Alcaligenes eutrophus H850, a strain similar to LB400 in PCB-degrading capability. These two organisms showed a strong conservation of restriction sites in the region of DNA encoding PCB metabolism. No other sequence similarities were detected in the two genomes. DNA from the other PCB-degrading strains showed no hybridization to the probe, which demonstrated the existence of at least two distinct classes of genes encoding PCB degradation.

  3. Systematic Global Analysis of Genes Encoding Protein Phosphatases in Aspergillus fumigatus

    PubMed Central

    Winkelströter, Lizziane K.; Dolan, Stephen K.; Fernanda dos Reis, Thaila; Bom, Vinícius Leite Pedro; Alves de Castro, Patrícia; Hagiwara, Daisuke; Alowni, Raneem; Jones, Gary W.; Doyle, Sean; Brown, Neil Andrew; Goldman, Gustavo H.

    2015-01-01

    Aspergillus fumigatus is a fungal pathogen that causes several invasive and noninvasive diseases named aspergillosis. This disease is generally regarded as multifactorial, considering that several pathogenicity determinants are present during the establishment of this illness. It is necessary to obtain an increased knowledge of how, and which, A. fumigatus signal transduction pathways are engaged in the regulation of these processes. Protein phosphatases are essential to several signal transduction pathways. We identified 32 phosphatase catalytic subunit-encoding genes in A. fumigatus, of which we were able to construct 24 viable deletion mutants. The role of nine phosphatase mutants in the HOG (high osmolarity glycerol response) pathway was evaluated by measuring phosphorylation of the p38 MAPK (SakA) and expression of osmo-dependent genes. We were also able to identify 11 phosphatases involved in iron assimilation, six that are related to gliotoxin resistance, and three implicated in gliotoxin production. These results present the creation of a fundamental resource for the study of signaling in A. fumigatus and its implications in the regulation of pathogenicity determinants and virulence in this important pathogen. PMID:25943523

  4. Glyceraldehyde-3-Phosphate Dehydrogenase-Encoding Gene as a Useful Taxonomic Tool for Staphylococcus spp.

    PubMed Central

    Yugueros, Javier; Temprano, Alejandro; Berzal, Beatriz; Sánchez, María; Hernanz, Carmen; Luengo, José María; Naharro, Germán

    2000-01-01

    The gap gene of Staphylococcus aureus, encoding glyceraldehyde-3-phosphate dehydrogenase, was used as a target to amplify a 933-bp DNA fragment by PCR with a pair of primers 26 and 25 nucleotides in length. PCR products, detected by agarose gel electrophoresis, were also amplified from 12 Staphylococcus spp. analyzed previously. Hybridization with an internal 279-bp DNA fragment probe was positive in all PCR-positive samples. No PCR products were amplified when other gram-positive and gram-negative bacterial genera were analyzed using the same pair of primers. AluI digestion of PCR-generated products gave 12 different restriction fragment length polymorphism (RFLP) patterns, one for each species analyzed. However, we could detect two intraspecies RFLP patterns in Staphylococcus epidermidis, Staphylococcus hominis, and Staphylococcus simulans which were different from the other species. An identical RFLP pattern was observed for 112 S. aureus isolates from humans, cows, and sheep. The sensitivity of the PCR assays was very high, with a detection limit for S. aureus cells of 20 CFU when cells were suspended in saline. PCR amplification of the gap gene has the potential for rapid identification of at least 12 species belonging to the genus Staphylococcus, as it is highly specific. PMID:11101563

  5. Glyceraldehyde-3-phosphate dehydrogenase-encoding gene as a useful taxonomic tool for Staphylococcus spp.

    PubMed

    Yugueros, J; Temprano, A; Berzal, B; Sánchez, M; Hernanz, C; Luengo, J M; Naharro, G

    2000-12-01

    The gap gene of Staphylococcus aureus, encoding glyceraldehyde-3-phosphate dehydrogenase, was used as a target to amplify a 933-bp DNA fragment by PCR with a pair of primers 26 and 25 nucleotides in length. PCR products, detected by agarose gel electrophoresis, were also amplified from 12 Staphylococcus spp. analyzed previously. Hybridization with an internal 279-bp DNA fragment probe was positive in all PCR-positive samples. No PCR products were amplified when other gram-positive and gram-negative bacterial genera were analyzed using the same pair of primers. AluI digestion of PCR-generated products gave 12 different restriction fragment length polymorphism (RFLP) patterns, one for each species analyzed. However, we could detect two intraspecies RFLP patterns in Staphylococcus epidermidis, Staphylococcus hominis, and Staphylococcus simulans which were different from the other species. An identical RFLP pattern was observed for 112 S. aureus isolates from humans, cows, and sheep. The sensitivity of the PCR assays was very high, with a detection limit for S. aureus cells of 20 CFU when cells were suspended in saline. PCR amplification of the gap gene has the potential for rapid identification of at least 12 species belonging to the genus Staphylococcus, as it is highly specific.

  6. Structure and chromosomal localization of the genomic locus encoding the Kiz1 LIM-kinase gene

    SciTech Connect

    Bernard, O.; Burkitt, V.; Webb, G.C.

    1996-08-01

    We have cloned and characterized the mouse gene encoding Kiz1/Limk1, a new member of the zinc-finger LIM family that also has a kinase domain. The gene encompasses 25 kb of the mouse genome, and the organization of its 16 exons does not correlate with its functional domains. The promoter region of Kiz1/Limk1 was identified by cloning a 1.06-kb genomic fragment upstream from the first ATG in a promotorless CAT vector. This construct was demonstrated to drive CAT expression in Jurkat cells. The promoter sequence lacks conventional TATA and CAAT motifs but contains consensus binding sequences for several transcriptional regulators implicated in control of transcription in many different cell types, including Sp1, Ets, and E2A. Analysis of the chromosomal localization of KIZ1/LIMK1 indicates that it lies on human chromosome 17 in the region 17q25 and on mouse Chromosome 5, band G2. 15 refs., 3 figs., 1 tab.

  7. The lumazine protein-encoding gene in Photobacterium leiognathi is linked to the lux operon.

    PubMed

    Lin, J W; Chao, Y F; Weng, S F

    1993-04-15

    The nucleotide (nt) sequence of the lumP (EMBL accession No. X65612) gene of Photobacterium leiognathi PL741 was determined and the amino acid (aa) sequence deduced. The encoded aa sequence of lumP was identified as that of the lumazine protein (LumP) by homology with that of Photobacterium phosphoreum (56%). This small protein has a calculated M(r) of 19,997 and comprises 186 aa residues. Biochemical studies suggested that LumP is the protein which, when combined with luciferase, is responsible for the bioluminescent spectrum shift from blue-green light (490-505 nm) to blue (470 nm) in P. leiognathi. The nt sequence of the flanking region showed that lumP is linked to the lux operon but runs in the opposite direction. The gene order of the lumP and lux operon is as follows: <--lumP-R&R-luxC-luxD-luxA-luxB-luxN-lu xE-->; the R&R regulatory region sequence included two promoter systems, PR for the lux operon and PL for the lumP or the lum operon.

  8. Toxic peptides and genes encoding toxin gamma of the Brazilian scorpions Tityus bahiensis and Tityus stigmurus.

    PubMed

    Becerril, B; Corona, M; Coronas, F I; Zamudio, F; Calderon-Aranda, E S; Fletcher, P L; Martin, B M; Possani, L D

    1996-02-01

    Seven toxic peptides from the venom of Tityus bahiensis and Tityus stigmurus was isolated and sequenced, five of them to completion. The most abundant peptide from each of these two species of scorpion was 95% identical with that of toxin gamma from the venom of Tityus serrulatus. They were consequently named gamma-b and gamma-st respectively. The genes encoding these new gamma-like peptides were cloned and sequenced by utilizing oligonucleotides synthesized according to known cDNA sequences of toxin gamma, and amplified by PCR on templates of DNA purified from both T. bahiensis and T. stigmurus. They contain an intron of approx. 470 bp. Possible mechanisms of processing and expressing these peptides are discussed, in view of the fact that glycine is the first residue of the N-terminal sequence of T. stigmurus, whereas lysine is the residue at position 1 of toxin gamma from T. serrulatus and T. bahiensis. In addition, chemical characterization of the less abundant toxic peptides showed the presence of at least four distinct families of peptides in all three species of the genus Tityus studied. There is a large degree of similarity among peptides from different venoms of the same family. By using specific horse and rabbit antisera, the venoms of T. bahiensis, T. serrulatus and T. stigmurus were compared. They showed an extended degree of cross-reactivity. Thus these three species of scorpion have similar toxic components, the genes of which are similarly organized, processed and expressed.

  9. Toxic peptides and genes encoding toxin gamma of the Brazilian scorpions Tityus bahiensis and Tityus stigmurus.

    PubMed Central

    Becerril, B; Corona, M; Coronas, F I; Zamudio, F; Calderon-Aranda, E S; Fletcher, P L; Martin, B M; Possani, L D

    1996-01-01

    Seven toxic peptides from the venom of Tityus bahiensis and Tityus stigmurus was isolated and sequenced, five of them to completion. The most abundant peptide from each of these two species of scorpion was 95% identical with that of toxin gamma from the venom of Tityus serrulatus. They were consequently named gamma-b and gamma-st respectively. The genes encoding these new gamma-like peptides were cloned and sequenced by utilizing oligonucleotides synthesized according to known cDNA sequences of toxin gamma, and amplified by PCR on templates of DNA purified from both T. bahiensis and T. stigmurus. They contain an intron of approx. 470 bp. Possible mechanisms of processing and expressing these peptides are discussed, in view of the fact that glycine is the first residue of the N-terminal sequence of T. stigmurus, whereas lysine is the residue at position 1 of toxin gamma from T. serrulatus and T. bahiensis. In addition, chemical characterization of the less abundant toxic peptides showed the presence of at least four distinct families of peptides in all three species of the genus Tityus studied. There is a large degree of similarity among peptides from different venoms of the same family. By using specific horse and rabbit antisera, the venoms of T. bahiensis, T. serrulatus and T. stigmurus were compared. They showed an extended degree of cross-reactivity. Thus these three species of scorpion have similar toxic components, the genes of which are similarly organized, processed and expressed. PMID:8611151

  10. Molecular cloning and analysis of the gene encoding the thermostable penicillin G acylase from Alcaligenes faecalis.

    PubMed Central

    Verhaert, R M; Riemens, A M; van der Laan, J M; van Duin, J; Quax, W J

    1997-01-01

    Alcaligenes faecalis penicillin G acylase is more stable than the Escherichia coli enzyme. The activity of the A. faecalis enzyme was not affected by incubation at 50 degrees C for 20 min, whereas more than 50% of the E. coli enzyme was irreversibly inactivated by the same treatment. To study the molecular basis of this higher stability, the A. faecalis enzyme was isolated and its gene was cloned and sequenced. The gene encodes a polypeptide that is characteristic of periplasmic penicillin G acylase (signal peptide-alpha subunit-spacer-beta subunit). Purification, N-terminal amino acid analysis, and molecular mass determination of the penicillin G acylase showed that the alpha and beta subunits have molecular masses of 23.0 and 62.7 kDa, respectively. The length of the spacer is 37 amino acids. Amino acid sequence alignment demonstrated significant homology with the penicillin G acylase from E. coli A unique feature of the A. faecalis enzyme is the presence of two cysteines that form a disulfide bridge. The stability of the A. faecalis penicillin G acylase, but not that of the E. coli enzyme, which has no cysteines, was decreased by a reductant. Thus, the improved thermostability is attributed to the presence of the disulfide bridge. PMID:9292993

  11. Genes encoding norcoclaurine synthase occur as tandem fusions in the Papaveraceae

    PubMed Central

    Li, Jing; Lee, Eun-Jeong; Chang, Limei; Facchini, Peter J.

    2016-01-01

    Norcoclaurine synthase (NCS) catalyzes the enantioselective Pictet-Spengler condensation of dopamine and 4-hydroxyphenylacetaldehyde as the first step in benzylisoquinoline alkaloid (BIA) biosynthesis. NCS orthologs in available transcriptome databases were screened for variants that might improve the low yield of BIAs in engineered microorganisms. Databases for 21 BIA-producing species from four plant families yielded 33 assembled contigs with homology to characterized NCS genes. Predicted translation products generated from nine contigs consisted of two to five sequential repeats, each containing most of the sequence found in single-domain enzymes. Assembled contigs containing tandem domain repeats were detected only in members of the Papaveraceae family, including opium poppy (Papaver somniferum). Fourteen cDNAs were generated from 10 species, five of which encoded NCS orthologs with repeated domains. Functional analysis of corresponding recombinant proteins yielded six active NCS enzymes, including four containing either two, three or four repeated catalytic domains. Truncation of the first 25 N-terminal amino acids from the remaining polypeptides revealed two additional enzymes. Multiple catalytic domains correlated with a proportional increase in catalytic efficiency. Expression of NCS genes in Saccharomyces cereviseae also produced active enzymes. The metabolic conversion capacity of engineered yeast positively correlated with the number of repeated domains. PMID:27991536

  12. Genes encoding norcoclaurine synthase occur as tandem fusions in the Papaveraceae.

    PubMed

    Li, Jing; Lee, Eun-Jeong; Chang, Limei; Facchini, Peter J

    2016-12-19

    Norcoclaurine synthase (NCS) catalyzes the enantioselective Pictet-Spengler condensation of dopamine and 4-hydroxyphenylacetaldehyde as the first step in benzylisoquinoline alkaloid (BIA) biosynthesis. NCS orthologs in available transcriptome databases were screened for variants that might improve the low yield of BIAs in engineered microorganisms. Databases for 21 BIA-producing species from four plant families yielded 33 assembled contigs with homology to characterized NCS genes. Predicted translation products generated from nine contigs consisted of two to five sequential repeats, each containing most of the sequence found in single-domain enzymes. Assembled contigs containing tandem domain repeats were detected only in members of the Papaveraceae family, including opium poppy (Papaver somniferum). Fourteen cDNAs were generated from 10 species, five of which encoded NCS orthologs with repeated domains. Functional analysis of corresponding recombinant proteins yielded six active NCS enzymes, including four containing either two, three or four repeated catalytic domains. Truncation of the first 25 N-terminal amino acids from the remaining polypeptides revealed two additional enzymes. Multiple catalytic domains correlated with a proportional increase in catalytic efficiency. Expression of NCS genes in Saccharomyces cereviseae also produced active enzymes. The metabolic conversion capacity of engineered yeast positively correlated with the number of repeated domains.

  13. AtGDI2, a novel Arabidopsis gene encoding a Rab GDP dissociation inhibitor.

    PubMed

    Ueda, T; Yoshizumi, T; Anai, T; Matsui, M; Uchimiya, H; Nakano, A

    1998-01-05

    The GTPase cycle of Rab/Ypt proteins is strictly controlled by several classes of regulators to ensure their proper roles in membrane traffic. GDP dissociation inhibitor (GDI) is known to play essential roles in regulating nucleotide states and subcellular localizations of Rab/Ypt proteins. To obtain further knowledge on this regulator molecule in plants, we isolated and characterized two genes of Arabidopsis thaliana that encode different GDIs. AtGDI1 has been identified by a novel functional cloning in yeast [Ueda et al. (1996) Plant Cell, 8, 2079-2091] and AtGDI2 was isolated by cross-hybridization in this study. AtGDI2, as well as AtGDI1, complements the yeast sec19/gdi1 mutant, indicating that they can replace the function of yeast GDI. Evidence is shown that both AtGDI1 and AtGDI2 can interact with Ara4, an Arabidopsis Rab protein, in the yeast ypt1 mutant cells. AtGDI2 is ubiquitously expressed in Arabidopsis tissues with some difference from AtGDI1 in expression level. Genomic DNA hybridization using specific probes reveals the presence of one more GDI gene in Arabidopsis. This may imply differentiated roles of GDI in higher plants.

  14. The yeast VAS1 gene encodes both mitochondrial and cytoplasmic valyl-tRNA synthetases.

    PubMed

    Chatton, B; Walter, P; Ebel, J P; Lacroute, F; Fasiolo, F

    1988-01-05

    S1 mapping on the VAS1 structural gene indicates the existence of two classes of transcripts initiating at distinct in-frame translation start codons. The longer class of VAS1 transcripts initiates upstream of both ATG codons located 138 base pairs away and the shorter class downstream of the first ATG. A mutation that destroys the first AUG on the long message results in respiratory deficiency but does not affect viability. Mutation of the ATG at position 139 leads to lethality because the initiating methionine codon of the essential cytoplasmic valyl-tRNA synthetase has been destroyed. N-terminal protein sequence data further confirm translation initiation at ATG-139 for the cytoplasmic valyl-tRNA synthetase. From these results, we conclude that the VAS1 single gene encodes both mitochondrial and cytoplasmic valyl-tRNA synthetases. The presequence of the mitochondrial valyl-tRNA synthetase shows amino acid composition but not the amphiphilic character of imported mitochondrial proteins. From mutagenesis of the ATG-139 we conclude that the presequence specifically targets the cytoplasmically synthesized mitochondrial valyl-tRNA synthetase to the mitochondrial outer membrane and prevents binding of the enzyme core to cytoplasmic tRNAVal.

  15. Identification and isolation of three proteasome subunits and their encoding genes from Trypanosoma brucei.

    PubMed

    Huang, L; Shen, M; Chernushevich, I; Burlingame, A L; Wang, C C; Robertson, C D

    1999-08-20

    We have determined peptide sequences of three Trypanosoma brucei proteasome subunit proteins by mass spectrometry of tryptic digests of the proteins purified by two-dimensional (2-D) polyacrylamide gel electrophoresis. Three genes identified by the sequence of their cDNA encode the peptides identified in these three proteins. The three proteins predicted from the gene sequences have significant similarity to other known proteasome subunits and represent an alpha6 type subunit (TbPSA6), and two beta-type subunits belonging to the beta1-type (TbPSB1) and beta2 type (TbPSB2). The sequences of both beta-subunits predict formation of catalytically active subunits through proteolytic processing. The prediction is supported by the presence in each of the two beta-subunits of a tryptic peptide that has the correctly processed N-terminus that creates the threonine nucleophile of the mature protein. This peptide cannot be generated by trypsin because of the required cleavage of a glycine-threonine bond. It is thus likely that there are at least two catalytically active beta-subunits, TbPSB1 and TbPSB2, present in the mature 20S proteasome from T. brucei.

  16. High nucleosome occupancy is encoded at X-linked gene promoters in C. elegans

    PubMed Central

    Ercan, Sevinç; Lubling, Yaniv; Segal, Eran; Lieb, Jason D.

    2011-01-01

    We mapped nucleosome occupancy by paired-end Illumina sequencing in C. elegans embryonic cells, adult somatic cells, and a mix of adult somatic and germ cells. In all three samples, the nucleosome occupancy of gene promoters on the X chromosome differed from autosomal promoters. While both X and autosomal promoters exhibit a typical nucleosome-depleted region upstream of transcript start sites and a well-positioned +1 nucleosome, X-linked gene promoters on average exhibit higher nucleosome occupancy relative to autosomal promoters. We show that the difference between X and autosomes does not depend on the somatic dosage compensation machinery. Instead, the chromatin difference at promoters is partly encoded by DNA sequence, because a model trained on nucleosome sequence preferences from S. cerevisiae in vitro data recapitulate nearly completely the experimentally observed difference between X and autosomal promoters. The model predictions also correlate very well with experimentally determined occupancy values genome-wide. The nucleosome occupancy differences observed on X promoters may bear on mechanisms of X chromosome dosage compensation in the soma, and chromosome-wide repression of X in the germline. PMID:21177966

  17. Differential expression of nuclear- and organelle-encoded genes during tomato fruit development.

    PubMed

    Piechulla, B

    1988-12-01

    Steady-state mRNA levels of nuclear-and organelle-encoded genes were determined during fruit development and ripening. Transcripts specific for subunits of the mitochondrial and chloroplast ATPase complexes appear simultaneously and reach high levels two to three weeks after anthesis, but follow a different expression pattern during the ripening period. While the chloroplast-specific mRNA levels continuously decrease to low levels in ripe tomato fruits, the transcripts specific for two mitochondrial ATPase subunits continue to be present at relative high levels in red fruits. Transcript levels for the fructose-1,6-bisphosphate aldolase increase significantly during ripening. Structural proteins such as the alpha-subunit of tubulin and the hydroxyproline-rich glycoprotein extensin are expressed during maximal fruit growth. In addition, comparisons of mRNA levels of different genes in several plant organs (leaf, fruit, stem, and root) show characteristic differences. The results presented in this paper demonstrate that changes at the transcriptional or post-transcriptional level during fruit development can be correlated with morphological and physiological alterations.

  18. Identification and characterization of the genes encoding a unique surface (S-) layer of Tannerella forsythia.

    PubMed

    Lee, Seok-Woo; Sabet, Mojgan; Um, Heung-Sik; Yang, Jun; Kim, Hyeong C; Zhu, Weidong

    2006-04-12

    A newly emerged periodontopathic pathogen Tannerella forsythia (formerly Bacteroides forsythus), a Gram-negative, filament-shaped, strict anaerobic, non-pigmented oral bacterium, possesses a surface (S-) layer. In our previous studies, the S-layer has been isolated, and shown to mediate hemagglutination, adhesion/invasion of epithelial cell, and murine subcutaneous abscess formation. In the present study, biochemical and molecular genetic characterization of the S-layer are reported. Amino acid sequencing and mass spectrometry indicated that the S-layer is composed of two different proteins, termed 200 and 210 kDa proteins. It was also shown that these proteins are glycosylated. The genes encoding the core proteins of these glycoproteins, designated as tfsA and tfsB, have been identified in silico, cloned, and their sequences have been determined. The tfsA (3.5 kb) and tfsB (4.1 kb) genes are located in tandem, and encode for 135 and 152 kDa proteins, respectively. An apparent discrepancy in molecular weights, 135 vs. 200 kDa and 152 vs. 210 kDa, is accounted for carbohydrate residues attached to the core proteins. Amino acid sequence comparison exhibited a 24% similarity between the 200 and 210 kDa proteins. Further sequence analyses showed that TfsA and TfsB possess putative signal peptide sequences with cleavage sites at alanine residues, and transmembrane domains on the C-terminal region. Northern blot and RT-PCR analyses confirmed an operon structure of tfsAB, suggesting co-regulation of these genes in producing the S-layer. Putative promoter sequences and transcription termination sequences for this operon have also been identified. Comparison with database indicates that the S-layer of T. forsythia has a unique structure exhibiting no homology to other known S-layers of prokaryotic organisms. The present study shows that the T. forsythia S-layer is very unique, since it appears to be composed of two large glycoproteins, and it does not reveal any homology to

  19. 21 CFR 172.315 - Nicotinamide-ascorbic acid complex.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Nicotinamide-ascorbic acid complex. 172.315... HUMAN CONSUMPTION Special Dietary and Nutritional Additives § 172.315 Nicotinamide-ascorbic acid complex. Nicotinamide-ascorbic acid complex may be safely used in accordance with the following prescribed...

  20. 21 CFR 172.315 - Nicotinamide-ascorbic acid complex.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Nicotinamide-ascorbic acid complex. 172.315... HUMAN CONSUMPTION Special Dietary and Nutritional Additives § 172.315 Nicotinamide-ascorbic acid complex. Nicotinamide-ascorbic acid complex may be safely used in accordance with the following prescribed...

  1. 21 CFR 172.315 - Nicotinamide-ascorbic acid complex.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Nicotinamide-ascorbic acid complex. 172.315 Section... HUMAN CONSUMPTION Special Dietary and Nutritional Additives § 172.315 Nicotinamide-ascorbic acid complex. Nicotinamide-ascorbic acid complex may be safely used in accordance with the following prescribed...

  2. Macrolide resistance gene mreA of Streptococcus agalactiae encodes a flavokinase.

    PubMed

    Clarebout, G; Villers, C; Leclercq, R

    2001-08-01

    The mreA gene from Streptococcus agalactiae COH31 gamma/delta, resistant to macrolides and clindamycin by active efflux, has recently been cloned in Escherichia coli, where it was reported to confer macrolide resistance (J. Clancy, F. Dib-Hajj, J. W. Petitpas, and W. Yuan, Antimicrob. Agents Chemother. 41:2719--2723, 1997). Cumulative data suggested that the mreA gene was located on the chromosome of S. agalactiae COH31 gamma/delta. Analysis of the deduced amino acid sequence of mreA revealed significant homology with several bifunctional flavokinases/(flavin adenine dinucleotide (FAD) synthetases, which convert riboflavin to flavin mononucleotide (FMN) and FMN to FAD, respectively. High-performance liquid chromatography experiments showed that the mreA gene product had a monofunctional flavokinase activity, similar to that of RibR from Bacillus subtilis. Sequences identical to those of the mreA gene and of a 121-bp upstream region containing a putative promoter were detected in strains of S. agalactiae UCN4, UCN5, and UCN6 susceptible to macrolides. mreA and its allele from S. agalactiae UCN4 were cloned on the shuttle vector pAT28. Both constructs were introduced into E. coli, where they conferred a similar two- to fourfold increase in the MICs of erythromycin, spiramycin, and clindamycin. The MICs of a variety of other molecules, including crystal violet, acriflavin, sodium dodecyl sulfate, and antibiotics, such as certain cephalosporins, chloramphenicol, doxycycline, nalidixic acid, novobiocin, and rifampin, were also increased. In contrast, resistance to these compounds was not detected when the constructs were introduced into E. faecalis JH2-2. In conclusion, the mreA gene was probably resident in S. agalactiae and may encode a metabolic function. We could not provide any evidence that it was responsible for macrolide resistance in S. agalactiae COH31 gamma/delta; broad-spectrum resistance conferred by the gene in E. coli could involve multidrug efflux pumps

  3. Sequence and genetic organization of a Zymomonas mobilis gene cluster that encodes several enzymes of glucose metabolism

    SciTech Connect

    Barnell, W.O.; Kyung Cheol Yi; Conway, T. )

    1990-12-01

    The Zymomonas mobilis genes that encode glucose-6-phosphate dehydrogenase (zwf), 6-phosphogluconate dehydratase (edd), and glucokinase (glk) were cloned independently by genetic complementation of specific defects in Escherichia coli metabolism. The identify of these cloned genes was confirmed by various biochemical means. Nucleotide sequence analysis established that these three genes are clustered on the genome and revealed an additional open reading frame in this region that has significant amino acid identity to the E.coli xylose-proton symporter and the human glucose transporter. On the basis of this evidence and structural analysis of the deduced primary amino acid sequence, this gene is believed to encode the Z. mobilis glucose-facilitated diffusion protein, glf. The four genes in the 6-kb cluster are organized in the order glf, zwf, edd, glk. The glf and zwf genes are separated by 146 bp. The zwf and edd genes overlap by 8 bp, and their expression may be translationally coupled. The edd and glk genes are separated by 203 bp. The glk gene is followed by tandem transcriptional terminators. The four genes appear to be organized in an operon. Such an arrangement of the genes that govern glucose uptake and the first three steps of the Entner-Doudoroff glycolytic pathway provides the organism with a mechanism for carefully regulating the levels of the enzymes that control carbon flux into the pathway.

  4. Transcriptional analysis and functional characterization of XCC1294 gene encoding a GGDEF domain protein in Xanthomonas campestris pv. campestris.

    PubMed

    Hsiao, Yi-Min; Song, Wan-Ling; Liao, Chao-Tsai; Lin, I-Hsuan; Pan, Mei-Ying; Lin, Ching-Fen

    2012-04-01

    The nucleotide cyclic di-GMP is a second messenger in bacteria that regulates a range of cellular functions including the virulence of pathogens. GGDEF is a protein domain involved in the synthesis of cyclic di-GMP. The genome of the crucifer pathogen Xanthomonas campestris pv. campestris (Xcc) encodes 21 proteins with a GGDEF domain. Clp, a homolog of the model transcription factor Crp of Escherichia coli, is a global regulator in Xcc. The aim of this study is to identify genes encoding GGDEF domain proteins whose expression is regulated by Clp. Results of reporter assay and RT-PCR analysis suggested that Clp regulates the expression of a set of genes encoding proteins harboring GGDEF domain. The transcription initiation site of XCC1294, one of the Clp regulated gene encoding a GGDEF domain protein, was mapped. Promoter analysis and gel retardation assay indicated that the transcription of XCC1294 is positively and directly regulated by Clp. Furthermore, transcription of XCC1294 was subject to catabolite repression and affected by several stress conditions. We also showed that mutation of XCC1294 results in enhanced surface attachment. In addition, transcription of three putative adhesin genes (xadA, fhaC, and yapH) was increased in the XCC1294 mutant. Taken together, the data presented here indicate that Clp positively regulates expression of XCC1294, and that XCC1294 serves a regulator of bacterial attachment and regulates different adhesin genes expression.

  5. Differential expression of genes encoding neuronal ion-channel subunits in major depression, bipolar disorder and schizophrenia: implications for pathophysiology.

    PubMed

    Smolin, Bella; Karry, Rachel; Gal-Ben-Ari, Shunit; Ben-Shachar, Dorit

    2012-08-01

    Evidence concerning ion-channel abnormalities in the pathophysiology of common psychiatric disorders is still limited. Given the significance of ion channels in neuronal activity, neurotransmission and neuronal plasticity we hypothesized that the expression patterns of genes encoding different ion channels may be altered in schizophrenia, bipolar and unipolar disorders. Frozen samples of striatum including the nucleus accumbens (Str-NAc) and the lateral cerebellar hemisphere of 60 brains from depressed (MDD), bipolar (BD), schizophrenic and normal subjects, obtained from the Stanley Foundation Brain Collection, were assayed. mRNA of 72 different ion-channel subunits were determined by qRT-PCR and alteration in four genes were verified by immunoblotting. In the Str-NAc the prominent change was observed in the MDD group, in which there was a significant up-regulation in genes encoding voltage-gated potassium-channel subunits. However, in the lateral cerebellar hemisphere (cerebellum), the main change was observed in schizophrenia specimens, as multiple genes encoding various ion-channel subunits were significantly down-regulated. The impaired expression of genes encoding ion channels demonstrates a disease-related neuroanatomical pattern. The alterations observed in Str-NAc of MDD may imply electrical hypo-activity of this region that could be of relevance to MDD symptoms and treatment. The robust unidirectional alteration of both excitatory and inhibitory ion channels in the cerebellum may suggests cerebellar general hypo-transcriptional activity in schizophrenia.

  6. Genes encoding Δ(8)-sphingolipid desaturase from various plants: identification, biochemical functions, and evolution.

    PubMed

    Li, Shu-Fen; Zhang, Guo-Jun; Zhang, Xue-Jin; Yuan, Jin-Hong; Deng, Chuan-Liang; Hu, Zan-Min; Gao, Wu-Jun

    2016-09-01

    ∆(8)-sphingolipid desaturase catalyzes the C8 desaturation of a long chain base, which is the characteristic structure of various complex sphingolipids. The genes of 20 ∆(8)-sphingolipid desaturases from 12 plants were identified and functionally detected by using Saccharomyces cerevisiae system to elucidate the relationship between the biochemical function and evolution of this enzyme. Results showed that the 20 genes all can encode a functional ∆(8)-sphingolipid desaturase, which catalyzes different ratios of two products, namely, 8(Z) and 8(E)-C18-phytosphingenine. The coded enzymes could be divided into two groups on the basis of biochemical functions: ∆(8)-sphingolipid desaturase with a preference for an E-isomer product and ∆(8)-sphingolipid desaturase with a preference for a Z-isomer product. The conversion rate of the latter was generally lower than that of the former. Phylogenetic analysis revealed that the 20 desaturases could also be clustered into two groups, and this grouping is consistent with that of the biochemical functions. Thus, the biochemical function of ∆(8)-sphingolipid desaturase is correlated with its evolution. The two groups of ∆(8)-sphingolipid desaturases could arise from distinct ancestors in higher plants. However, they might have initially evolved from ∆(8)-sphingolipid desaturases in lower organisms, such as yeasts, which can produce E-isomer products only. Furthermore, almost all of the transgenic yeasts harboring ∆(8)-sphingolipid desaturase genes exhibit an improvement in aluminum tolerance. Our study provided new insights into the biochemical function and evolution of ∆(8)-sphingolipid desaturases in plants.

  7. Characterization of a Thioredoxin-1 Gene from Taenia solium and Its Encoding Product.

    PubMed

    Jiménez, Lucía; Rodríguez-Lima, Oscar; Ochoa-Sánchez, Alicia; Landa, Abraham

    2015-01-01

    Taenia solium thioredoxin-1 gene (TsTrx-1) has a length of 771 bp with three exons and two introns. The core promoter gene presents two putative stress transcription factor binding sites, one putative TATA box, and a transcription start site (TSS). TsTrx-1 mRNA is expressed higher in larvae than in adult. This gene encodes a protein of 107 amino acids that presents the Trx active site (CGPC), the classical secondary structure of the thioredoxin fold, and the highest degree of identity with the Echinococcus granulosus Trx. A recombinant TsTrx-1 (rTsTrx-1) was produced in Escherichia coli with redox activity. Optimal activity for rTsTrx-1 was at pH 6.5 in the range of 15 to 25°C. The enzyme conserved activity for 3 h and lost it in 24 h at 37°C. rTsTrx-1 lost 50% activity after 1 h and lost activity completely in 24 h at temperatures higher than 55°C. Best storage temperature for rTsTrx-1 was at -70°C. It was inhibited by high concentrations of H₂O₂ and methylglyoxal (MG), but it was inhibited neither by NaCl nor by anti-rTsTrx-1 rabbit antibodies that strongly recognized a ~12 kDa band in extracts from several parasites. These TsTrx-1 properties open the opportunity to study its role in relationship T. solium-hosts.

  8. Characterization of a Thioredoxin-1 Gene from Taenia solium and Its Encoding Product

    PubMed Central

    Jiménez, Lucía; Rodríguez-Lima, Oscar; Ochoa-Sánchez, Alicia; Landa, Abraham

    2015-01-01

    Taenia solium thioredoxin-1 gene (TsTrx-1) has a length of 771 bp with three exons and two introns. The core promoter gene presents two putative stress transcription factor binding sites, one putative TATA box, and a transcription start site (TSS). TsTrx-1 mRNA is expressed higher in larvae than in adult. This gene encodes a protein of 107 amino acids that presents the Trx active site (CGPC), the classical secondary structure of the thioredoxin fold, and the highest degree of identity with the Echinococcus granulosus Trx. A recombinant TsTrx-1 (rTsTrx-1) was produced in Escherichia coli with redox activity. Optimal activity for rTsTrx-1 was at pH 6.5 in the range of 15 to 25°C. The enzyme conserved activity for 3 h and lost it in 24 h at 37°C. rTsTrx-1 lost 50% activity after 1 h and lost activity completely in 24 h at temperatures higher than 55°C. Best storage temperature for rTsTrx-1 was at −70°C. It was inhibited by high concentrations of H2O2 and methylglyoxal (MG), but it was inhibited neither by NaCl nor by anti-rTsTrx-1 rabbit antibodies that strongly recognized a ~12 kDa band in extracts from several parasites. These TsTrx-1 properties open the opportunity to study its role in relationship T. solium-hosts. PMID:26090410

  9. Dioxygenase-encoding AtDAO1 gene controls IAA oxidation and homeostasis in Arabidopsis

    PubMed Central

    Porco, Silvana; Pěnčík, Aleš; Rashed, Afaf; Voß, Ute; Casanova-Sáez, Rubén; Bishopp, Anthony; Golebiowska, Agata; Swarup, Ranjan; Swarup, Kamal; Peňáková, Pavlína; Novák, Ondřej; Staswick, Paul; Hedden, Peter; Phillips, Andrew L.; Vissenberg, Kris

    2016-01-01

    Auxin represents a key signal in plants, regulating almost every aspect of their growth and development. Major breakthroughs have been made dissecting the molecular basis of auxin transport, perception, and response. In contrast, how plants control the metabolism and homeostasis of the major form of auxin in plants, indole-3-acetic acid (IAA), remains unclear. In this paper, we initially describe the function of the Arabidopsis thaliana gene DIOXYGENASE FOR AUXIN OXIDATION 1 (AtDAO1). Transcriptional and translational reporter lines revealed that AtDAO1 encodes a highly root-expressed, cytoplasmically localized IAA oxidase. Stable isotope-labeled IAA feeding studies of loss and gain of function AtDAO1 lines showed that this oxidase represents the major regulator of auxin degradation to 2-oxoindole-3-acetic acid (oxIAA) in Arabidopsis. Surprisingly, AtDAO1 loss and gain of function lines exhibited relatively subtle auxin-related phenotypes, such as altered root hair length. Metabolite profiling of mutant lines revealed that disrupting AtDAO1 regulation resulted in major changes in steady-state levels of oxIAA and IAA conjugates but not IAA. Hence, IAA conjugation and catabolism seem to regulate auxin levels in Arabidopsis in a highly redundant manner. We observed that transcripts of AtDOA1 IAA oxidase and GH3 IAA-conjugating enzymes are auxin-inducible, providing a molecular basis for their observed functional redundancy. We conclude that the AtDAO1 gene plays a key role regulating auxin homeostasis in Arabidopsis, acting in concert with GH3 genes, to maintain auxin concentration at optimal levels for plant growth and development. PMID:27651491

  10. Identification and functional characterization of K(+) transporters encoded by Legionella pneumophila kup genes.

    PubMed

    Hori, Juliana I; Pereira, Marcelo S F; Roy, Craig R; Nagai, Hiroki; Zamboni, Dario S

    2013-12-01

    Legionnaires' disease is an emerging, severe, pneumonia-like illness caused by the Gram-negative intracellular bacteria Legionella pneumophila, which are able to infect and replicate intracellularly in macrophages. Little is known regarding the mechanisms used by intracellular L. pneumophila for the acquisition of specific nutrients that are essential for bacterial replication. Here, we investigate three L. pneumophila genes with high similarity to the Escherichia coli K(+) transporters. These three genes were expressed by L. pneumophila and have been designated kupA, kupB and kupC. Investigation using the L. pneumophila kup mutants revealed that kupA is involved in K(+) acquisition during axenic growth. The kupA mutants replicated efficiently in rich axenic media, but poorly in a chemically defined medium. The kupA mutants were defective in the recruitment of polyubiquitinated proteins to the Legionella-containing vacuole that is formed in macrophages and displayed an intracellular multiplication defect during the replication in Acanthamoeba castellanii and in mouse macrophages. We found that bafilomycin treatment of macrophages was able to rescue the growth defects of kupA mutants, but itdid not influence the replication of wild-type bacteria. The defects identified in kupA mutants of L. pneumophila were complemented by the expression E. coli trkD/Kup gene in trans, a bona fide K(+) transporter encoded by E. coli. Collectively, our data indicate that KupA is a functional K(+) transporter expressed by L. pneumophila that facilitates the bacterial replication intracellularly and in nutrient-limited conditions.

  11. Identification and functional characterization of K+ transporters encoded by Legionella pneumophila kup genes

    PubMed Central

    Hori, Juliana I.; Pereira, Marcelo S.F.; Roy, Craig R.; Nagai, Hiroki; Zamboni, Dario S.

    2013-01-01

    Summary Legionnaires’ disease is an emerging, severe, pneumonia-like illness caused by the Gram-negative intracellular bacteria Legionella pneumophila, which are able to infect and replicate intracellularly in macrophages. Little is known regarding the mechanisms used by intracellular L. pneumophila for the acquisition of specific nutrients that are essential for bacterial replication. Here, we investigate three L. pneumophila genes with high similarity to the E. coli K+ transporters. These three genes were expressed by L. pneumophila and have been designated kupA, kupB and kupC. Investigation using the L. pneumophila kup mutants revealed that kupA is involved in K+ acquisition during axenic growth. The kupA mutants replicated efficiently in rich axenic media, but poorly in a chemically defined medium. The kupA mutants were defective in the recruitment of polyubiquitinated proteins to the Legionella-containing vacuole that is formed in macrophages and displayed an intracellular multiplication defect during the replication in Acanthamoeba castellanii and in mouse macrophages. We found that bafilomycin treatment of macrophages was able to rescue the growth defects of kupA mutants, but it did not influence the replication of wild-type bacteria. The defects identified in kupA mutants of L. pneumophila were complemented by the expression E. coli trkD/Kup gene in trans, a bona fide K+ transporter encoded by E. coli. Collectively, our data indicate that KupA is a functional K+ transporter expressed by L. pneumophila that facilitates the bacterial replication intracellularly and in nutrient-limited conditions. PMID:23848378

  12. Characterization of a novel gene encoding ankyrin repeat domain from Cotesia vestalis polydnavirus (CvBV)

    SciTech Connect

    Shi Min; Chen Yafeng; Huang Fang; Liu Pengcheng; Zhou Xueping; Chen Xuexin

    2008-06-05

    Cotesia vestalis (Haliday) is an endoparasitoid of Plutella xylostella (L.) larvae and injects a polydnavirus (CvBV) into its host during oviposition. In this report we describe the characterization of a gene (CvBV805) and its products. CvBV805 is located on the segment S8 of CvBV genome; it has a size of 909 bp and encodes a predicted protein of 125 amino acids. This protein contains an ankyrin repeat domain with a high degree of similarity with I{kappa}B-like genes. Gene transcripts were detected in extracts of the host as early as 2 h post-parasitization (p.p.) and continued to be detected through 24 h. Tissue-specific expression patterns showed that CvBV805 might be involved in early host immunosuppression. CvBV805 was detected in parasitized hosts at 12 h p.p. and in rBac-eGFP-CvBV805-infected Tn-5B1-4 cells at 72 h.p.i. by using western blots analysis. The size of the protein expressed in the host hemocytes and infected Tn-5B1-4 cells was 17 kDa and 56 kDa (including eGFP), respectively, which nearly corresponded with the predicted molecular weight (14.31 kDa) of CvBV805, suggesting that the protein did not undergo extensive post-translational modification. The protein was confirmed to be present within the nuclear region in hemocytes of the parasitized P. xylostella larvae at 48 h p.p. using confocal laser scanning microscopy.

  13. TOM1, an Arabidopsis gene required for efficient multiplication of a tobamovirus, encodes a putative transmembrane protein.

    PubMed

    Yamanaka, T; Ohta, T; Takahashi, M; Meshi, T; Schmidt, R; Dean, C; Naito, S; Ishikawa, M

    2000-08-29

    Host-encoded factors play an important role in virus multiplication, acting in concert with virus-encoded factors. However, information regarding the host factors involved in this process is limited. Here we report the map-based cloning of an Arabidopsis thaliana gene, TOM1, which is necessary for the efficient multiplication of tobamoviruses, positive-strand RNA viruses infecting a wide variety of plants. The TOM1 mRNA is suggested to encode a 291-aa polypeptide that is predicted to be a multipass transmembrane protein. The Sos recruitment assay supported the hypothesis that TOM1 is associated with membranes, and in addition, that TOM1 interacts with the helicase domain of tobamovirus-encoded replication proteins. Taken into account that the tobamovirus replication complex is associated with membranes, we propose that TOM1 participates in the in vivo formation of the replication complex by serving as a membrane anchor.

  14. Positional cloning of rice semidwarfing gene, sd-1: rice "green revolution gene" encodes a mutant enzyme involved in gibberellin synthesis.

    PubMed

    Monna, Lisa; Kitazawa, Noriyuki; Yoshino, Rika; Suzuki, Junko; Masuda, Haruka; Maehara, Yumiko; Tanji, Masao; Sato, Mizuho; Nasu, Shinobu; Minobe, Yuzo

    2002-02-28

    A rice semidwarfing gene, sd-1, known as the "green revolution gene," was isolated by positional cloning and revealed to encode gibberellin 20-oxidase, the key enzyme in the gibberellin biosynthesis pathway. Analysis of 3477 segregants using several PCR-based marker technologies, including cleaved amplified polymorphic sequence, derived-CAPS, and single nucleotide polymorphisms revealed 1 ORF in a 6-kb candidate interval. Normal-type rice cultivars have an identical sequence in this region, consisting of 3 exons (558, 318, and 291 bp) and 2 introns (105 and 1471 bp). Dee-Geo-Woo-Gen-type sd-1 mutants have a 383-bp deletion from the genome (278-bp deletion from the expressed sequence), from the middle of exon 1 to upstream of exon 2, including a 105-bp intron, resulting in a frame-shift that produces a termination codon after the deletion site. The radiation-induced sd-1 mutant Calrose 76 has a 1-bp substitution in exon 2, causing an amino acid substitution (Leu [CTC] to Phe [TTC]). Expression analysis suggests the existence of at least one more locus of gibberellin 20-oxidase which may prevent severe dwarfism from developing in sd-1 mutants.

  15. Killing of cancer cells through the use of eukaryotic expression vectors harbouring genes encoding nucleases and ribonuclease inhibitor.

    PubMed

    Glinka, Elena M

    2015-05-01

    Cancer gene therapy vectors are promising tools for killing cancer cells with the purpose of eradicating malignant tumours entirely. Different delivery methods of vectors into the cancer cells, including both non-viral and viral, as well as promoters for the targeted expression of genes encoding anticancer proteins were developed for effective and selective killing of cancer cells without harming healthy cells. Many vectors have been created to kill cancer cells, and some vectors suppress malignant tumours with high efficiency. This review is focused on vectors bearing genes for nucleases such as deoxyribonucleases (caspase-activated DNase, deoxyribonuclease I-like 3, endonuclease G) and ribonucleases (human polynucleotide phosphorylase, ribonuclease L, α-sarcin, barnase), as well as vectors harbouring gene encoding ribonuclease inhibitor. The data concerning the functionality and the efficacy of such vectors are presented.

  16. Six mouse alpha-tubulin mRNAs encode five distinct isotypes: testis-specific expression of two sister genes.

    PubMed Central

    Villasante, A; Wang, D; Dobner, P; Dolph, P; Lewis, S A; Cowan, N J

    1986-01-01

    Five mouse alpha-tubulin isotypes are described, each distinguished by the presence of unique amino acid substitutions within the coding region. Most, though not all of these isotype-specific amino acids, are clustered at the carboxy terminus. One of the alpha-tubulin isotypes described is expressed exclusively in testis and is encoded by two closely related genes (M alpha 3 and M alpha 7) which have homologous 3' untranslated regions but which differ at multiple third codon positions and in their 5' untranslated regions. We show that a subfamily of alpha-tubulin genes encoding the same testis-specific isotype also exists in humans. Thus, we conclude that the duplication event leading to a pair of genes encoding a testis-specific alpha-tubulin isotype predated the mammalian radiation, and both members of the duplicated sequence have been maintained since species divergence. A second alpha-tubulin gene, M alpha 6, is expressed ubiquitously at a low level, whereas a third gene, M alpha 4, is unique in that it does not encode a carboxy-terminal tyrosine residue. This gene yields two transcripts: a 1.8-kilobase (kb) mRNA that is abundant in muscle and a 2.4-kb mRNA that is abundant in testis. Whereas the 1.8-kb mRNA encodes a distinct alpha-tubulin isotype, the 2.4-kb mRNA is defective in that the methionine residue required for translational initiation is missing. Patterns of developmental expression of the various alpha-tubulin isotypes are presented. Our data support the view that individual tubulin isotypes are capable of conferring functional specificity on different kinds of microtubules. Images PMID:3785200

  17. Identification of stsC, the gene encoding the L-glutamine:scyllo-inosose aminotransferase from streptomycin-producing Streptomycetes.

    PubMed

    Ahlert, J; Distler, J; Mansouri, K; Piepersberg, W

    1997-08-01

    Eight new genes, strO-stsABCDEFG, were identified by sequencing DNA in the gene cluster that encodes proteins for streptomycin production of Streptomyces griseus N2-3-11. The StsA (calculated molecular mass 43.5 kDa) and StsC (45.5 kDa) proteins - together with another gene product, StrS (39.8 kDa), encoded in another operon of the same gene cluster - show significant sequence identity and are members of a new class of pyridoxal-phosphate-dependent aminotransferases that have been observed mainly in the biosynthetic pathways for secondary metabolites. The aminotransferase activity was demonstrated for the first time by identification of the overproduced and purified StsC protein as the L-glutamine:scyllo-inosose aminotransferase, which catalyzes the first amino transfer in the biosynthesis of the streptidine subunit of streptomycin. The stsC and stsA genes each hybridized specifically to distinct fragments in the genomic DNA of most actinomycetes tested that produce diaminocyclitolaminoglycosides. In contrast, only stsC, but not stsA, hybridized to the DNA of Streptomyces hygroscopicus ssp. glebosus, which produces the monoaminocyclitol antibiotic bluensomycin; this suggests that both genes are specifically used in the first and second steps of the cyclitol transamination reactions. Sequence comparison studies performed with the deduced polypeptides of the genes adjacent to stsC suggest that the enzymes encoded by some of these genes [strO (putative phosphatase gene), stsB (putative oxidoreductase gene), and stsE (putative phosphotransferase gene)] also could be involved in (di-)aminocyclitol synthesis.

  18. A novel strategy for obtaining kanamycin resistance in Arabidopsis thaliana by silencing an endogenous gene encoding a putative chloroplast transporter.

    PubMed

    Aufsatz, Werner; Nehlin, Lilian; Voronin, Viktor; Schmidt, Agnes; Matzke, Antonius J M; Matzke, Marjori

    2009-02-01

    The use of bacterial antibiotic resistance markers in transgenic plants raises concerns about horizontal gene transfer to soil bacteria. We report here that kanamycin resistance in Arabidopsis thaliana can be achieved by silencing an endogenous gene encoding a putative chloroplast transporter, which presumably imports kanamycin into chloroplasts to interfere with ribosomal RNA. Homologs of the transporter exist in other plant species, suggesting this strategy may be generally useful for selecting transformed plant cells.

  19. Influence of energy supply on expression of genes encoding for lipogenic enzymes and regulatory proteins in growing beef steers

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Forty crossbred beef steers were used to determine the effects metabolizable energy (ME) intake and of site and complexity of carbohydrate (CHO) infusion on expression of genes encoding lipogenic enzymes and regulatory proteins in subcutaneous (SC), mesenteric (MES) and omental (OM) adipose. Treatm...

  20. The bglA Gene of Aspergillus kawachii Encodes Both Extracellular and Cell Wall-Bound β-Glucosidases

    PubMed Central

    Iwashita, Kazuhiro; Nagahara, Tatsuya; Kimura, Hitoshi; Takano, Makoto; Shimoi, Hitoshi; Ito, Kiyoshi

    1999-01-01

    We cloned the genomic DNA and cDNA of bglA, which encodes β-glucosidase in Aspergillus kawachii, based on a partial amino acid sequence of purified cell wall-bound β-glucosidase CB-1. The nucleotide sequence of the cloned bglA gene revealed a 2,933-bp open reading frame with six introns that encodes an 860-amino-acid protein. Based on the deduced amino acid sequence, we concluded that the bglA gene encodes cell wall-bound β-glucosidase CB-1. The amino acid sequence exhibited high levels of homology with the amino acid sequences of fungal β-glucosidases classified in subfamily B. We expressed the bglA cDNA in Saccharomyces cerevisiae and detected the recombinant β-glucosidase in the periplasm fraction of the recombinant yeast. A. kawachii can produce two extracellular β-glucosidases (EX-1 and EX-2) in addition to the cell wall-bound β-glucosidase. A. kawachii in which the bglA gene was disrupted produced none of the three β-glucosidases, as determined by enzyme assays and a Western blot analysis. Thus, we concluded that the bglA gene encodes both extracellular and cell wall-bound β-glucosidases in A. kawachii. PMID:10584016

  1. The Bacillus subtilis ydjL (bdhA) Gene Encodes Acetoin Reductase/2,3-Butanediol Dehydrogenase ▿

    PubMed Central

    Nicholson, Wayne L.

    2008-01-01

    Bacillus subtilis is capable of producing 2,3-butanediol from acetoin by fermentation, but to date, the gene encoding the enzyme responsible, acetoin reductase/2,3-butanediol dehydrogenase (AR/BDH), has remained unknown. A search of the B. subtilis genome database with the amino acid sequences of functional AR/BDHs from Saccharomyces cerevisiae and Bacillus cereus resulted in the identification of a highly similar protein encoded by the B. subtilis ydjL gene. A knockout strain carrying a ydjL::cat insertion mutation was constructed, which (i) abolished 2,3-butanediol production in early stationary phase, (ii) produced no detectable AR or BDH activity in vitro, and (iii) accumulated the precursor acetoin in early stationary phase. The ydjL::cat mutation also affected the kinetics of lactate but not acetate production during stationary-phase cultivation with glucose under oxygen limitation. A very small amount of 2,3-butanediol was detected in very-late-stationary-phase (96-hour) cultures of the ydjL::cat mutant, suggesting the existence of a second gene encoding a minor AR activity. From the data, it is proposed that the major AR/BDH-encoding gene ydjL be renamed bdhA. PMID:18820069

  2. Expression of the Immediate-Early Gene-Encoded Protein Egr-1 ("zif268") during in Vitro Classical Conditioning

    ERIC Educational Resources Information Center

    Mokin, Maxim; Keifer, Joyce

    2005-01-01

    Expression of the immediate-early genes (IEGs) has been shown to be induced by activity-dependent synaptic plasticity or behavioral training and is thought to play an important role in long-term memory. In the present study, we examined the induction and expression of the IEG-encoded protein Egr-1 during an in vitro neural correlate of eyeblink…

  3. Identification of the gene encoding the 65-kilodalton DNA-binding protein of herpes simplex virus type 1

    SciTech Connect

    Parris, D.S. Institute of Virology, Glasgow ); Cross, A.; Orr, A.; Frame, M.C.; Murphy, M.; McGeoch, D.J.; Marsden, H.S. ); Haarr, L. )

    1988-03-01

    Hybrid arrest of in vitro translation was used to localize the region of the herpes simplex virus type 1 genome encoding the 65-kilodalton DNA-binding protein (65K{sub DBP}) to between genome coordinates 0.592 and 0.649. Knowledge of the DNA sequence of this region allowed us to identify three open reading frames as likely candidates for the gene encoding 65K{sub DBP}. Two independent approaches were used to determine which of these three open reading frames encoded the protein. For the first approach a monoclonal antibody, MAb 6898, which reacted specifically with 65K{sub DBP}, was isolated. This antibody was used, with the techniques of hybrid arrest of in vitro translation and in vitro translation of selected mRNA, to identify the gene encoding 65K{sub DBP}. The second approach involved preparation of antisera directed against oligopeptides corresponding to regions of the predicted amino acid sequence of this gene. These antisera reacted specifically with 65K{sub DBP}, thus confirming the gene assignment.

  4. The mae1 gene of Schizosaccharomyces pombe encodes a permease for malate and other C4 dicarboxylic acids.

    PubMed

    Grobler, J; Bauer, F; Subden, R E; Van Vuuren, H J

    1995-12-01

    The mae1 gene of the yeast Schizosaccharomyces pombe was identified on the basis of its ability to complement a mutant defective in the transport of malic acid. Analysis of the DNA sequence revealed an open reading frame of 1314 base pairs, encoding a polypeptide of 438 amino acids with a predicted molecular weight of 49 kDa. A hydropathy profile of the predicted amino acid sequence revealed a protein with ten membrane-spanning or associated domains and hydrophilic N- and C- termini. The predicted secondary structure of the protein in similar to models proposed for other integral membrane proteins from both prokaryotes and eukaryotes. The S. pombe mae1 gene encodes a single mRNA of 1.5 kb. The mea1 gene is expressed constitutively and is not subject to catabolite repression as was previously reported for the malate permease systems of Candida utilis and Hansenula anomala. The mae1 gene was mapped 2842 bp 5' to the MFml gene on chromosome I. Transport assays revealed that the mae1 gene encodes a permease involved in the uptake of L-malate, succinate and malonic acid.

  5. The genome of the mustard leaf beetle encodes two active xylanases originally acquired from bacteria through horizontal gene transfer

    PubMed Central

    Pauchet, Yannick; Heckel, David G.

    2013-01-01

    The primary plant cell wall comprises the most abundant polysaccharides on the Earth and represents a rich source of energy for organisms which have evolved the ability to digest them. Enzymes able to degrade plant cell wall polysaccharides are widely distributed in micro-organisms but are generally absent in animals, although their presence in insects, especially phytophagous beetles from the superfamilies Chrysomeloidea and Curculionoidea, has recently begun to be appreciated. The observed patchy distribution of endogenous genes encoding these enzymes in animals has raised questions about their evolutionary origins. Recent evidence suggests that endogenous plant cell wall degrading enzymes-encoding genes have been acquired by animals through a mechanism known as horizontal gene transfer (HGT). HGT describes how genetic material is moved by means other than vertical inheritance from a parent to an offspring. Here, we provide evidence that the mustard leaf beetle, Phaedon cochleariae, possesses in its genome genes encoding active xylanases from the glycoside hydrolase family 11 (GH11). We also provide evidence that these genes were originally acquired by P. cochleariae from a species of gammaproteobacteria through HGT. This represents the first example of the presence of genes from the GH11 family in animals. PMID:23698014

  6. Transcriptional regulation of YWHAZ, the gene encoding 14-3-3ζ.

    PubMed

    Kasinski, Andrea; Dong, Xueyuan; Khuri, Fadlo R; Boss, Jeremy; Fu, Haian

    2014-01-01

    Aberrant expression of oncogenic 14-3-3 proteins is correlated with poor survival of cancer patients. While the underlying mechanism of the abnormal expression in tumors remains elusive for the six oncogenic 14-3-3 isoforms; the potential involvement of a transcriptional component has been suggested. Unfortunately, little experimental data has been reported to support this hypothesis. In this study we describe the genetic structure of YWHAZ, the gene encoding 14-3-3ζ, including the identification of previously unreported transcript variants. In total, five transcript variants were revealed and their expressions confirmed in a panel of cell lines. Expressed sequence tag (EST) database mining and in vitro rapid-amplification of cDNA ends (RACE) confirmed that one variant, 1c, represents >80% of the expressed transcripts, which is also the most efficiently translated. An analysis of the proximal promoter of this variant revealed a functional Cyclic-AMP Response Element (CRE). Factors that bound to the CRE element were recognized through fractionation and subsequent EMSAs. This analysis identified CREB and ATF-1 as the trans-interacting factors. Cell-based assays confirm that ATF-1, and to a lesser extent CREB, bind the endogenous YWHAZ promoter especially under TNF-α stimulating conditions. In support of a role of ATF-1 in the regulation of YWHAZ, silencing of ATF-1 resulted in a marked reduction in two of the five YWHAZ transcripts. These data suggest a novel mechanism for the transcriptional regulation of a major pro-survival gene, YWHAZ, by ATF-1.

  7. The life-extending gene Indy encodes an exchanger for Krebs-cycle intermediates.

    PubMed

    Knauf, Felix; Mohebbi, Nilufar; Teichert, Carsten; Herold, Diana; Rogina, Blanka; Helfand, Stephen; Gollasch, Maik; Luft, Friedrich C; Aronson, Peter S

    2006-07-01

    A longevity gene called Indy (for 'I'm not dead yet'), with similarity to mammalian genes encoding sodium-dicarboxylate cotransporters, was identified in Drosophila melanogaster. Functional studies in Xenopus oocytes showed that INDY mediates the flux of dicarboxylates and citrate across the plasma membrane, but the specific transport mechanism mediated by INDY was not identified. To test whether INDY functions as an anion exchanger, we examined whether substrate efflux is stimulated by transportable substrates added to the external medium. Efflux of [14C]citrate from INDY-expressing oocytes was greatly accelerated by the addition of succinate to the external medium, indicating citrate-succinate exchange. The succinate-stimulated [14C]citrate efflux was sensitive to inhibition by DIDS (4,4'-di-isothiocyano-2,2'-disulphonic stilbene), as demonstrated previously for INDY-mediated succinate uptake. INDY-mediated efflux of [14C]citrate was also stimulated by external citrate and oxaloacetate, indicating citrate-citrate and citrate-oxaloacetate exchange. Similarly, efflux of [14C]succinate from INDY-expressing oocytes was stimulated by external citrate, alpha-oxoglutarate and fumarate, indicating succinate-citrate, succinate-alpha-oxoglutarate and succinate-fumarate exchange respectively. Conversely, when INDY-expressing Xenopus oocytes were loaded with succinate and citrate, [14C]succinate uptake was markedly stimulated, confirming succinate-succinate and succinate-citrate exchange. Exchange of internal anion for external citrate was markedly pH(o)-dependent, consistent with the concept that citrate is co-transported with a proton. Anion exchange was sodium-independent. We conclude that INDY functions as an exchanger of dicarboxylate and tricarboxylate Krebs-cycle intermediates. The effect of decreasing INDY activity, as in the long-lived Indy mutants, may be to alter energy metabolism in a manner that favours lifespan extension.

  8. StAR enhances transcription of genes encoding the mitochondrial proteases involved in its own degradation.

    PubMed

    Bahat, Assaf; Perlberg, Shira; Melamed-Book, Naomi; Lauria, Ines; Langer, Thomas; Orly, Joseph

    2014-02-01

    Steroidogenic acute regulatory protein (StAR) is essential for steroid hormone synthesis in the adrenal cortex and the gonads. StAR activity facilitates the supply of cholesterol substrate into the inner mitochondrial membranes where conversion of the sterol to a steroid is catalyzed. Mitochondrial import terminates the cholesterol mobilization activity of StAR and leads to mounting accumulation of StAR in the mitochondrial matrix. Our studies suggest that to prevent mitochondrial impairment, StAR proteolysis is executed by at least 2 mitochondrial proteases, ie, the matrix LON protease and the inner membrane complexes of the metalloproteases AFG3L2 and AFG3L2:SPG7/paraplegin. Gonadotropin administration to prepubertal rats stimulated ovarian follicular development associated with increased expression of the mitochondrial protein quality control system. In addition, enrichment of LON and AFG3L2 is evident in StAR-expressing ovarian cells examined by confocal microscopy. Furthermore, reporter studies of the protease promoters examined in the heterologous cell model suggest that StAR expression stimulates up to a 3.5-fold increase in the protease gene transcription. Such effects are StAR-specific, are independent of StAR activity, and failed to occur upon expression of StAR mutants that do not enter the matrix. Taken together, the results of this study suggest the presence of a novel regulatory loop, whereby acute accumulation of an apparent nuisance protein in the matrix provokes a mitochondria to nucleus signaling that, in turn, activates selected transcription of genes encoding the enrichment of mitochondrial proteases relevant for enhanced clearance of StAR.

  9. The Drosophila Stubble-stubbloid gene encodes an apparent transmembrane serine protease required for epithelial morphogenesis.

    PubMed Central

    Appel, L F; Prout, M; Abu-Shumays, R; Hammonds, A; Garbe, J C; Fristrom, D; Fristrom, J

    1993-01-01

    The Stubble-stubbloid (Sb-sbd) gene is required for hormone-dependent epithelial morphogenesis of imaginal discs of Drosophila, including the formation of bristles, legs, and wings. The gene has been cloned by using Sb-sbd-associated DNA lesions in a 20-kilobase (kb) region of a 263-kb genomic walk. The region specifies an approximately 3.8-kb transcript that is induced by the steroid hormone 20-hydroxyecdysone in imaginal discs cultured in vitro. The conceptually translated protein is an apparent 786-residue type II transmembrane protein (N terminus in, C terminus out), including an intracellular N-terminal domain of at least 35 residues and an extracellular C-terminal trypsin-like serine protease domain of 244 residues. Sequence analyses indicate that the Sb-sbd-encoded protease could activate itself by proteolytic cleavage. Consistent with the cell-autonomous nature of the Sb-sbd bristle phenotype, a disulfide bond between cysteine residues in the noncatalytic N-terminal fragment and the C-terminal catalytic fragment could tether the protease to the membrane after activation. Both dominant Sb and recessive sbd mutations affect the organization of microfilament bundles during bristle morphogenesis. We propose that the Sb-sbd product has a dual function. (i) It acts through its proteolytic extracellular domain to detach imaginal disc cells from extracellular matrices, and (ii) it transmits an outside-to-inside signal to its intracellular domain to modify the cytoskeleton and facilitate cell shape changes underlying morphogenesis. Images Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:7685111

  10. Cloning and expression of a gene encoding the flagellin of Clostridium chauvoei.

    PubMed

    Kojima, A; Uchida, I; Sekizaki, T; Sasaki, Y; Ogikubo, Y; Kijima, M; Tamura, Y

    2000-10-20

    Clostridium chauvoei is a causative agent of blackleg and the major protective antigen of the organism is the flagellar protein. Using an Escherichia coli expression library of the C. chauvoei Okinawa strain, we isolated the fliC gene encoding the flagellin protein. DNA sequence analysis revealed an open reading frame of 413 amino acid residues with a calculated molecular mass of 43819Da. Comparison of the sequence with those of flagellins from other bacteria showed considerable homology in the N-terminal and C-terminal domains. The glutathione-S-transferase (GST)-flagellin fusion protein and the purified FliC protein after removing the GST part with thrombin reacted with both polyclonal antisera and the non-protective monoclonal antibody (Mab), Mo-114. However, the protective Mab, Mo-41, which may recognize its conformational epitope, failed to react with both the GST-flagellin fusion protein and the purified FliC. Furthermore, the GST-flagellin fusion protein and the purified FliC induced very little protective immunity in mice. These results suggested that a conformation-dependent epitope play an important role in the development of immunity against blackleg.

  11. Brd1 gene in maize encodes a brassinosteroid C-6 oxidase.

    PubMed

    Makarevitch, Irina; Thompson, Addie; Muehlbauer, Gary J; Springer, Nathan M

    2012-01-01

    The role of brassinosteroids in plant growth and development has been well-characterized in a number of plant species. However, very little is known about the role of brassinosteroids in maize. Map-based cloning of a severe dwarf mutant in maize revealed a nonsense mutation in an ortholog of a brassinosteroid C-6 oxidase, termed brd1, the gene encoding the enzyme that catalyzes the final steps of brassinosteroid synthesis. Homozygous brd1-m1 maize plants have essentially no internode elongation and exhibit no etiolation response when germinated in the dark. These phenotypes could be rescued by exogenous application of brassinolide, confirming the molecular defect in the maize brd1-m1 mutant. The brd1-m1 mutant plants also display alterations in leaf and floral morphology. The meristem is not altered in size but there is evidence for differences in the cellular structure of several tissues. The isolation of a maize mutant defective in brassinosteroid synthesis will provide opportunities for the analysis of the role of brassinosteroids in this important crop system.

  12. The Caenorhabditis elegans odr-2 gene encodes a novel Ly-6-related protein required for olfaction.

    PubMed Central

    Chou, J H; Bargmann, C I; Sengupta, P

    2001-01-01

    Caenorhabditis elegans odr-2 mutants are defective in the ability to chemotax to odorants that are recognized by the two AWC olfactory neurons. Like many other olfactory mutants, they retain responses to high concentrations of AWC-sensed odors; we show here that these residual responses are caused by the ability of other olfactory neurons (the AWA neurons) to be recruited at high odor concentrations. odr-2 encodes a membrane-associated protein related to the Ly-6 superfamily of GPI-linked signaling proteins and is the founding member of a C. elegans gene family with at least seven other members. Alternative splicing of odr-2 yields three predicted proteins that differ only at the extreme amino terminus. The three isoforms have different promoters, and one isoform may have a unique role in olfaction. An epitope-tagged ODR-2 protein is expressed at high levels in sensory neurons, motor neurons, and interneurons and is enriched in axons. The AWC neurons are superficially normal in their development and structure in odr-2 mutants, but their function is impaired. Our results suggest that ODR-2 may regulate AWC signaling within the neuronal network required for chemotaxis. PMID:11139503

  13. A novel GDNF-inducible gene, BMZF3, encodes a transcriptional repressor associated with KAP-1

    SciTech Connect

    Suzuki, Chikage; Murakumo, Yoshiki Kawase, Yukari; Sato, Tomoko; Morinaga, Takatoshi; Fukuda, Naoyuki; Enomoto, Atsushi; Ichihara, Masatoshi; Takahashi, Masahide

    2008-02-01

    The Krueppel-associated box (KRAB)-containing zinc finger proteins (ZFPs) comprise the largest family of zinc finger transcription factors that function as transcriptional repressors. In the study of glial cell line-derived neurotrophic factor (GDNF)-RET signaling, we have identified bone marrow zinc finger 3 (BMZF3), encoding a KRAB-ZFP, as a GDNF-inducible gene by differential display analysis. The expression of BMZF3 transcripts in the human neuroblastoma cell line TGW increased 1 h after GDNF stimulation, as determined by Northern blotting and quantitative reverse-transcriptase polymerase chain reaction. The BMZF3 possesses transcriptional repressor activity in the KRAB domain. BMZF3 interacts with a co-repressor protein, KRAB-associated protein 1 (KAP-1), through the KRAB domain and siRNA-mediated knockdown of KAP-1 abolished the transcriptional repressor activity of BMZF3, indicating that KAP-1 is necessary for BMZF3 function. Furthermore, siRNA-mediated silencing of BMZF3 inhibited cell proliferation. These findings suggest that BMZF3 is a transcriptional repressor induced by GDNF that plays a role in cell proliferation.

  14. Identification and characterization of a cellulase-encoding gene from the buffalo rumen metagenomic library.

    PubMed

    Nguyen, Nhung Hong; Maruset, Lalita; Uengwetwanit, Tanaporn; Mhuantong, Wuttichai; Harnpicharnchai, Piyanun; Champreda, Verawat; Tanapongpipat, Sutipa; Jirajaroenrat, Kanya; Rakshit, Sudip K; Eurwilaichitr, Lily; Pongpattanakitshote, Somchai

    2012-01-01

    Microorganisms residing in the rumens of cattle represent a rich source of lignocellulose-degrading enzymes, since their diet consists of plant-based materials that are high in cellulose and hemicellulose. In this study, a metagenomic library was constructed from buffalo rumen contents using pCC1FOS fosmid vector. Ninety-three clones from the pooled library of approximately 10,000 clones showed degrading activity against AZCL-HE-Cellulose, whereas four other clones showed activity against AZCL-Xylan. Contig analysis of pyrosequencing data derived from the selected strongly positive clones revealed 15 ORFs that were closely related to lignocellulose-degrading enzymes belonging to several glycosyl hydrolase families. Glycosyl hydrolase family 5 (GHF5) was the most abundant glycosyl hydrolase found, and a majority of the GHF5s in our metagenomes were closely related to several ruminal bacteria, especially ones from other buffalo rumen metagenomes. Characterization of BT-01, a selected clone with highest cellulase activity from the primary plate screening assay, revealed a cellulase encoding gene with optimal working conditions at pH 5.5 at 50 °C. Along with its stability over acidic pH, the capability efficiently to hydrolyze cellulose in feed for broiler chickens, as exhibited in an in vitro digestibility test, suggests that BT-01 has potential application as a feed supplement.

  15. Proteogenomic Analysis of Human Chromosome 9-Encoded Genes from Human Samples and Lung Cancer Tissues

    PubMed Central

    Ahn, Jung-Mo; Kim, Min-Sik; Kim, Yong-In; Jeong, Seul-Ki; Lee, Hyoung-Joo; Lee, Sun Hee; Paik, Young-Ki; Pandey, Akhilesh; Cho, Je-Yoel

    2014-01-01

    The Chromosome-centric Human Proteome Project (C-HPP) was recently initiated as an international collaborative effort. Our team adopted chromosome 9 (Chr 9) and performed a bioinformatics and proteogenomic analysis to catalog Chr 9-encoded proteins from normal tissues, lung cancer cell lines and lung cancer tissues. Approximately 74.7% of the Chr 9 genes of the human genome were identified, which included approximately 28% of missing proteins (46 of 162) on Chr 9 compared with the list of missing proteins from the neXtProt master table (2013-09). In addition, we performed a comparative proteomics analysis between normal lung and lung cancer tissues. Based on the data analysis, 15 proteins from Chr 9 were detected only in lung cancer tissues. Finally, we conducted a proteogenomic analysis to discover Chr 9-residing single nucleotide polymorphisms (SNP) and mutations described in the COSMIC cancer mutation database. We identified 21 SNPs and 4 mutations containing peptides on Chr 9 from normal human cells/tissues and lung cancer cell lines, respectively. In summary, this study provides valuable information of the human proteome for the scientific community as part of C-HPP. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium with the data set identifier PXD. PMID:24274035

  16. Identification and characterization of a gene encoding a vertebrate-type carbonic anhydrase in cyanobacteria.

    PubMed Central

    Soltes-Rak, E; Mulligan, M E; Coleman, J R

    1997-01-01

    A gene (designated ecaA) encoding a vertebrate-like (alpha-type) carbonic anhydrase (CA) has been isolated from two disparate cyanobacteria, Anabaena sp. strain PCC 7120 and Synechococcus sp. strain PCC 7942. The deduced amino acid sequences correspond to proteins of 29 and 26 kDa, respectively, and revealed significant sequence similarity to human CAI and CAII, as well as Chlamydomonas CAHI, including conservation of most active-site residues identified in the animal enzymes. Structural similarities between the animal and cyanobacterial enzymes extend to the levels of antigenicity, as the Anabaena protein cross-reacts with antisera derived against chicken CAII. Expression of the cyanobacterial ecaA is regulated by CO2 concentration and is highest in cells grown at elevated levels of CO2. Immunogold localization using an antibody derived against the ecaA protein indicated an extracellular location. Preliminary analysis of Synechococcus mutants in which ecaA has been inactivated by insertion of a drug resistance cassette suggests that extracellular carbonic anhydrase plays a role in inorganic-carbon accumulation by maintaining equilibrium levels of CO2 and HCO3- in the periplasm. PMID:9006032

  17. Evolution-guided functional analyses reveal diverse antiviral specificities encoded by IFIT1 genes in mammals

    PubMed Central

    Daugherty, Matthew D; Schaller, Aaron M; Geballe, Adam P; Malik, Harmit S

    2016-01-01

    IFIT (interferon-induced with tetratricopeptide repeats) proteins are critical mediators of mammalian innate antiviral immunity. Mouse IFIT1 selectively inhibits viruses that lack 2'O-methylation of their mRNA 5' caps. Surprisingly, human IFIT1 does not share this antiviral specificity. Here, we resolve this discrepancy by demonstrating that human and mouse IFIT1 have evolved distinct functions using a combination of evolutionary, genetic and virological analyses. First, we show that human IFIT1 and mouse IFIT1 (renamed IFIT1B) are not orthologs, but are paralogs that diverged >100 mya. Second, using a yeast genetic assay, we show that IFIT1 and IFIT1B proteins differ in their ability to be suppressed by a cap 2'O-methyltransferase. Finally, we demonstrate that IFIT1 and IFIT1B have divergent antiviral specificities, including the discovery that only IFIT1 proteins inhibit a virus encoding a cap 2'O-methyltransferase. These functional data, combined with widespread turnover of mammalian IFIT genes, reveal dramatic species-specific differences in IFIT-mediated antiviral repertoires. DOI: http://dx.doi.org/10.7554/eLife.14228.001 PMID:27240734

  18. Sequence and expression analysis of the gene encoding inducible cAMP early repressor in tilapia.

    PubMed

    Chen, Ming; Wang, Rui; Gan, Xi; Lei, Aiying; Li, Chao; Yu, Xiaoli; Huang, Jun; Huang, Ting; Liang, Wanwen

    2010-06-01

    Suppression subtractive hybridization library was generated by comparison of cDNA populations isolated from peripheral leukocytes of pre- and post-immunized tilapia. One cDNA sequence encoding complete inducible cAMP early repressor was obtained from the library. The sequence was characterized by the presence of the basic structure of ICER IIgamma. Expression of ICER was in the tissues of four types of tilapia was decreased after infection with Streptococcus. After immunization, expression of ICER was initially decreased and then increased after 7 days. In addition, the order for the overall expression of ICER gene after infection and the increases of ICER expression later after immunization in these four types of tilapia was positively correlated to the disease resistance and productivity of these four species of tilapia. Our results provided molecular mechanisms for the different disease resistance capability in different species of tilapia. In addition, our results also provided reference molecular marker for breeding disease resistant tilapia, cAMP responsive element modulator.

  19. Transcriptional analysis of pmeA gene encoding a pectin methylesterase in Xanthomonas campestris pv. campestris.

    PubMed

    Hsiao, Yi-Min; Liu, Yu-Fan; Huang, Yi-Ling; Lee, Pei-Yu

    2011-04-01

    Exopolysaccharides and several extracellular enzymes of Xanthomonas campestris pv. campestris (Xcc), the causative agent of black rot in crucifers, are virulence determinants. It is known that Clp (cAMP receptor protein-like protein) and RpfF (an enoyl-CoA hydratase homologue required for the synthesis of the diffusible signal factor, DSF) regulate production of these factors. In this study, plate assay revealed that Xcc possesses pectin methylesterase activity and that its expression is controlled by Clp and RpfF. Mutational analysis has demonstrated that pmeA encodes a pectin methylesterase. Using the 5' RACE method, the pmeA transcription initiation site was mapped. Transcriptional fusion assays showed that pmeA transcription is positively regulated by Clp and RpfF, subject to catabolite repression which is independent of Clp or RpfF, and repressed under conditions of high osmolarity or oxygen limitation. This study not only extends previous work on Clp and RpfF regulation by showing that they both influence the expression of pmeA in Xcc, but also, for the first time, characterizes pectin methylesterase gene expression in Xanthomonas.

  20. Tenascin-X: a novel extracellular matrix protein encoded by the human XB gene overlapping P450c21B

    PubMed Central

    1993-01-01

    A human gene termed XB overlaps the P450c21B gene encoding steroid 21- hydroxylase and encodes a protein that closely resembles extracellular matrix proteins. Sequencing of phage and cosmid clones and of cDNA fragments shows that the XB gene spans 65 kb of DNA, consisting of 39 exons that encode a 12-kb mRNA. The predicted protein of over 400 kD consists of five distinct domains: a signal peptide, a hydrophobic domain containing three heptad repeats, a series of 18.5 EGF-like repeats, 29 fibronectin type III repeats, and a carboxy-terminal fibrinogen-like domain. Because the structure of the protein encoded by the XB gene closely resembles tenascin, we term this protein tenascin-X (TN-X), and propose a simplified nomenclature system for the family of tenascins. RNase protection experiments show that the TN-X transcript is expressed ubiquitously in human fetal tissues, with the greatest expression in the fetal testis and in fetal skeletal, cardiac, and smooth muscle. Two adrenal-specific transcripts, P450c21B (steroid 21- hydroxylase) and Y (an untranslated transcript) overlap the XB gene on the complementary strand of DNA, yielding a unique array of overlapping transcripts: a "polygene." In situ hybridization histochemistry experiments show that the TN-X transcript and the P450c21 and Y transcripts encoded on the complementary DNA strand are all expressed in the same cells of the human adrenal cortex. Genetic data suggest that TN-X may be essential for life. PMID:7686164

  1. [Cloning and analysis of three genes encoding type II CHH family neuropeptides from Fennropenaeus chinensis].

    PubMed

    Wang, Zai-Zhao; Xiang, Jian-Hai

    2003-10-01

    On the basis of sequence similarity, the crustean hyperglycemic hormone (CHH) family peptides have been classified into two types of hormones: type I and type II. Molt-inhibiting hormone (MIH) is a neuropeptide member of type II CHH family. Molting in shrimp is controlled by MIH and ecdysone. By inhibiting the synthesis of ecdysone in the Y-organ, MIH indirectly suppresses the molting activity of shrimp. In this study, we reported the cloning and characterization of 3 gene fragments encoding type II CHH family neuropeptides of the shrimp Fennropenaeus chinensis. According to the complementary DNA sequence of the mult-inhibiting hormone of Fennropenaeus chinensis, 3 primers were designed and synthesized. MP1 and MP2 are sense primers, and MP3 is anti-sense primer. Polymerase chain reaction was performed using genomic DNA of Fennropenaeus chinensis as template. Three PCR products were obtained using primers MP1 and MP3. Their sizes are about 600 bp, 850 bp, 1050 bp, respectively. A 580 bp PCR product was obtained using primers MP2 and MP3. All the 4 PCR products were cloned into pMD18-T vector. The recombinant clones were sequenced using ABI 310 Genetic Analyzer. After sequencing, all the DNA sequences were searched in the GenBank by Blast program to find similar gene sequences. The searching results revealed 3 DNA fragment sequences were of high similarity with CHH family neuropeptide genes from various crustean species. The 3 DNA fragments were named as NP1, NP2, and NP3. Their sizes were 540 bp, 601 bp, and 826 bp, respectively. Using the mRNA sequences with the most similarity to the 3 sequence fragments as reference, the gene structure of the 3 DNA fragment sequences was analyzed. The exons of 3 sequence fragments were aligned with their similar sequences by Clustal W program. Both NP1 and NP2 consisted of 1 intron and 2 exons. NP3 consisted of 2 introns and 3 exons. Sequence analysis suggested that these 3 products belonged to sequence fragments of neuropeptide

  2. CAT8, a new zinc cluster-encoding gene necessary for derepression of gluconeogenic enzymes in the yeast Saccharomyces cerevisiae.

    PubMed Central

    Hedges, D; Proft, M; Entian, K D

    1995-01-01

    The expression of gluconeogenic fructose-1,6-bisphosphatase (encoded by the FBP1 gene) depends on the carbon source. Analysis of the FBP1 promoter revealed two upstream activating elements, UAS1FBP1 and UAS2FBP1, which confer carbon source-dependent regulation on a heterologous reporter gene. On glucose media neither element was activated, whereas after transfer to ethanol a 100-fold derepression was observed. This gene activation depended on the previously identified derepression genes CAT1 (SNF1) (encoding a protein kinase) and CAT3 (SNF4) (probably encoding a subunit of Cat1p [Snf1p]). Screening for mutations specifically involved in UAS1FBP1 derepression revealed the new recessive derepression mutation cat8. The cat8 mutants also failed to derepress UAS2FBP1, and these mutants were unable to grow on nonfermentable carbon sources. The CAT8 gene encodes a zinc cluster protein related to Saccharomyces cerevisiae Gal4p. Deletion of CAT8 caused a defect in glucose derepression which affected all key gluconeogenic enzymes. Derepression of glucose-repressible invertase and maltase was still normally regulated. A CAT8-lacZ promoter fusion revealed that the CAT8 gene itself is repressed by Cat4p (Mig1p). These results suggest that gluconeogenic genes are derepressed upon binding of Cat8p, whose synthesis depends on the release of Cat4p (Mig1p) from the CAT8 promoter. However, gluconeogenic promoters are still glucose repressed in cat4 mutants, which indicates that in addition to its transcription, the Cat8p protein needs further activation. The observation that multicopy expression of CAT8 reverses the inability of cat1 and cat3 mutants to grow on ethanol indicates that Cat8p might be the substrate of the Cat1p/Cat3p protein kinase. PMID:7891685

  3. Localization of eight additional genes in the human major histocompatibility complex, including the gene encoding the casein kinase II {beta} subunit (CSNK2B)

    SciTech Connect

    Albertella, M.R.; Jones, H.; Thomson, W.

    1996-09-01

    A wide range of autoimmune and other diseases are known to be associated with the major histocompatibility complex. Many of these diseases are linked to the genes encoding the polymorphic histocompatibility complex. Many of these diseases are linked to the genes encoding the polymorphic histocompatibility antigens in the class I and class II regions, but some appear to be more strongly associated with genes in the central 1100-kb class III region, making it important to characterize this region fully for the presence of novel genes. An {approximately}220-kb segment of DNA in the class III region separating the Hsp70 (HSPA1L) and BAT1 (D6S8IE) genes, which was previously known to contain 14 genes. Genomic DNA fragments spanning the gaps between the known genes were used as probes to isolate cDNAs corresponding to five new genes within this region. Evidence from Northern blot analysis and exon trapping experiments that suggested the presence of at least two more new genes was also obtained. Partial cDNA and complete exonic genomic sequencing of one of the new genes has identified it as the casein kinase II{beta} subunit (CSNK2B). Two of the other novel genes lie within a region syntenic to that implicated in susceptibility to experimental allergic orchitis in the mouse, an autoimmune disease of the testis, and represent additional candidates for the Orch-1 locus associated with this disease. In addition, characterization of the 13-kb intergenic gap separating the RD (D6545) and G11 (D6S60E) genes has revealed the presence of a gene encoding a 1246-amino-acid polypeptide that shows significant sequence similarity to the yeast anti-viral Ski2p gene product. 49 refs., 8 figs.

  4. Identification of the gene for fly non-muscle myosin heavy chain: Drosophila myosin heavy chains are encoded by a gene family.

    PubMed Central

    Kiehart, D P; Lutz, M S; Chan, D; Ketchum, A S; Laymon, R A; Nguyen, B; Goldstein, L S

    1989-01-01

    In contrast to vertebrate species Drosophila has a single myosin heavy chain gene that apparently encodes all sarcomeric heavy chain polypeptides. Flies also contain a cytoplasmic myosin heavy chain polypeptide that by immunological and peptide mapping criteria is clearly different from the major thoracic muscle isoform. Here, we identify the gene that encodes this cytoplasmic isoform and demonstrate that it is distinct from the muscle myosin heavy chain gene. Thus, fly myosin heavy chains are the products of a gene family. Our data suggest that the contractile function required to power myosin based movement in non-muscle cells requires myosin diversity beyond that available in a single heavy chain gene. In addition, we show, that accumulation of cytoplasmic myosin transcripts is regulated in a developmental stage specific fashion, consistent with a key role for this protein in the movements of early embryogenesis. Images PMID:2498088

  5. Qa-region class I gene expression: identification of a second class I gene, Q9, encoding a Qa-2 polypeptide.

    PubMed Central

    Soloski, M J; Hood, L; Stroynowski, I

    1988-01-01

    A feature of the expression of the tissue-specific class I antigen Qa-2 is the quantitative variation among mouse strains. Recently, the class I gene Q7 has been shown to encode a protein product that is biochemically indistinguishable from the lymphocyte-bound Qa-2 molecule. Utilizing gene transfection, we have identified a second Qa-2 subregion class I gene (Q9), in H-2b mice, which encodes a polypeptide biochemically similar to the Q7 and the Qa-2 polypeptides. Furthermore, we have observed that cell lines transfected with the allelic forms of the Q7 gene from C57BL/10 (Qa-2hi) or BALB/C (Qa-2low) display quantitative differences in cell-surface expression. Based on these studies, we suggest that gene dosage and allele-specific variation in cell-surface expression contribute to the strain-specific variation in the levels of Qa-2 antigen expression. Images PMID:3362864

  6. Inactivation of the Neurospora Crassa Gene Encoding the Mitochondrial Protein Import Receptor Mom19 by the Technique of ``sheltered Rip''

    PubMed Central

    Harkness, TAA.; Metzenberg, R. L.; Schneider, H.; Lill, R.; Neupert, W.; Nargang, F. E.

    1994-01-01

    We have used a technique referred to as ``sheltered RIP'' (repeat induced point mutation) to create mutants of the mom-19 gene of Neurospora crassa, which encodes an import receptor for nuclear encoded mitochondrial precursor proteins. Sheltered RIP permits the isolation of a mutant gene in one nucleus, even if that gene is essential for the survival of the organism, by sheltering the nucleus carrying the mutant gene in a heterokaryon with an unaffected nucleus. Furthermore, the nucleus harboring the RIPed gene contains a selectable marker so that it is possible to shift nuclear ratios in the heterokaryons to a state in which the nucleus containing the RIPed gene predominates in cultures grown under selective conditions. This results in a condition where the target gene product should be present at very suboptimal levels and allows the study of the mutant phenotype. One allele of mom-19 generated by this method contains 44 transitions resulting in 18 amino acid substitutions. When the heterokaryon containing this allele was grown under conditions favoring the RIPed nucleus, no MOM19 protein was detectable in the mitochondria of the strain. Homokaryotic strains containing the RIPed allele exhibit a complex and extremely slow growth phenotype suggesting that the product of the mom-19 gene is important in N. crassa. PMID:8138148

  7. Mutations of the Corynebacterium glutamicum NCgl1221 gene, encoding a mechanosensitive channel homolog, induce L-glutamic acid production.

    PubMed

    Nakamura, Jun; Hirano, Seiko; Ito, Hisao; Wachi, Masaaki

    2007-07-01

    Corynebacterium glutamicum is a biotin auxotroph that secretes L-glutamic acid in response to biotin limitation; this process is employed in industrial L-glutamic acid production. Fatty acid ester surfactants and penicillin also induce L-glutamic acid secretion, even in the presence of biotin. However, the mechanism of L-glutamic acid secretion remains unclear. It was recently reported that disruption of odhA, encoding a subunit of the 2-oxoglutarate dehydrogenase complex, resulted in L-glutamic acid secretion without induction. In this study, we analyzed odhA disruptants and found that those which exhibited constitutive L-glutamic acid secretion carried additional mutations in the NCgl1221 gene, which encodes a mechanosensitive channel homolog. These NCgl1221 gene mutations lead to constitutive L-glutamic acid secretion even in the absence of odhA disruption and also render cells resistant to an L-glutamic acid analog, 4-fluoroglutamic acid. Disruption of the NCgl1221 gene essentially abolishes L-glutamic acid secretion, causing an increase in the intracellular L-glutamic acid pool under biotin-limiting conditions, while amplification of the wild-type NCgl1221 gene increased L-glutamate secretion, although only in response to induction. These results suggest that the NCgl1221 gene encodes an L-glutamic acid exporter. We propose that treatments that induce L-glutamic acid secretion alter membrane tension and trigger a structural transformation of the NCgl1221 protein, enabling it to export L-glutamic acid.

  8. Cloning, expression, and nucleotide sequence of the Lactobacillus helveticus 481 gene encoding the bacteriocin helveticin J.

    PubMed Central

    Joerger, M C; Klaenhammer, T R

    1990-01-01

    Lactobacillus helveticus 481 produces a 37-kDa bacteriocin called helveticin J. Libraries of chromosomal DNA from L. helveticus were prepared in lambda gt11 and probed for phage-producing fusion proteins that could react with polyclonal helveticin J antibody. Two recombinant phage, HJ1 and HJ4, containing homologous inserts of 350 and 600 bp, respectively, produced proteins that reacted with antibody. These two phage clones specifically hybridized to L. helveticus 481 total genomic DNA but not to DNA from strains that did not produce helveticin J or strains producing unrelated bacteriocins. HJ1 and HJ4 lysogens produced beta-galactosidase fusion proteins that shared similar epitopes with each other and helveticin J. The intact helveticin J gene (hlv) was isolated by screening a library of L. helveticus chromosomal DNA in lambda EMBL3 with the insert DNA from phage HJ4 as a probe. The DNA sequence of a contiguous 3,364-bp region was determined. Two complete open reading frames (ORF), designated ORF2 and ORF3, were identified within the sequenced fragment. The 3' end of another open reading frame, ORF1, was located upstream of ORF2. A noncoding region and a putative promoter were located between ORF1 and ORF2. ORF2 could encode an 11,808-Da protein. The L. helveticus DNA inserts of the HJ1 and HJ4 clones reside within ORF3, which begins 30 bp downstream from the termination codon of ORF2. ORF3 could encode a 37,511-Da protein. Downstream from ORF3, the 5' end of another ORF (ORF4) was found. A Bg/II fragment containing ORF2 and ORF3 was cloned into pGK12, and the recombinant plasmid, pTRK135, was transformed into Lactobacillus acidophilus via electroporation. Transformants carrying pTRK135 produced a bacteriocin that was heat labile and exhibited an acitivity spectrum that was the same as that of helveticin J. Images PMID:2228964

  9. The ULTRACURVATA2 Gene of Arabidopsis Encodes an FK506-Binding Protein Involved in Auxin and Brassinosteroid Signaling1

    PubMed Central

    Pérez-Pérez, José Manuel; Ponce, María Rosa; Micol, José Luis

    2004-01-01

    The dwarf ucu (ultracurvata) mutants of Arabidopsis display vegetative leaves that are spirally rolled downwards and show reduced expansion along the longitudinal axis. We have previously determined that the UCU1 gene encodes a SHAGGY/GSK3-like kinase that participates in the signaling pathways of auxins and brassinosteroids. Here, we describe four recessive alleles of the UCU2 gene, whose homozygotes display helical rotation of several organs in addition to other phenotypic traits shared with ucu1 mutants. Following a map-based strategy, we identified the UCU2 gene, which was found to encode a peptidyl-prolyl cis/trans-isomerase of the FK506-binding protein family, whose homologs in metazoans are involved in cell signaling and protein trafficking. Physiological and double mutant analyses suggest that UCU2 is required for growth and development and participates in auxin and brassinosteroid signaling. PMID:14730066

  10. Changes in mitochondrial DNA alter expression of nuclear encoded genes associated with tumorigenesis

    SciTech Connect

    Jandova, Jana; Janda, Jaroslav; Sligh, James E

    2012-10-15

    We previously reported the presence of a mtDNA mutation hotspot in UV-induced premalignant and malignant skin tumors in hairless mice. We have modeled this change (9821insA) in murine cybrid cells and demonstrated that this alteration in mtDNA associated with mtBALB haplotype can alter the biochemical characteristics of cybrids and subsequently can contribute to significant changes in their behavioral capabilities. This study shows that changes in mtDNA can produce differences in expression levels of specific nuclear-encoded genes, which are capable of triggering the phenotypes such as seen in malignant cells. From a potential list of differentially expressed genes discovered by microarray analysis, we selected MMP-9 and Col1a1 for further studies. Real-time PCR confirmed up-regulation of MMP-9 and down-regulation of Col1a1 in cybrids harboring the mtDNA associated with the skin tumors. These cybrids also showed significantly increased migration and invasion abilities compared to wild type. The non-specific MMP inhibitor, GM6001, was able to inhibit migratory and invasive abilities of the 9821insA cybrids confirming a critical role of MMPs in cellular motility. Nuclear factor-{kappa}B (NF-{kappa}B) is a key transcription factor for production of MMPs. An inhibitor of NF-{kappa}B activation, Bay 11-7082, was able to inhibit the expression of MMP-9 and ultimately decrease migration and invasion of mutant cybrids containing 9821insA. These studies confirm a role of NF-{kappa}B in the regulation of MMP-9 expression and through this regulation modulates the migratory and invasive capabilities of cybrids with mutant mtDNA. Enhanced migration and invasion abilities caused by up-regulated MMP-9 may contribute to the tumorigenic phenotypic characteristics of mutant cybrids. -- Highlights: Black-Right-Pointing-Pointer Cybrids are useful models to study the role of mtDNA changes in cancer development. Black-Right-Pointing-Pointer mtDNA changes affect the expression of nuclear

  11. Translational Control of Host Gene Expression by a Cys-Motif Protein Encoded in a Bracovirus

    PubMed Central

    Kim, Eunseong; Kim, Yonggyun

    2016-01-01

    Translational control is a strategy that various viruses use to manipulate their hosts to suppress acute antiviral response. Polydnaviruses, a group of insect double-stranded DNA viruses symbiotic to some endoparasitoid wasps, are divided into two genera: ichnovirus (IV) and bracovirus (BV). In IV, some Cys-motif genes are known as host translation-inhibitory factors (HTIF). The genome of endoparasitoid wasp Cotesia plutellae contains a Cys-motif gene (Cp-TSP13) homologous to an HTIF known as teratocyte-secretory protein 14 (TSP14) of Microplitis croceipes. Cp-TSP13 consists of 129 amino acid residues with a predicted molecular weight of 13.987 kDa and pI value of 7.928. Genomic DNA region encoding its open reading frame has three introns. Cp-TSP13 possesses six conserved cysteine residues as other Cys-motif genes functioning as HTIF. Cp-TSP13 was expressed in Plutella xylostella larvae parasitized by C. plutellae. C. plutellae bracovirus (CpBV) was purified and injected into non-parasitized P. xylostella that expressed Cp-TSP13. Cp-TSP13 was cloned into a eukaryotic expression vector and used to infect Sf9 cells to transiently express Cp-TSP13. The synthesized Cp-TSP13 protein was detected in culture broth. An overlaying experiment showed that the purified Cp-TSP13 entered hemocytes. It was localized in the cytosol. Recombinant Cp-TSP13 significantly inhibited protein synthesis of secretory proteins when it was added to in vitro cultured fat body. In addition, the recombinant Cp-TSP13 directly inhibited the translation of fat body mRNAs in in vitro translation assay using rabbit reticulocyte lysate. Moreover, the recombinant Cp-TSP13 significantly suppressed cellular immune responses by inhibiting hemocyte-spreading behavior. It also exhibited significant insecticidal activities by both injection and feeding routes. These results indicate that Cp-TSP13 is a viral HTIF. PMID:27598941

  12. Structure of the CD59-encoding gene: further evidence of a relationship to murine lymphocyte antigen Ly-6 protein.

    PubMed Central

    Petranka, J G; Fleenor, D E; Sykes, K; Kaufman, R E; Rosse, W F

    1992-01-01

    The gene for CD59 [membrane inhibitor of reactive lysis (MIRL), protectin], a phosphatidylinositol-linked surface glycoprotein that regulates the formation of the polymeric C9 complex of complement and that is deficient on the abnormal hematopoietic cells of patients with paroxysmal nocturnal hemoglobinuria, consists of four exons spanning 20 kilobases. The untranslated first exon is preceded by a G+C-rich promoter region that lacks a consensus TATA or CAAT motif. The second exon encodes the hydrophobic leader sequence of the protein, and the third exon encodes the amino-terminal portion of the mature protein. The fourth exon encodes the remainder of the mature protein, including the hydrophobic sequence necessary for glycosyl-phosphatidylinositol anchor attachment. The structure of the CD59 gene is very similar to that encoding Ly-6, a murine glycoprotein with which CD59 has some structural similarity. The striking similarity in gene structure is further evidence that the two proteins belong to a superfamily of proteins that may also include the urokinase plasminogen-activator receptor and a squid glycoprotein of unknown function. Images PMID:1381503

  13. A microRNA encoded by Autographa californica nucleopolyhedrovirus regulates expression of viral gene ODV-E25.

    PubMed

    Zhu, Mengxiao; Wang, Jinwen; Deng, Riqiang; Xiong, Peiwen; Liang, Hai; Wang, Xunzhang

    2013-12-01

    Baculovirus-encoded microRNAs (miRNAs) have been described in Bombyx mori nucleopolyhedrovirus; however, most of their functions remain unclear. Here we report the identification and characterization of an miRNA encoded by Autographa californica nucleopolyhedrovirus. The identified miRNA, AcMNPV-miR-1, perfectly matched a segment in the coding sequence of the viral gene ODV-E25 and downregulated ODV-E25 mRNA expression, which likely resulted in a reduction of infectious budded virions and accelerated the formation of occlusion-derived virions.

  14. Cloning and Expression of Genes for Dengue Virus Type-2 Encoded Antigens for Rapid Diagnosis and Vaccine Development

    DTIC Science & Technology

    1986-11-26

    SIE cop AD nCloning and Expression of Genes for Dengue Virus ,4. CJ Type 2 Encoded Antigens for Rapid Diagnosis and Vaccine Development 0ANNUAL...Type 2 Encoded Antigens for Rapid Diagnosis and Vaccine Development 12. PERSONAL AUTHOR(S) Radha K. Padmanabhan, Ph.D. 13a. TYPE OF REPORT 13b. TIME...pVVI and pVVI7 cDNA clones, synthetic peptides homologous to NS5 and NSI regions were synthesized. These peptides are being used at Walter Reed Army

  15. Structure and heterologous expression of the gene encoding the cell surface glycoprotein from Haloarcula japonica strain TR-1.

    PubMed

    Wakai, H; Takada, K; Nakamura, S; Horikoshi, K

    1995-01-01

    The gene encoding the cell surface glycoprotein (CSG) of Haloarcula japonica strain TR-1 was cloned and sequenced. The structural gene consisted from an open reading frame of 2,586 bp. A potential promoter sequence was found about 150 bp upstream of the ATG initiation codon. N-terminal amino acid sequence of the Ha. japonica CSG revealed that the mature CSG consisted of 828 amino acids. Five potential N-glycosylation sites were found in the mature sequence. The cloned CSG gene of Ha. japonica was expressed in closely-related halophilic archaea.

  16. Predominance of a versatile-peroxidase-encoding gene, mnp4, as demonstrated by gene replacement via a gene targeting system for Pleurotus ostreatus.

    PubMed

    Salame, Tomer M; Knop, Doriv; Tal, Dana; Levinson, Dana; Yarden, Oded; Hadar, Yitzhak

    2012-08-01

    Pleurotus ostreatus (the oyster mushroom) and other white rot filamentous basidiomycetes are key players in the global carbon cycle. P. ostreatus is also a commercially important edible fungus with medicinal properties and is important for biotechnological and environmental applications. Efficient gene targeting via homologous recombination (HR) is a fundamental tool for facilitating comprehensive gene function studies. Since the natural HR frequency in Pleurotus transformations is low (2.3%), transformed DNA is predominantly integrated ectopically. To overcome this limitation, a general gene targeting system was developed by producing a P. ostreatus PC9 homokaryon Δku80 strain, using carboxin resistance complemented by the development of a protocol for hygromycin B resistance protoplast-based DNA transformation and homokaryon isolation. The Δku80 strain exhibited exclusive (100%) HR in the integration of transforming DNA, providing a high efficiency of gene targeting. Furthermore, the Δku80 strains produced showed a phenotype similar to that of the wild-type PC9 strain, with similar growth fitness, ligninolytic functionality, and capability of mating with the incompatible strain PC15 to produce a dikaryon which retained its resistance to the corresponding selection and was capable of producing typical fruiting bodies. The applicability of this system is demonstrated by inactivation of the versatile peroxidase (VP) encoded by mnp4. This enzyme is part of the ligninolytic system of P. ostreatus, being one of the nine members of the manganese-peroxidase (MnP) gene family, and is the predominantly expressed VP in Mn(2+)-deficient media. mnp4 inactivation provided a direct proof that mnp4 encodes a key VP responsible for the Mn(2+)-dependent and Mn(2+)-independent peroxidase activity under Mn(2+)-deficient culture conditions.

  17. Thermal and acid tolerant beta-xylosidases, genes encoding, related organisms, and methods

    DOEpatents

    Thompson, David N.; Thompson, Vicki S.; Schaller, Kastli D.; Apel, William A.; Lacey, Jeffrey A.; Reed, David W.

    2011-04-12

    Isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius and variations thereof are provided. Further provided are methods of at least partially degrading xylotriose and/or xylobiose using isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius and variations thereof.

  18. Thermal and acid tolerant beta xylosidases, arabinofuranosidases, genes encoding, related organisms, and methods

    SciTech Connect

    Thompson, David N; Thompson, Vicki S; Schaller, Kastli D; Apel, William A; Reed, David W; Lacey, Jeffrey A

    2013-04-30

    Isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius and variations thereof are provided. Further provided are methods of at least partially degrading xylotriose, xylobiose, and/or arabinofuranose-substituted xylan using isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius and variations thereof.

  19. Cloning, characterization, expression analysis and inhibition studies of a novel gene encoding Bowman-Birk type protease inhibitor from rice bean

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This paper presents the first study describing the isolation, cloning and characterization of a full length gene encoding Bowman-Birk protease inhibitor (RbTI) from rice bean (Vigna umbellata). A full-length protease inhibitor gene with complete open reading frame of 327bp encoding 109 amino acids w...

  20. Regulation of three genes encoding cell-wall-degrading enzymes of Trichoderma aggressivum during interaction with Agaricus bisporus.

    PubMed

    Abubaker, Kamal S; Sjaarda, Calvin; Castle, Alan J

    2013-06-01

    Members of the genus Trichoderma are very effective competitors of a variety of fungi. Cell-wall-degrading enzymes, including proteinases, glucanases, and chitinases, are commonly secreted as part of the competitive process. Trichoderma aggressivum is the causative agent of green mould disease of the button mushroom, Agaricus bisporus. The structures of 3 T. aggressivum genes, prb1 encoding a proteinase, ech42 encoding an endochitinase, and a β-glucanase gene, were determined. Promoter elements in the prb1 and ech42 genes suggested that transcription is regulated by carbon and nitrogen levels and by stress. Both genes had mycoparasitism-related elements indicating potential roles for the protein products in competition. The promoter of the β-glucanase gene contained CreA and AreA binding sites indicative of catabolite regulation but contained no mycoparasitism elements. Transcription of the 3 genes was measured in mixed cultures of T. aggressivum and A. bisporus. Two A. bisporus strains, U1, which is sensitive to green mould disease, and SB65, which shows some resistance, were used in co-cultivation tests to assess possible roles of the genes in disease production and severity. prb1 and ech42 were coordinately upregulated after 5 days, whereas β-glucanase transcription was upregulated from day 0 with both Agaricus strains. Upregulation was much less pronounced in mixed cultures of T. aggressivum with the resistant strain, SB65, than with the sensitive strain, U1. These observations suggested that the proteins encoded by these genes have roles in both nutrition and in severity of green mould disease.

  1. MYB98 Positively Regulates a Battery of Synergid-Expressed Genes Encoding Filiform Apparatus–Localized Proteins[W

    PubMed Central

    Punwani, Jayson A.; Rabiger, David S.; Drews, Gary N.

    2007-01-01

    The synergid cells within the female gametophyte are essential for reproduction in angiosperms. MYB98 encodes an R2R3-MYB protein required for pollen tube guidance and filiform apparatus formation by the synergid cells. To test the predicted function of MYB98 as a transcriptional regulator, we determined its subcellular localization and examined its DNA binding properties. We show that MYB98 binds to a specific DNA sequence (TAAC) and that a MYB98–green fluorescent protein fusion protein localizes to the nucleus, consistent with a role in transcriptional regulation. To identify genes regulated by MYB98, we tested previously identified synergid-expressed genes for reduced expression in myb98 female gametophytes and identified 16 such genes. We dissected the promoter of one of the downstream genes, DD11, and show that it contains a MYB98 binding site required for synergid expression, suggesting that DD11 is regulated directly by MYB98. To gain insight into the functions of the downstream genes, we chose five genes and determined the subcellular localization of the encoded proteins. We show that these five proteins are secreted into the filiform apparatus, suggesting that they play a role in either the formation or the function of this unique structure. Together, these data suggest that MYB98 functions as a transcriptional regulator in the synergid cells and activates the expression of genes required for pollen tube guidance and filiform apparatus formation. PMID:17693534

  2. scratch, a pan-neural gene encoding a zinc finger protein related to snail, promotes neuronal development.

    PubMed

    Roark, M; Sturtevant, M A; Emery, J; Vaessin, H; Grell, E; Bier, E

    1995-10-01

    The Drosophila scratch (scrt) gene is expressed in most or all neuronal precursor cells and encodes a predicted zinc finger transcription factor closely related to the product of the mesoderm determination gene snail (sna). Adult flies homozygous for scrt null alleles have a reduced number of photoreceptors in the eye, and embryos lacking the function of both scrt and the pan-neural gene deadpan (dpn), which encodes a basic helix-loop-helix (bHLH) protein, exhibit a significant loss of neurons. Conversely, ectopic expression of a scrt transgene during embryonic and adult development leads to the production of supernumerary neurons. Consistent with scrt functioning as a transcription factor, various genes are more broadly expressed than normal in scrt null mutants. Reciprocally, these same genes are expressed at reduced levels in response to ectopic scrt expression. We propose that scrt promotes neuronal cell fates by suppressing expression of genes promoting non-neuronal cell fates. We discuss the similarities between the roles of the ancestrally related scrt, sna, and escargot (esc) genes in regulating cell fate choices.

  3. Characterization and Functional Analysis of atl, a Novel Gene Encoding Autolysin in Streptococcus suis

    PubMed Central

    Ju, Cun-Xiang; Gu, Hong-Wei

    2012-01-01

    Streptococcus suis serotype 2 (S. suis 2) is an important swine and human pathogen responsible for septicemia and meningitis. A novel gene, designated atl and encoding a major autolysin of S. suis 2 virulent strain HA9801, was identified and characterized in this study. The Atl protein contains 1,025 amino acids with a predicted molecular mass of 113 kDa and has a conserved N-acetylmuramoyl-l-alanine amidase domain. Recombinant Atl was expressed in Escherichia coli, and its bacteriolytic and fibronectin-binding activities were confirmed by zymography and Western affinity blotting. Two bacteriolytic bands were shown in the sodium dodecyl sulfate extracts of HA9801, while both were absent from the atl inactivated mutant. Cell chains of the mutant strain became longer than that of the parental strain. In the autolysis assay, HA9801 decreased to 20% of the initial optical density (OD) value, while the mutant strain had almost no autolytic activity. The biofilm capacity of the atl mutant was reduced ∼30% compared to the parental strain. In the zebrafish infection model, the 50% lethal dose of the mutant strain was increased up to 5-fold. Furthermore, the adherence to HEp-2 cells of the atl mutant was 50% less than that of the parental strain. Based on the functional analysis of the recombinant Atl and observed effects of atl inactivation on HA9801, we conclude that Atl is a major autolysin of HA9801. It takes part in cell autolysis, separation of daughter cells, biofilm formation, fibronectin-binding activity, cell adhesion, and pathogenesis of HA9801. PMID:22228730

  4. The mouse and human genes encoding the recognition component of the N-end rule pathway

    PubMed Central

    Kwon, Yong Tae; Reiss, Yuval; Fried, Victor A.; Hershko, Avram; Yoon, Jeong Kyo; Gonda, David K.; Sangan, Pitchai; Copeland, Neal G.; Jenkins, Nancy A.; Varshavsky, Alexander

    1998-01-01

    The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. The N-end rule pathway is one proteolytic pathway of the ubiquitin system. The recognition component of this pathway, called N-recognin or E3, binds to a destabilizing N-terminal residue of a substrate protein and participates in the formation of a substrate-linked multiubiquitin chain. We report the cloning of the mouse and human Ubr1 cDNAs and genes that encode a mammalian N-recognin called E3α. Mouse UBR1p (E3α) is a 1,757-residue (200-kDa) protein that contains regions of sequence similarity to the 225-kDa Ubr1p of the yeast Saccharomyces cerevisiae. Mouse and human UBR1p have apparent homologs in other eukaryotes as well, thus defining a distinct family of proteins, the UBR family. The residues essential for substrate recognition by the yeast Ubr1p are conserved in the mouse UBR1p. The regions of similarity among the UBR family members include a putative zinc finger and RING-H2 finger, another zinc-binding domain. Ubr1 is located in the middle of mouse chromosome 2 and in the syntenic 15q15-q21.1 region of human chromosome 15. Mouse Ubr1 spans ≈120 kilobases of genomic DNA and contains ≈50 exons. Ubr1 is ubiquitously expressed in adults, with skeletal muscle and heart being the sites of highest expression. In mouse embryos, the Ubr1 expression is highest in the branchial arches and in the tail and limb buds. The cloning of Ubr1 makes possible the construction of Ubr1-lacking mouse strains, a prerequisite for the functional understanding of the mammalian N-end rule pathway. PMID:9653112

  5. High affinity of acid phosphatase encoded by PHO3 gene in Saccharomyces cerevisiae for thiamin phosphates.

    PubMed

    Nosaka, K

    1990-02-09

    The enzymatic properties of acid phosphatase (orthophosphoric-monoester phosphohydrolase, EC 3.1.3.2) encoded by PHO3 gene in Saccharomyces cerevisiae, which is repressed by thiamin and has thiamin-binding activity at pH 5.0, were investigated to study physiological functions. The following results led to the conclusion that thiamin-repressible acid phosphatase physiologically catalyzes the hydrolysis of thiamin phosphates in the periplasmic space of S. cerevisiae, thus participating in utilization of the thiamin moiety of the phosphates by yeast cells: (a) thiamin-repressible acid phosphatase showed Km values of 1.6 and 1.7 microM at pH 5.0 for thiamin monophosphate and thiamin pyrophosphate, respectively. These Km values were 2-3 orders of magnitude lower than those (0.61 and 1.7 mM) for p-nitrophenyl phosphate; (b) thiamin exerted remarkable competitive inhibition in the hydrolysis of thiamin monophosphate (Ki 2.2 microM at pH 5.0), whereas the activity for p-nitrophenyl phosphate was slightly affected by thiamin; (c) the inhibitory effect of inorganic phosphate, which does not repress the thiamin-repressible enzyme, on the hydrolysis of thiamin monophosphate was much smaller than that of p-nitrophenyl phosphate. Moreover, the modification of thiamin-repressible acid phosphatase of S. cerevisiae with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide resulted in the complete loss of thiamin-binding activity and the Km value of the modified enzyme for thiamin monophosphate increased nearly to the value of the native enzyme for p-nitrophenyl phosphate. These results also indicate that the high affinity of the thiamin-repressible acid phosphatase for thiamin phosphates is due to the thiamin-binding properties of this enzyme.

  6. Characterization of mouse UDP-glucose pyrophosphatase, a Nudix hydrolase encoded by the Nudt14 gene

    SciTech Connect

    Heyen, Candy A.; Tagliabracci, Vincent S.; Zhai, Lanmin; Roach, Peter J.

    2009-12-25

    Recombinant mouse UDP-glucose pyrophosphatase (UGPPase), encoded by the Nudt14 gene, was produced in Escherichia coli and purified close to homogeneity. The enzyme catalyzed the conversion of [{beta}-{sup 32}P]UDP-glucose to [{sup 32}P]glucose-1-P and UMP, confirming that it hydrolyzed the pyrophosphate of the nucleoside diphosphate sugar to generate glucose-1-P and UMP. The enzyme was also active toward ADP-ribose. Activity is dependent on the presence of Mg{sup 2+} and was greatest at alkaline pH above 8. Kinetic analysis indicated a K{sub m} of {approx}4 mM for UDP-glucose and {approx}0.3 mM for ADP-ribose. Based on V{sub max}/K{sub m} values, the enzyme was {approx}20-fold more active toward ADP-ribose. UGPPase behaves as a dimer in solution and can be cross-linked to generate a species of M{sub r} 54,000 from a monomer of 30,000 as judged by SDS-PAGE. The dimerization was not affected by the presence of glucose-1-P or UDP-glucose. Using antibodies raised against the recombinant protein, Western analysis indicated that UGPPase was widely expressed in mouse tissues, including skeletal muscle, liver, kidney, heart, lung, fat, heart and pancreas with a lower level in brain. It was generally present as a doublet when analyzed by SDS-PAGE, suggesting the occurrence of some form of post-translational modification. Efforts to interconvert the species by adding or inhibiting phosphatase activity were unsuccessful, leaving the nature of the modification unknown. Sequence alignments and database searches revealed related proteins in species as distant as Drosophila melanogaster and Caenorhabditis elegans.

  7. Identification of a gene sequence encoding a putative pyruvate oxidoreductase in Serpulina pilosicoli.

    PubMed

    Rayment, S J; Lee, B J; Hampson, D J; Livesley, M A

    1998-09-01

    Serpulina pilosicoli is a recently described species of intestinal spirochaete which can be identified using a species-specific monoclonal antibody BJL/AC1 reactive with a 29-kDa protein located in the cell envelope. A genomic library of the type strain of S. pilosicoli P43/6/78T was created in lambda zap express and screened using BJL/AC1. Single positive clones were isolated and excised into the phagemid vector pBK-CMV. Phagemid DNA was purified and a single clone was selected for sequencing. The size of spirochaetal DNA insert was determined by digestion with restriction endonucleases EcoRI and PstI as being approximately 2.6 kb. The nucleotide sequence of the gene encoding the protein with which the antibody reacted was determined by cycle sequencing. The insert contained an open reading frame of 285 nucleotides. Translation of the nucleotide sequence into amino acid (aa) residues showed a sequence of 275 aa. Comparison of this sequence with databases revealed homology to pyruvate oxidoreductases from various organisms found in the gastroinestinal tract. These included the pyruvate ferredoxin oxidoreductase (POR) alpha submit of Helicobacter pylori (38.8% identity in 250 aa), pyruvate-flavodoxin oxidoreductase of Escherichia coli (28.7% identify in 258 aa) and Giardia intestinalis (25.1% identity in 251 aa). A significant level of homology was also observed with hyperthermophilic bacteria such as the POR of Thermatoga maritima (38.6% in 254 aa) and the 2-ketovalerate-ferredoxin oxidoreductase of Pyrococcus furiosus (34% in 262 aa).

  8. Crystal structure of human nicotinamide riboside kinase.

    PubMed

    Khan, Javed A; Xiang, Song; Tong, Liang

    2007-08-01

    Nicotinamide riboside kinase (NRK) has an important role in the biosynthesis of NAD(+) as well as the activation of tiazofurin and other NR analogs for anticancer therapy. NRK belongs to the deoxynucleoside kinase and nucleoside monophosphate (NMP) kinase superfamily, although the degree of sequence conservation is very low. We report here the crystal structures of human NRK1 in a binary complex with the reaction product nicotinamide mononucleotide (NMN) at 1.5 A resolution and in a ternary complex with ADP and tiazofurin at 2.7 A resolution. The active site is located in a groove between the central parallel beta sheet core and the LID and NMP-binding domains. The hydroxyl groups on the ribose of NR are recognized by Asp56 and Arg129, and Asp36 is the general base of the enzyme. Mutation of residues in the active site can abolish the catalytic activity of the enzyme, confirming the structural observations.

  9. Crystal Structure of Human Nicotinamide Riboside Kinase

    SciTech Connect

    Khan,J.; Xiang, S.; Tong, L.

    2007-01-01

    Nicotinamide riboside kinase (NRK) has an important role in the biosynthesis of NAD{sup +} as well as the activation of tiazofurin and other NR analogs for anticancer therapy. NRK belongs to the deoxynucleoside kinase and nucleoside monophosphate (NMP) kinase superfamily, although the degree of sequence conservation is very low. We report here the crystal structures of human NRK1 in a binary complex with the reaction product nicotinamide mononucleotide (NMN) at 1.5 {angstrom} resolution and in a ternary complex with ADP and tiazofurin at 2.7 {angstrom} resolution. The active site is located in a groove between the central parallel {beta} sheet core and the LID and NMP-binding domains. The hydroxyl groups on the ribose of NR are recognized by Asp56 and Arg129, and Asp36 is the general base of the enzyme. Mutation of residues in the active site can abolish the catalytic activity of the enzyme, confirming the structural observations.

  10. Exons I and VII of the gene (Ker10) encoding human keratin 10 undergo structural rearrangements within repeats.

    PubMed

    Tkachenko, A V; Buchman, V L; Bliskovsky, V V; Shvets YuP; Kisselev, L L

    1992-07-15

    A genomic fragment containing the K51 gene previously isolated from a rat genomic library by hybridization with the v-mos probe in nonstringent conditions [Chumakov et al., Dokl. Akad. Nauk SSSR 290 (1986) 1252-1254], resembles a human keratin type-I-encoding gene [Shvets et al., Mol. Biol. 24 (1990) 663-677]. This genomic clone, K51, has been used as a probe to search for related human genes. A recombinant clone, HK51, with a 1.5-kb insert, was isolated from a human embryonic skin cDNA library, and its nucleotide (nt) sequence was determined. Analysis has shown that the cloned cDNA encodes human keratin 10 (Ker10). All presently known nt sequences of the human Ker10-encoding gene (Ker10) are not identical. Differences are concentrated in the 5'-end of the first exon and in the middle of the seventh exon within repeats. In spite of structural rearrangements in two of eight exons, the reading frame and position of the stop codon are preserved. The genetic rearrangements cause changes in hydrophobicity profiles of the N and C termini of Ker10. It was also noticed that insertion of one nt leads to the formation of an unusual 3'-end of the transcript.

  11. Cloning and expression of a novel, moderately thermostable xylanase-encoding gene (Cflxyn11A) from Cellulomonas flavigena.

    PubMed

    Amaya-Delgado, Lorena; Mejía-Castillo, Teresa; Santiago-Hernández, Alejandro; Vega-Estrada, Jesús; Amelia, Farrés-G-S; Xoconostle-Cázares, Beatriz; Ruiz-Medrano, Roberto; Montes-Horcasitas, María Del Carmen; Hidalgo-Lara, María Eugenia

    2010-07-01

    The Cfl xyn11A gene, encoding the endo-1,4-beta-xylanase Cfl Xyn11A from Cellulomonas flavigena, was isolated from a genomic DNA library. The open reading frame of the Cfl xyn11A gene was 999 base pairs long and encoded a polypeptide (Cfl Xyn11A) of 332 amino acids with a calculated molecular mass of 35,110Da. The Cfl xyn11A gene was expressed in Escherichia coli and the recombinant enzyme, with an estimated molecular weight of 31kDa was purified and xylanase activity was measured. Cfl Xyn11A showed optimal activity at pH 6.5 and 55 degrees C. The enzyme demonstrated moderate thermal stability as Cfl Xyn11A maintained 50% of its activity when incubated at 55 degrees C for 1h or at 45 degrees C for 6h. This is the first report describing the cloning, expression and functional characterization of an endo-1,4-beta-xylanase-encoding gene from C. flavigena. Cfl Xyn11A may be suitable for industrial applications in the food and feed industries, or in the pre-treatment of lignocellulosic biomass required to improve the yields of fermentable sugars for bioethanol production.

  12. Molecular cloning and expression of key gene encoding hypothetical DNA polymerase from B. mori parvo-like virus.

    PubMed

    Zhang, Junhong; Li, Guohui; Chen, Huiqing; Li, Xiaogang; Lv, Meng; Chen, Keping; Yao, Qin

    2010-10-01

    BmPLV-Z is the abbreviation for Bombyx mori parvo-like virus (China isolate). This is a novel virus with two single-stranded linear DNA molecules, viz., VD1 (6543 bp) and VD2 (6022 bp), which are encapsidated respectively into separate virions. Analysis of the deduced amino acid sequence of VD1-ORF4 indicated the existence of a putative DNA-polymerase with exonuclease activity, possibly involved in the replication of BmPLV-Z. In the present study, a recombinant baculovirus was constructed to express the full length of the protein encoded by the VD1-ORF4 gene (3318 bp). In addition, a 2163-bp fragment amplified from the very same gene was cloned into prokaryotic expression vector pET-30a and expressed in E.coli Rosetta 2 (DE3) pLysS. The expressed fusion protein was employed to immunize New Zealand white rabbits for the production of an antiserum, afterwards used for examining the expression of the protein encoded by VD1-ORF4 gene in Sf-9 cells infected with recombinant baculovirus. Western blot analysis of extracts from thus cells infected revealed a specific band of about 120 kDa, thereby indicating that the full length protein encoded by the VD1-ORF4 gene had been successfully and stably expressed in Sf-9 cells.

  13. Genomic organization and chromosomal localization of the human and mouse genes encoding the alpha receptor component for ciliary neurotrophic factor.

    PubMed

    Valenzuela, D M; Rojas, E; Le Beau, M M; Espinosa, R; Brannan, C I; McClain, J; Masiakowski, P; Ip, N Y; Copeland, N G; Jenkins, N A

    1995-01-01

    Ciliary neurotrophic factor (CNTF) has recently been found to share receptor components with, and to be structurally related to, a family of broadly acting cytokines, including interleukin-6, leukemia inhibitory factor, and oncostatin M. However, the CNTF receptor complex also includes a CNTF-specific component known as CNTF receptor alpha (CNTFR alpha). Here we describe the molecular cloning of the human and mouse genes encoding CNTFR. We report that the human and mouse genes have an identical intron-exon structure that correlates well with the domain structure of CNTFR alpha. That is, the signal peptide and the immunoglobulin-like domain are each encoded by single exons, the cytokine receptor-like domain is distributed among 4 exons, and the C-terminal glycosyl phosphatidylinositol recognition domain is encoded by the final coding exon. The position of the introns within the cytokine receptor-like domain corresponds to those found in other members of the cytokine receptor superfamily. Confirming a recent study using radiation hybrids, we have also mapped the human CNTFR gene to chromosome band 9p13 and the mouse gene to a syntenic region of chromosome 4.

  14. Genome organisation and expression profiling of ABC protein-encoding genes in Heterobasidion annosum s.l. complex.

    PubMed

    Baral, Bikash; Kovalchuk, Andriy; Asiegbu, Fred O

    2016-03-01

    Members of Heterobasidion annosum species complex are widely regarded as the most destructive fungal pathogens of conifer trees in the boreal and temperate zones of Northern hemisphere. To invade and colonise their host trees, Heterobasidion fungi must overcome components of host chemical defence, including terpenoid oleoresin and phenolic compounds. ABC transporters may play an important role in this process participating in the export of toxic host metabolites and maintaining their intracellular concentration below the critical level. We have identified and phylogenetically classified Heterobasidion genes encoding ABC transporters and closely related ABC proteins. The number of ABC proteins in the Heterobasidion genome is one of the lowest among analysed species of Agaricomycotina. Using quantitative RT-PCR, we have analysed transcriptional response of Heterobasidion ABC transporter-encoding genes to monoterpenes as well as their expression profile during growth on pine wood in comparison to the growth on defined media. Several ABC transporters were up-regulated during growth on pine wood. The ABC-transporter encoding gene ABCG1.1 was induced both during growth of H. annosum on pine wood and upon exposure to monoterpenes. Our experimental data demonstrate the differential responses of Heterobasidion ABC genes to growth conditions and chemical stressors. The presented results suggest a potential role of Heterobasidion ABC-G transporters in the resistance to the components of conifer chemical defence.

  15. A single gene encodes two different transcripts for the ADP-glucose pyrophosphorylase small subunit from barley (Hordeum vulgare).

    PubMed Central

    Thorbjørnsen, T; Villand, P; Kleczkowski, L A; Olsen, O A

    1996-01-01

    ADP-glucose pyrophosphorylase (AGPase), a heterotetrameric enzyme composed of two small and two large subunits, catalyses the first committed step of starch synthesis in plant tissues. In an attempt to learn more about the organization and expression of the small-subunit gene of AGPase, we have studied the small-subunit transcripts as well as the structure of the gene encoding these transcripts in barley (Hordeum vulgare L. cv. Bomi). Two different transcripts (bepsF1 and blps14) were identified: bepF1 was abundantly expressed in the starchy endosperm but not in leaves, whereas blps14 was isolated from leaves but was also found to be present at a moderate level in the starchy endosperm. The sequences for the two transcripts are identical over approx. 90% of the length, with differences being confined solely to their 5' ends. In blps14, the unique 5' end is 259 nt long and encodes a putative plastid transit peptide sequence. For the 178-nt 5' end of bepsF1, on the other hand, no transit peptide sequence could be recognized. A lambda clone that hybridized to the AGPase transcripts was isolated from a barley genomic library and characterized. The restriction map has suggested a complex organization of the gene, with alternative exons encoding the different 5' ends of the two transcripts followed by nine exons coding for the common part of the transcripts. The sequence of a portion of the genomic clone, covering the alternative 5'-end exons as well as upstream regions, has verified that both transcripts are encoded by the gene. The results suggest that the small-subunit gene of barley AGPase transcribes two different mRNAs by a mechanism classified as alternative splicing. PMID:8546676

  16. A novel prophenoloxidase, hemocyanin encoded copper containing active enzyme from prawn: gene characterization.

    PubMed

    Arockiaraj, Jesu; Gnanam, Annie J; Pothikasalam, Gopi; Milton, James; Pasupuleti, Mukesh; Bhatt, Prasanth; Palanisamy, Rajesh; Kumaresan, Venkatesh; Thirumalai, Muthukumaresan Kuppusamy; Arasu, Abirami; Sathyamoorthi, Akila; Prabha, Nagaram

    2013-07-25

    The copper containing prophenoloxidase enzyme plays a crucial role in the defense system of arthropods, especially crustaceans and insects. In this study, we have reported a full length cDNA of prophenoloxidase identified from the constructed cDNA library of freshwater prawn Macrobrachium rosenbergii by genome sequence FLX technology. The identified full length M. rosenbergii prophenoloxidase (MrProPO) consists of 3378 base pairs (bp) with an open reading frame (ORF) of 2099 bp. This ORF encoded a polypeptide of 700 amino acids (aa) with an estimated molecular mass of 80 kDa and a predicted isoelectric point (pI) of 6.7. The motif analysis of MrProPO shows two copper binding sites (CuA and CuB) along with hemocyanin signatures and a thiol-ester like motif. MrProPO exhibited the maximum similarity (97%) with ProPO from Macrobrachium nipponense and is closely clustered with other crustacean ProPO in the phylogenetic tree. Bioinformatics analysis suggests that MrProPO is a member of the prophenoloxidase family, due to the conserved domains, motifs and similarity with other known ProPOs. The 3D structural analysis of MrProPO reveals that it has more random coils, moderate α-helices, few extended β-sheets and a very few β-turns. Among the 700 aa of MrProPO, 355 (50.71%), 206 (29.43%), 110 (15.71%) and 29 (4.14%) amino acids are responsible for random coils, α-helices, extended β-sheets and β-turns respectively. The gene expression results indicate MrProPO is widely distributed in all the tissues studied, but significantly (P<0.05) highest expression was observed in hepatopancreas. The relative expression of mRNA was quantified in hepatopancreas after being infected with virus [white spot syndrome baculovirus (WSBV) and M. rosenbergii nodovirus (MrNV)] and bacteria (Aeromonas hydrophila and Vibrio harveyi) using real-time PCR. MrProPO mRNA transcription significantly (P<0.05) increased at 24h post injection (p.i.) with subsequent decrease at 48 h p.i. in both viral

  17. Naturally occurring anti-i/I cold agglutinins may be encoded by different VH3 genes as well as the VH4.21 gene segment.

    PubMed Central

    Jefferies, L C; Carchidi, C M; Silberstein, L E

    1993-01-01

    In the current study, we wished to determine if the V regions encoding the naturally occurring anti-i/I Cold Agglutinins (anti-i/I CA) differ from pathogenic anti-i/I CA that are exclusively encoded by the VH4.21 gene. After EBV transformation of B lymphocytes, we generated one anti-I secreting clone from each of two individuals; clone 4G (individual CM, PBL) and clone Sp1 (individual SC, spleen). Clone 4G expresses a VH3 gene sequence that is 92% homologous to the germline gene WHG26. Clone Sp1 also expresses a VH3 gene that is 98% homologous to the fetally rearranged M85/20P1 gene. Another clone, Sp2 (anti-i specificity), from individual SC is 98% homologous to the germline gene VH4.21. For correlation, we studied anti-i/I CA fractions purified from 15 normal sera and found no or relatively small amounts of 9G4 (VH4.21 related idiotype) reactive IgM. Five cold agglutinin fractions contained large amounts of VH3-encoded IgM (compared to pooled normal IgM) by virtue of their binding to modified protein Staph A (SPA), and absorption of three CA fractions with modified SPA specifically removed anti-i/I binding specificity entirely. Collectively, the data indicate that naturally occurring anti-i/I CA may be encoded to a large extent by non-VH4.21-related genes, and that the VH4.21 gene is not uniquely required for anti-i/I specificity. Images PMID:8254037

  18. Characterization of the norCBQD genes, encoding nitric oxide reductase, in the nitrogen fixing bacterium Bradyrhizobium japonicum.

    PubMed

    Mesa, Socorro; Velasco, Leonardo; Manzanera, Maximino E; Delgado, María J; Bedmar, Eulogio J

    2002-11-01

    The genes norCBQD that encode the bc-type nitric oxide reductase from Bradyrhizobium japonicum USDA110 have been isolated and characterized. norC and norB encode the cytochrome c-containing subunit II and cytochrome b-containing subunit I of nitric oxide reductase, respectively. norQ encodes a protein with an ATP/GTP-binding motif, and the predicted norD gene product shows similarity with NorD from other denitrifiers. Mutational analysis indicates that the two structural norC and norB genes are required for microaerobic growth under nitrate-respiring conditions. A mutant strain lacking a functional norC gene also lacked the 16 kDa c-type cytochrome that is normally detectable by haem-staining of proteins from membranes of microaerobically grown wild-type cells. Expression of a transcriptional fusion of the nor promoter region to the reporter gene lacZ (P(norC)-lacZ) was not detected in aerobically grown cells of USDA110, but the fusion was induced threefold when the cells were cultured under microaerobic conditions (1% O(2)) with either nitrite or nitric oxide, and about 18-fold when nitrate was the N oxide present in the medium. The P(norC)-lacZ fusion was not expressed in the B. japonicum fixK(2) mutant strain 9043, but complementation of the mutant with the fixK(2) gene restored beta-galactosidase activity to levels similar to those found in the parental strain. The promoter region of the norCBQD genes has been characterized by primer extension. A major transcript initiates 45.5 bp downstream of the centre of a putative binding site for the transcription factor FixK(2).

  19. Overexpression of genes encoding glycolytic enzymes in Corynebacterium glutamicum enhances glucose metabolism and alanine production under oxygen deprivation conditions.

    PubMed

    Yamamoto, Shogo; Gunji, Wataru; Suzuki, Hiroaki; Toda, Hiroshi; Suda, Masako; Jojima, Toru; Inui, Masayuki; Yukawa, Hideaki

    2012-06-01

    We previously reported that Corynebacterium glutamicum strain ΔldhAΔppc+alaD+gapA, overexpressing glyceraldehyde-3-phosphate dehydrogenase-encoding gapA, shows significantly improved glucose consumption and alanine formation under oxygen deprivation conditions (T. Jojima, M. Fujii, E. Mori, M. Inui, and H. Yukawa, Appl. Microbiol. Biotechnol. 87:159-165, 2010). In this study, we employ stepwise overexpression and chromosomal integration of a total of four genes encoding glycolytic enzymes (herein referred to as glycolytic genes) to demonstrate further successive improvements in C. glutamicum glucose metabolism under oxygen deprivation. In addition to gapA, overexpressing pyruvate kinase-encoding pyk and phosphofructokinase-encoding pfk enabled strain GLY2/pCRD500 to realize respective 13% and 20% improved rates of glucose consumption and alanine formation compared to GLY1/pCRD500. Subsequent overexpression of glucose-6-phosphate isomerase-encoding gpi in strain GLY3/pCRD500 further improved its glucose metabolism. Notably, both alanine productivity and yield increased after each overexpression step. After 48 h of incubation, GLY3/pCRD500 produced 2,430 mM alanine at a yield of 91.8%. This was 6.4-fold higher productivity than that of the wild-type strain. Intracellular metabolite analysis showed that gapA overexpression led to a decreased concentration of metabolites upstream of glyceraldehyde-3-phosphate dehydrogenase, suggesting that the overexpression resolved a bottleneck in glycolysis. Changing ratios of the extracellular metabolites by overexpression of glycolytic genes resulted in reduction of the intracellular NADH/NAD(+) ratio, which also plays an important role on the improvement of glucose consumption. Enhanced alanine dehydrogenase activity using a high-copy-number plasmid further accelerated the overall alanine productivity. Increase in glycolytic enzyme activities is a promising approach to make drastic progress in growth-arrested bioprocesses.

  20. The maize brown midrib2 (bm2) gene encodes a methylenetetrahydrofolate reductase that contributes to lignin accumulation.

    PubMed

    Tang, Ho Man; Liu, Sanzhen; Hill-Skinner, Sarah; Wu, Wei; Reed, Danielle; Yeh, Cheng-Ting; Nettleton, Dan; Schnable, Patrick S

    2014-02-01

    The midribs of maize brown midrib (bm) mutants exhibit a reddish-brown color associated with reductions in lignin concentration and alterations in lignin composition. Here, we report the mapping, cloning, and functional and biochemical analyses of the bm2 gene. The bm2 gene was mapped to a small region of chromosome 1 that contains a putative methylenetetrahydrofolate reductase (MTHFR) gene, which is down-regulated in bm2 mutant plants. Analyses of multiple Mu-induced bm2-Mu mutant alleles confirmed that this constitutively expressed gene is bm2. Yeast complementation experiments and a previously published biochemical characterization show that the bm2 gene encodes a functional MTHFR. Quantitative RT-PCR analyses demonstrated that the bm2 mutants accumulate substantially reduced levels of bm2 transcript. Alteration of MTHFR function is expected to influence accumulation of the methyl donor S-adenosyl-L-methionine (SAM). Because SAM is consumed by two methyltransferases in the lignin pathway (Ye et al., ), the finding that bm2 encodes a functional MTHFR is consistent with its lignin phenotype. Consistent with this functional assignment of bm2, the expression patterns of genes in a variety of SAM-dependent or -related pathways, including lignin biosynthesis, are altered in the bm2 mutant. Biochemical assays confirmed that bm2 mutants accumulate reduced levels of lignin with altered composition compared to wild-type. Hence, this study demonstrates a role for MTHFR in lignin biosynthesis.

  1. Structural analysis of the genes encoding the molybdenum-iron protein of nitrogenase in the Parasponia rhizobium strain ANU289.

    PubMed Central

    Weinman, J J; Fellows, F F; Gresshoff, P M; Shine, J; Scott, K F

    1984-01-01

    The genes encoding the Molybdenum-Iron protein component of nitrogenase (nifD and nifK) have been identified and fully characterised in the Parasponia Rhizobium strain ANU289. The two genes are contiguous and are separated from the gene encoding the Fe-protein component of nitrogenase (nifH) by 21 kb of DNA. We present the entire DNA sequence of the nifD and nifK genes, thus completing the characterisation of the primary structure of the nitrogenase genes in this Rhizobium strain. Comparison of the sequence preceding the transcription initiation point of nifDK with that preceding nifH reveals a consensus promoter sequence 5'-PyTGGCAPyG-4 bp-TTGC(T/A)-10 bp-3'. This consensus promoter is found preceding nif genes in both fast-growing and slow-growing Rhizobium strains and shows a structural similarity to that preceding the coordinately-regulated nif operons in the asymbiotic organism Klebsiella pneumoniae. Images PMID:6095197

  2. Molecular Characterization and Evolution of a Gene Family Encoding Both Female- and Male-Specific Reproductive Proteins in Drosophila

    PubMed Central

    Sirot, Laura K.; Findlay, Geoffrey D.; Sitnik, Jessica L.; Frasheri, Dorina; Avila, Frank W.; Wolfner, Mariana F.

    2014-01-01

    Gene duplication is an important mechanism for the evolution of new reproductive proteins. However, in most cases, each resulting paralog continues to function within the same sex. To investigate the possibility that seminal fluid proteins arise through duplicates of female reproductive genes that become “co-opted” by males, we screened female reproductive genes in Drosophila melanogaster for cases of duplication in which one of the resulting paralogs produces a protein in males that is transferred to females during mating. We identified a set of three tandemly duplicated genes that encode secreted serine-type endopeptidase homologs, two of which are expressed primarily in the female reproductive tract (RT), whereas the third is expressed specifically in the male RT and encodes a seminal fluid protein. Evolutionary and gene expression analyses across Drosophila species suggest that this family arose from a single-copy gene that was female-specific; after duplication, one paralog evolved male-specific expression. Functional tests of knockdowns of each gene in D. melanogaster show that one female-expressed gene is essential for full fecundity, and both female-expressed genes contribute singly or in combination to a female’s propensity to remate. In contrast, knockdown of the male-expressed paralog had no significant effect on female fecundity or remating. These data are consistent with a model in which members of this gene family exert effects on females by acting on a common, female-expressed target. After duplication and male co-option of one paralog, the evolution of the interacting proteins could have resulted in differential strengths or effects of each paralog. PMID:24682282

  3. Positional cloning in Cryptococcus neoformans and its application for identification and cloning of the gene encoding methylenetetrahydrofolate reductase.

    PubMed

    Toh-E, Akio; Ohkusu, Misako; Shimizu, Kiminori; Kawamoto, Susumu

    2015-03-01

    Cryptococcus neoformans, a basidiomycetous human pathogenic yeast, has been widely used in research fields in medical mycology as well as basic biology. Gene cloning or identification of the gene responsible for a mutation of interest is a key step for functional analysis of a particular gene. The availability therefore, of the multiple methods for cloning is desirable. In this study, we proposed a method for a mapping-based gene identification/cloning (positional cloning) method in C. neoformans. To this end, we constructed a series of tester strains, one of whose chromosomes was labeled with the URA5 gene. A heterozygous diploid constructed by crossing one of the tester strains to a mutant strain of interest loses a chromosome(s) spontaneously, which is the basis for assigning a recessive mutant gene to a particular chromosome in the mitotic mapping method. Once the gene of interest is mapped to one of the 14 chromosomes, classical genetic crosses can then be performed to determine its more precise location. The positional information thus obtained can then be used to significantly narrow down candidate genes by referring to the Cryptococcus genome database. Each candidate gene is then examined whether it would complement the mutation. We successfully applied this method to identify CNA07390 encoding methylenetetrahydrofolate reductase as the gene responsible for a methionine-requiring mutant in our mutant collection.

  4. Multiple sulfatase deficiency is caused by mutations in the gene encoding the human C(alpha)-formylglycine generating enzyme.

    PubMed

    Dierks, Thomas; Schmidt, Bernhard; Borissenko, Ljudmila V; Peng, Jianhe; Preusser, Andrea; Mariappan, Malaiyalam; von Figura, Kurt

    2003-05-16

    C(alpha)-formylglycine (FGly) is the catalytic residue in the active site of eukaryotic sulfatases. It is posttranslationally generated from a cysteine in the endoplasmic reticulum. The genetic defect of FGly formation causes multiple sulfatase deficiency (MSD), a lysosomal storage disorder. We purified the FGly generating enzyme (FGE) and identified its gene and nine mutations in seven MSD patients. In patient fibroblasts, the activity of sulfatases is partially restored by transduction of FGE encoding cDNA, but not by cDNA carrying an MSD mutation. The gene encoding FGE is highly conserved among pro- and eukaryotes and has a paralog of unknown function in vertebrates. FGE is localized in the endoplasmic reticulum and is predicted to have a tripartite domain structure.

  5. Toward Understanding the Functional Role of Ss-riok-1, a RIO Protein Kinase-Encoding Gene of Strongyloides stercoralis

    PubMed Central

    Yuan, Wang; Lok, James B.; Stoltzfus, Jonathan D.; Gasser, Robin B.; Fang, Fang; Lei, Wei-Qiang; Fang, Rui; Zhou, Yan-Qin; Zhao, Jun-Long; Hu, Min

    2014-01-01

    Background Some studies of Saccharomyces cerevisiae and mammals have shown that RIO protein kinases (RIOKs) are involved in ribosome biogenesis, cell cycle progression and development. However, there is a paucity of information on their functions in parasitic nematodes. We aimed to investigate the function of RIOK-1 encoding gene from Strongyloides stercoralis, a nematode parasitizing humans and dogs. Methodology/Principal Findings The RIOK-1 protein-encoding gene Ss-riok-1 was characterized from S. stercoralis. The full-length cDNA, gDNA and putative promoter region of Ss-riok-1 were isolated and sequenced. The cDNA comprises 1,828 bp, including a 377 bp 5′-UTR, a 17 bp 3′-UTR and a 1,434 bp ORF encoding a protein of 477 amino acids containing a RIOK-1 signature motif. The genomic sequence of the Ss-riok-1 coding region is 1,636 bp in length and has three exons and two introns. The putative promoter region comprises 4,280 bp and contains conserved promoter elements, including four CAAT boxes, 12 GATA boxes, eight E-boxes (CANNTG) and 38 TATA boxes. The Ss-riok-1 gene is transcribed throughout all developmental stages with the highest transcript abundance in the infective third-stage larva (iL3). Recombinant Ss-RIOK-1 is an active kinase, capable of both phosphorylation and auto-phosphorylation. Patterns of transcriptional reporter expression in transgenic S. stercoralis larvae indicated that Ss-RIOK-1 is expressed in neurons of the head, body and tail as well as in pharynx and hypodermis. Conclusions/Significance The characterization of the molecular and the temporal and spatial expression patterns of the encoding gene provide first clues as to functions of RIOKs in the biological processes of parasitic nematodes. PMID:25101874

  6. The Bradyrhizobium japonicum napEDABC genes encoding the periplasmic nitrate reductase are essential for nitrate respiration.

    PubMed

    Delgado, María J; Bonnard, Nathalie; Tresierra-Ayala, Alvaro; Bedmar, Eulogio J; Müller, Peter

    2003-12-01

    The napEDABC gene cluster that encodes the periplasmic nitrate reductase from Bradyrhizobium japonicum USDA110 has been isolated and characterized. napA encodes the catalytic subunit, and the napB and napC gene products are predicted to be a soluble dihaem c and a membrane-anchored tetrahaem c-type cytochrome, respectively. napE encodes a transmembrane protein of unknown function, and the napD gene product is a soluble protein which is assumed to play a role in the maturation of NapA. Western blots of the periplasmic fraction from wild-type cells grown anaerobically with nitrate revealed the presence of a protein band with a molecular size of about 90 kDa corresponding to NapA. A B. japonicum mutant carrying an insertion in the napA gene was unable to grow under nitrate-respiring conditions, lacked nitrate reductase activity, and did not show the 90 kDa protein band. Complementation of the mutant with a plasmid bearing the napEDABC genes restored both nitrate-dependent anaerobic growth of the cells and nitrate reductase activity. A membrane-bound and a periplasmic c-type cytochrome, with molecular masses of 25 kDa and 15 kDa, respectively, were not detected in the napA mutant strain incubated anaerobically with nitrate, which identifies those proteins as the NapC and the NapB components of the B. japonicum periplasmic nitrate reductase enzyme. These results suggest that the periplasmic nitrate reductase is the enzyme responsible for anaerobic growth of B. japonicum under nitrate-respiring conditions. The promoter region of the napEDABC genes has been characterized by primer extension. A major transcript initiates 66.5 bp downstream of the centre of a putative FNR-like binding site.

  7. A Single Gene on the Staphylococcal Multiresistance Plasmid pSK1 Encodes a Novel Partitioning System

    PubMed Central

    Simpson, Alice E.; Skurray, Ronald A.; Firth, Neville

    2003-01-01

    The orf245 gene is located immediately upstream of, and divergently transcribed from, the replication initiation gene, rep, of the Staphylococcus aureus multiresistance plasmid pSK1, and related genes have been found in association with a range of evolutionarily distinct replication genes on plasmids from various gram-positive genera. orf245 has been shown previously to extend the segregational stability of a pSK1 minireplicon. Here we describe an investigation into the basis of orf245-mediated stabilization. orf245 was not found to influence transcription of pSK1 rep, indicating that it is not directly involved in plasmid replication. This was confirmed by demonstrating that orf245 is able to enhance the segregational stability of heterologous theta- and rolling-circle-replicating replicons, suggesting that it encodes a plasmid maintenance function. Evidence inconsistent with postsegregational killing and multimer resolution mechanisms was obtained; however, the intergenic region upstream of orf245 was found to mediate orf245-dependent incompatibility, as would be expected if it encodes a cis-acting centromere-like site. Taken together, these findings implicate active partitioning as the probable basis of the activity of orf245, which is therefore redesignated par. Since it is unrelated to any gene known to play a role in plasmid segregation, it seems likely that pSK1 par potentially represents the prototype of a novel class of active partitioning systems that are distinguished by their capacity to enhance plasmid segregational stability via a single protein-encoding gene. PMID:12644483

  8. Ribonucleotide Reduction in Mycobacterium tuberculosis: Function and Expression of Genes Encoding Class Ib and Class II Ribonucleotide Reductases

    PubMed Central

    Dawes, Stephanie S.; Warner, Digby F.; Tsenova, Liana; Timm, Juliano; McKinney, John D.; Kaplan, Gilla; Rubin, Harvey; Mizrahi, Valerie

    2003-01-01

    Mycobacterium tuberculosis, the causative agent of tuberculosis, possesses a class Ib ribonucleotide reductase (RNR), encoded by the nrdE and nrdF2 genes, in addition to a putative class II RNR, encoded by nrdZ. In this study we probed the relative contributions of these RNRs to the growth and persistence of M. tuberculosis. We found that targeted knockout of the nrdF2 gene could be achieved only in the presence of a complementing allele, confirming that this gene is essential under normal, in vitro growth conditions. This observation also implied that the alternate class Ib small subunit encoded by the nrdF1 gene is unable to substitute for nrdF2 and that the class II RNR, NrdZ, cannot substitute for the class Ib enzyme, NrdEF2. Conversely, a ΔnrdZ null mutant of M. tuberculosis was readily obtained by allelic exchange mutagenesis. Quantification of levels of nrdE, nrdF2, nrdF1, and nrdZ gene expression by real-time, quantitative reverse transcription-PCR with molecular beacons by using mRNA from aerobic and O2-limited cultures showed that nrdZ was significantly induced under microaerophilic conditions, in contrast to the other genes, whose expression was reduced by O2 restriction. However, survival of the ΔnrdZ mutant strain was not impaired under hypoxic conditions in vitro. Moreover, the lungs of B6D2/F1 mice infected with the ΔnrdZ mutant had bacterial loads comparable to those of lungs infected with the parental wild-type strain, which argues against the hypothesis that nrdZ plays a significant role in the virulence of M. tuberculosis in this mouse model. PMID:14573627

  9. C/EBPε mediates nicotinamide-enhanced clearance of Staphylococcus aureus in mice.

    PubMed

    Kyme, Pierre; Thoennissen, Nils H; Tseng, Ching Wen; Thoennissen, Gabriela B; Wolf, Andrea J; Shimada, Kenichi; Krug, Utz O; Lee, Kunik; Müller-Tidow, Carsten; Berdel, Wolfgang E; Hardy, W David; Gombart, Adrian F; Koeffler, H Phillip; Liu, George Y

    2012-09-01

    The myeloid-specific transcription factor, CCAAT/enhancer-binding protein ε (C/EBPε) is a critical mediator of myelopoiesis. Mutation of this gene is responsible for neutrophil-specific granule deficiency in humans, a condition that confers susceptibility to Staphylococcus aureus infection. We found that C/EBPε-deficient mice are severely affected by infection with S. aureus, and C/EBPε deficiency in neutrophils contributes to the infectious phenotype. Conversely, exposure to the epigenetic modulator nicotinamide (vitamin B3) increased expression of C/EBPε in WT myeloid cells. Further, nicotinamide increased the activity of C/EBPε and select downstream antimicrobial targets, particularly in neutrophils. In a systemic murine infection model as well as in murine and human peripheral blood, nicotinamide enhanced killing of S. aureus by up to 1,000 fold but had no effect when administered to either C/EBPε-deficient mice or mice depleted of neutrophils. Nicotinamide was efficacious in both prophylactic and therapeutic settings. Our findings suggest that C/EBPε is an important target to boost killing of bacteria by the innate immune system.

  10. C/EBPε mediates nicotinamide-enhanced clearance of Staphylococcus aureus in mice

    PubMed Central

    Kyme, Pierre; Thoennissen, Nils H.; Tseng, Ching Wen; Thoennissen, Gabriela B.; Wolf, Andrea J.; Shimada, Kenichi; Krug, Utz O.; Lee, Kunik; Müller-Tidow, Carsten; Berdel, Wolfgang E.; Hardy, W. David; Gombart, Adrian F.; Koeffler, H. Phillip; Liu, George Y.

    2012-01-01

    The myeloid-specific transcription factor, CCAAT/enhancer-binding protein ε (C/EBPε) is a critical mediator of myelopoiesis. Mutation of this gene is responsible for neutrophil-specific granule deficiency in humans, a condition that confers susceptibility to Staphylococcus aureus infection. We found that C/EBPε-deficient mice are severely affected by infection with S. aureus, and C/EBPε deficiency in neutrophils contributes to the infectious phenotype. Conversely, exposure to the epigenetic modulator nicotinamide (vitamin B3) increased expression of C/EBPε in WT myeloid cells. Further, nicotinamide increased the activity of C/EBPε and select downstream antimicrobial targets, particularly in neutrophils. In a systemic murine infection model as well as in murine and human peripheral blood, nicotinamide enhanced killing of S. aureus by up to 1,000 fold but had no effect when administered to either C/EBPε-deficient mice or mice depleted of neutrophils. Nicotinamide was efficacious in both prophylactic and therapeutic settings. Our findings suggest that C/EBPε is an important target to boost killing of bacteria by the innate immune system. PMID:22922257

  11. Genome-wide characterization and analysis of F-box protein-encoding genes in the Malus domestica genome.

    PubMed

    Cui, Hao-Ran; Zhang, Zheng-Rong; Lv, Wei; Xu, Jia-Ning; Wang, Xiao-Yun

    2015-08-01

    The F-box protein family is a large family that is characterized by conserved F-box domains of approximately 40-50 amino acids in the N-terminus. F-box proteins participate in diverse cellular processes, such as development of floral organs, signal transduction and response to stress, primarily as a component of the Skp1-cullin-F-box (SCF) complex. In this study, using a global search of the apple genome, 517 F-box protein-encoding genes (F-box genes for short) were identified and further subdivided into 12 groups according to the characterization of known functional domains, which suggests the different potential functions or processes that they were involved in. Among these domains, the galactose oxidase domain was analyzed for the first time in plants, and this domain was present with or without the Kelch domain. The F-box genes were distributed in all 17 apple chromosomes with various densities and tended to form gene clusters. Spatial expression profile analysis revealed that F-box genes have organ-specific expression and are widely expressed in all organs. Proteins that contained the galactose oxidase domain were highly expressed in leaves, flowers and seeds. From a fruit ripening expression profile, 166 F-box genes were identified. The expressions of most of these genes changed little during maturation, but five of them increased significantly. Using qRT-PCR to examine the expression of F-box genes encoding proteins with domains related to stress, the results revealed that F-box proteins were up- or down-regulated, which suggests that F-box genes were involved in abiotic stress. The results of this study helped to elucidate the functions of F-box proteins, especially in Rosaceae plants.

  12. Molecular Cloning, Nucleotide Sequence, and Expression of Genes Encoding a Polycyclic Aromatic Ring Dioxygenase from Mycobacterium sp. Strain PYR-1

    PubMed Central

    Khan, Ashraf A.; Wang, Rong-Fu; Cao, Wei-Wen; Doerge, Daniel R.; Wennerstrom, David; Cerniglia, Carl E.

    2001-01-01

    Mycobacterium sp. strain PYR-1 degrades high-molecular-weight polycyclic hydrocarbons (PAHs) primarily through the introduction of both atoms of molecular oxygen by a dioxygenase. To clone the dioxygenase genes involved in PAH degradation, two-dimensional (2D) gel electrophoresis of PAH-induced proteins from cultures of Mycobacterium sp. strain PYR-1 was used to detect proteins that increased after phenanthrene, dibenzothiophene, and pyrene exposure. Comparison of proteins from induced and uninduced cultures on 2D gels indicated that at least six major proteins were expressed (105, 81, 52, 50, 43, and 13 kDa). The N-terminal sequence of the 50-kDa protein was similar to those of other dioxygenases. A digoxigenin-labeled oligonucleotide probe designed from this protein sequence was used to screen dioxygenase-positive clones from a genomic library of Mycobacterium sp. strain PYR-1. Three clones, each containing a 5,288-bp DNA insert with three genes of the dioxygenase system, were obtained. The genes in the DNA insert, from the 5′ to the 3′ direction, were a dehydrogenase, the dioxygenase small (β)-subunit, and the dioxygenase large (α)-subunit genes, arranged in a sequence different from those of genes encoding other bacterial dioxygenase systems. Phylogenetic analysis showed that the large α subunit did not cluster with most of the known α-subunit sequences but rather with three newly described α subunits of dioxygenases from Rhodococcus spp. and Nocardioides spp. The genes from Mycobacterium sp. strain PYR-1 were subcloned and overexpressed in Escherichia coli with the pBAD/ThioFusion system. The functionality of the genes for PAH degradation was confirmed in a phagemid clone containing all three genes, as well as in plasmid subclones containing the two genes encoding the dioxygenase subunits. PMID:11472934

  13. PGC-1α encoded by the PPARGC1A gene regulates oxidative energy metabolism in equine skeletal muscle during exercise.

    PubMed

    Eivers, S S; McGivney, B A; Gu, J; MacHugh, D E; Katz, L M; Hill, E W

    2012-04-01

    Peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) has emerged as a critical control factor in skeletal muscle adaptation to exercise, acting via transcriptional control of genes responsible for angiogenesis, fatty acid oxidation, oxidative phosphorylation, mitochondrial biogenesis and muscle fibre type composition. In a previous study, we demonstrated a significant increase in mRNA expression for the gene encoding PGC-1α (PPARGC1A) in Thoroughbred horse skeletal muscle following a single bout of endurance exercise. In this study, we investigated mRNA expression changes in genes encoding transcriptional coactivators of PGC-1α and genes that function upstream and downstream of PGC-1α in known canonical pathways. We used linear regression to determine the associations between PPARGC1A mRNA expression and expression of the selected panel of genes. Biopsy samples were obtained from the gluteus medius pre-exercise (T(0) ), immediately post-exercise (T(1) ) and 4 h post-exercise (T(2) ). Significant (P < 0.05) expression fold change differences relative to T(0) were detected for genes functioning in angiogenesis (ANGP2 and VEGFA); Ca(2+) -dependent signalling pathway (PPP3CA); carbohydrate/glucose metabolism (PDK4); fatty acid metabolism/mitochondrial biogenesis (PPPARGC1B); haem biosynthetic process (ALAS1); insulin signalling (FOXO1, PPPARGC1A and SLC2A4); mitogen-activated protein kinase signalling (MAPK14 and MEF2A); and myogenesis (HDAC9). Gene expression associations were identified between PPARGC1A and genes involved in angiogenesis, mitochondrial respiration, glucose transport, insulin signalling and transcriptional regulation. These results suggest that PGC-1α and genes regulated by PGC-1α play significant roles in the skeletal muscle response to exercise and therefore may contribute to performance potential in Thoroughbred horses.

  14. fra-1: a serum-inducible, cellular immediate-early gene that encodes a fos-related antigen.

    PubMed Central

    Cohen, D R; Curran, T

    1988-01-01

    A set of proteins antigenically related to the c-fos protein (Fos) are induced by serum in fibroblasts. To isolate cDNA clones of genes encoding such proteins, a lambda gt11 expression cDNA library constructed from serum-stimulated rat fibroblasts was screened with antibodies raised against a hydrophilic region (amino acids 127 to 152) of Fos. One of the positive clones identified, termed fra-1 (Fos-related antigen) was characterized. It encoded a protein that shared several regions of extensive amino acid homology with Fos (including the region that showed similarity to both the yeast GCN4 regulatory protein and the protein encoded by the jun oncogene), although its nucleotide sequence was considerably diverged from that of the c-fos gene. Only a subset of the agents and conditions that activated c-fos also induced fra-1. Induction of fra-1 expression following serum stimulation was delayed compared with that of c-fos. However, like c-fos, fra-1 was induced rapidly by serum in the presence of protein synthesis inhibitors. Thus, a family of Fos-related, inducible genes are involved in the cellular immediate-early transcriptional response to extracellular stimuli. Images PMID:3133553

  15. Isolation and DNA sequence of ADH3, a nuclear gene encoding the mitochondrial isozyme of alcohol dehydrogenase in Saccharomyces cerevisiae.

    PubMed Central

    Young, E T; Pilgrim, D

    1985-01-01

    The Saccharomyces cerevisiae nuclear gene, ADH3, that encodes the mitochondrial alcohol dehydrogenase isozyme ADH III was cloned by virtue of its nucleotide homology to ADH1 and ADH2. Both chromosomal and plasmid-encoded ADH III isozymes were repressed by glucose and migrated heterogeneously on nondenaturing gels. Nucleotide sequence analysis indicated 73 and 74% identity for ADH3 with ADH1 and ADH2, respectively. The amino acid identity between the predicted ADH III polypeptide and ADH I and ADH II was 79 and 80%, respectively. The open reading frame encoding ADH III has a highly basic 27-amino-acid amino-terminal extension relative to ADH I and ADH II. The nucleotide sequence of the presumed leader peptide has a high degree of identity with the untranslated leader regions of ADH1 and ADH2 mRNAs. A strain containing a null allele of ADH3 did not have a detectably altered phenotype. The cloned gene integrated at the ADH3 locus, indicating that this is the structural gene for ADH III. Images PMID:2943982

  16. Low-Concentration Ciprofloxacin Selects Plasmid-Mediated Quinolone Resistance Encoding Genes and Affects Bacterial Taxa in Soil Containing Manure

    PubMed Central

    Huang, Ting; Xu, Ying; Zeng, Jie; Zhao, Dong-Hao; Li, Liang; Liao, Xiao-Ping; Liu, Ya-Hong; Sun, Jian

    2016-01-01

    The spread of antimicrobial resistance in environment is promoted at least in part by the inappropriate use of antibiotics in animals and humans. The present study was designed to investigate the impact of different concentrations of ciprofloxacin in soil containing manure on the development of plasmid-mediated quinolone resistance (PMQR) – encoding genes and the abundance of soil bacterial communities. For these studies, high-throughput next-generation sequencing of 16S rRNA, real-time polymerase chain reaction and standard microbiologic culture methods were utilized. We demonstrated that the dissipate rate of relative abundances of some of PMQR-encoding genes, such as qnrS, oqxA and aac(6′)-Ib-cr, were significantly lower with ciprofloxacin 0.04 and 0.4 mg/kg exposure as compared to no-ciprofloxacin control and ciprofloxacin 4 mg/kg exposure during 2 month. Also, the number of ciprofloxacin resistant bacteria was significantly greater in ciprofloxacin 0.04 and 0.4 mg/kg exposure as compared with no-ciprofloxacin control and the ciprofloxacin 4 mg/kg exposure. In addition, lower ciprofloxacin concentration provided a selective advantage for the populations of Xanthomonadales and Bacillales in orders while Agrobacterium, Bacillus, Enterococcus, and Burkholderia in genera. These findings suggest that lower concentration of ciprofloxacin resulted in a slower rate of PMQR-encoding genes dissipation and selected development of ciprofloxacin-resistant bacteria in soil amended with manure. PMID:27847506

  17. Cloning and functional characterization of the gene encoding the transcription factor Ace1 in the basidiomycete Phanerochaete chrysosporium.

    PubMed

    Polanco, Rubén; Canessa, Paulo; Rivas, Alexis; Larrondo, Luis F; Lobos, Sergio; Vicuña, Rafael

    2006-01-01

    In this report we describe the isolation and characterization of a gene encoding the transcription factor Ace1 (Activation protein of cup 1 Expression) in the white rot fungus Phanerochaete chrysosporium. Pc-ace1 encodes a predicted protein of 633 amino acids containing the copper-fist DNA binding domain typically found in fungal transcription factors such as Ace1, Mac1 and Haa1 from Saccharomyces cerevisiae. The Pc-ace1 gene is localized in Scaffold 5, between coordinates 220841 and 222983. A S. cerevisiae ace1 null mutant strain unable to grow in high-copper medium was fully complemented by transformation with the cDNA of Pc-ace1. Moreover, Northern blot hybridization studies indicated that Pc-ace1 cDNA restores copper inducibility of the yeast cup 1 gene, which encodes the metal-binding protein metallothionein implicated in copper resistance. To our knowledge, this is first report describing an Ace1 transcription factor in basidiomycetes.

  18. PABPN1 overexpression leads to upregulation of genes encoding nuclear proteins that are sequestered in oculopharyngeal muscular dystrophy nuclear inclusions.

    PubMed

    Corbeil-Girard, Louis-Philippe; Klein, Arnaud F; Sasseville, A Marie-Josée; Lavoie, Hugo; Dicaire, Marie-Josée; Saint-Denis, Anik; Pagé, Martin; Duranceau, André; Codère, François; Bouchard, Jean-Pierre; Karpati, George; Rouleau, Guy A; Massie, Bernard; Langelier, Yves; Brais, Bernard

    2005-04-01

    Oculopharyngeal muscular dystrophy (OPMD) is an adult-onset disease caused by expanded (GCN)12-17 stretches encoding the N-terminal polyalanine domain of the poly(A) binding protein nuclear 1 (PABPN1). OPMD is characterized by intranuclear inclusions (INIs) in skeletal muscle fibers, which contain PABPN1, molecular chaperones, ubiquitin, proteasome subunits, and poly(A)-mRNA. We describe an adenoviral model of PABPN1 expression that produces INIs in most cells. Microarray analysis revealed that PABPN1 overexpression reproducibly changed the expression of 202 genes. Sixty percent of upregulated genes encode nuclear proteins, including many RNA and DNA binding proteins. Immunofluorescence microscopy revealed that all tested nuclear proteins encoded by eight upregulated genes colocalize with PABPN1 within the INIs: CUGBP1, SFRS3, FKBP1A, HMG2, HNRPA1, PRC1, S100P, and HSP70. In addition, CUGBP1, SFRS3, and FKBP1A were also found in OPMD muscle INIs. This study demonstrates that a large number of nuclear proteins are sequestered in OPMD INIs, which may compromise cellular function.

  19. ICGA-PSO-ELM approach for accurate multiclass cancer classification resulting in reduced gene sets in which genes encoding secreted proteins are highly represented.

    PubMed

    Saraswathi, Saras; Sundaram, Suresh; Sundararajan, Narasimhan; Zimmermann, Michael; Nilsen-Hamilton, Marit

    2011-01-01

    A combination of Integer-Coded Genetic Algorithm (ICGA) and Particle Swarm Optimization (PSO), coupled with the neural-network-based Extreme Learning Machine (ELM), is used for gene selection and cancer classification. ICGA is used with PSO-ELM to select an optimal set of genes, which is then used to build a classifier to develop an algorithm (ICGA_PSO_ELM) that can handle sparse data and sample imbalance. We evaluate the performance of ICGA-PSO-ELM and compare our results with existing methods in the literature. An investigation into the functions of the selected genes, using a systems biology approach, revealed that many of the identified genes are involved in cell signaling and proliferation. An analysis of these gene sets shows a larger representation of genes that encode secreted proteins than found in randomly selected gene sets. Secreted proteins constitute a major means by which cells interact with their surroundings. Mounting biological evidence has identified the tumor microenvironment as a critical factor that determines tumor survival and growth. Thus, the genes identified by this study that encode secreted proteins might provide important insights to the nature of the critical biological features in the microenvironment of each tumor type that allow these cells to thrive and proliferate.

  20. A gene from the cellulose synthase-like C family encodes a β-1,4 glucan synthase

    PubMed Central

    Cocuron, Jean-Christophe; Lerouxel, Olivier; Drakakaki, Georgia; Alonso, Ana P.; Liepman, Aaron H.; Keegstra, Kenneth; Raikhel, Natasha; Wilkerson, Curtis G.

    2007-01-01

    Despite the central role of xyloglucan (XyG) in plant cell wall structure and function, important details of its biosynthesis are not understood. To identify the gene(s) responsible for synthesizing the β-1,4 glucan backbone of XyG, we exploited a property of nasturtium (Tropaeolum majus) seed development. During the last stages of nasturtium seed maturation, a large amount of XyG is deposited as a reserve polysaccharide. A cDNA library was produced from mRNA isolated during the deposition of XyG, and partial sequences of 10,000 cDNA clones were determined. A single member of the C subfamily from the large family of cellulose synthase-like (CSL) genes was found to be overrepresented in the cDNA library. Heterologous expression of this gene in the yeast Pichia pastoris resulted in the production of a β-1,4 glucan, confirming that the CSLC protein has glucan synthase activity. The Arabidopsis CSLC4 gene, which is the gene with the highest sequence similarity to the nasturtium CSL gene, is coordinately expressed with other genes involved in XyG biosynthesis. These and other observations provide a compelling case that the CSLC gene family encode proteins that synthesize the XyG backbone. PMID:17488821

  1. A Lepidopteran-Specific Gene Family Encoding Valine-Rich Midgut Proteins

    PubMed Central

    Odman-Naresh, Jothini; Duevel, Margret; Muthukrishnan, Subbaratnam; Merzendorfer, Hans

    2013-01-01

    Many lepidopteran larvae are serious agricultural pests due to their feeding activity. Digestion of the plant diet occurs mainly in the midgut and is facilitated by the peritrophic matrix (PM), an extracellular sac-like structure, which lines the midgut epithelium and creates different digestive compartments. The PM is attracting increasing attention to control lepidopteran pests by interfering with this vital function. To identify novel PM components and thus potential targets for insecticides, we performed an immunoscreening with anti-PM antibodies using an expression library representing the larval midgut transcriptome of the tobacco hornworm, Manduca sexta. We identified three cDNAs encoding valine-rich midgut proteins of M. sexta (MsVmps), which appear to be loosely associated with the PM. They are members of a lepidopteran-specific family of nine VMP genes, which are exclusively expressed in larval stages in M. sexta. Most of the MsVMP transcripts are detected in the posterior midgut, with the highest levels observed for MsVMP1. To obtain further insight into Vmp function, we expressed MsVMP1 in insect cells and purified the recombinant protein. Lectin staining and glycosidase treatment indicated that MsVmp1 is highly O-glycosylated. In line with results from qPCR, immunoblots revealed that MsVmp1 amounts are highest in feeding larvae, while MsVmp1 is undetectable in starving and molting larvae. Finally using immunocytochemistry, we demonstrated that MsVmp1 localizes to the cytosol of columnar cells, which secrete MsVmp1 into the ectoperitrophic space in feeding larvae. In starving and molting larvae, MsVmp1 is found in the gut lumen, suggesting that the PM has increased its permeability. The present study demonstrates that lepidopteran species including many agricultural pests have evolved a set of unique proteins that are not found in any other taxon and thus may reflect an important adaptation in the highly specialized lepidopteran digestive tract facing

  2. Expression of a gene encoding β-ureidopropionase is critical for pollen germination in tomatoes.

    PubMed

    Shen, Yu-Hsing; Chen, Yu-Huei; Liu, Hao-Yun; Chiang, Fang-Yi; Wang, Yi-Chieh; Hou, Liang-Yu; Lin, Jeng-Shane; Lin, Chih-Ching; Lin, Hsin-Hung; Lai, Hsi-Mei; Jeng, Shih-Tong

    2014-03-01

    Global warming has seriously decreased world crop yield. High temperatures affect development, growth and, particularly, reproductive tissues in plants. A gene encoding β-ureidopropionase (SlUPB1, EC 3.5.1.6) was isolated from the stamens of a heat-tolerant tomato (CL5915) using suppression subtractive hybridization. SlUPB1 catalyzes the production of β-alanine, the only β-form amino acid in nature. In the anthesis stage, SlUPB1 expression in CL5915 stamens, growing at 35/30°C (day/night), was 2.16 and 2.93 times greater than that in a heat-sensitive tomato (L4783) cultivated at 30/25°C or 25/20°C, respectively. Transgenic tomatoes, upregulating SlUPB1 in L4783 and downregulating SlUPB1 in CL5915, were constructed, and the amount of β-alanine measured by liquid chromatography-electrospray ionization-mass spectrometry in the transgenic overexpression of SlUPB1 was higher than that of L4783. However, the β-alanine in the transgenics downregulating SlUPB1 was significantly lower than the β-alanine of CL5915. Pollen germination rates of these transgenics were analyzed under different developmental and germinating temperatures. The results indicated that germination rates of transgenics overexpressing SlUPB1 were higher than germination rates of the background tomato L4783. Germination rates of transgenics downregulating SlUPB1 were significantly lower than germination rates of background tomato CL5915, indicating the necessity of functional SlUPB1 for pollen germination. Pollen germinating in the buffer with the addition of β-alanine further indicated that β-alanine effectively enhanced pollen germination in tomatoes with low SlUPB1 expression. Together, these results showed that the expression of SlUPB1 is important for pollen germination, and β-alanine may play a role in pollen germination under both optimal and high temperatures.

  3. The Fungus Tremella mesenterica Encodes the Longest Metallothionein Currently Known: Gene, Protein and Metal Binding Characterization

    PubMed Central

    Lin, Weiyu; Calatayud, Sara; Palacios, Òscar; Capdevila, Mercè; Atrian, Sílvia

    2016-01-01

    Fungal Cu-thioneins, and among them, the paradigmatic Neurospora crassa metallothionein (MT) (26 residues), were once considered as the shortest MTs -the ubiquitous, versatile metal-binding proteins- among all organisms, and thus representatives of their primeval forms. Nowadays, fungal MTs of diverse lengths and sequence features are known, following the huge heterogeneity of the Kingdom of Fungi. At the opposite end of N. crassa MT, the recently reported Cryptococcus neoformans CnMT1 and CnMT2 (122 and 186 aa) constitute the longest reported fungal MTs, having been identified as virulence factors of this pathogen. CnMTs are high-capacity Cu-thioneins that appear to be built by tandem amplification of a basic unit, a 7-Cys segment homologous to N. crassa MT. Here, we report the in silico, in vivo and in vitro study of a still longer fungal MT, belonging to Tremella mesenterica (TmMT), a saprophytic ascomycete. The TmMT gene has 10 exons, and it yields a 779-bp mature transcript that encodes a 257 residue-long protein. This MT is also built by repeated fragments, but of variable number of Cys: six units of the 7-Cys building blocks-CXCX3CSCPPGXCXCAXCP-, two fragments of six Cys, plus three Cys at the N-terminus. TmMT metal binding abilities have been analyzed through the spectrophotometric and spectrometric characterization of its recombinant Zn-, Cd- and Cu-complexes. Results allow it to be unambiguous classified as a Cu-thionein, also of extraordinary coordinating capacity. According to this feature, when the TmMT cDNA is expressed in MT-devoid yeast cells, it is capable of restoring a high Cu tolerance level. Since it is not obvious that T. mesenterica shares the same physiological needs for a high capacity Cu-binding protein with C. neoformans, the existence of this peculiar MT might be better explained on the basis of a possible role in Cu-handling for the Cu-enzymes responsible in lignin degradation pathways. PMID:26882011

  4. The Fungus Tremella mesenterica Encodes the Longest Metallothionein Currently Known: Gene, Protein and Metal Binding Characterization.

    PubMed

    Iturbe-Espinoza, Paul; Gil-Moreno, Selene; Lin, Weiyu; Calatayud, Sara; Palacios, Òscar; Capdevila, Mercè; Atrian, Sílvia

    2016-01-01

    Fungal Cu-thioneins, and among them, the paradigmatic Neurospora crassa metallothionein (MT) (26 residues), were once considered as the shortest MTs--the ubiquitous, versatile metal-binding proteins--among all organisms, and thus representatives of their primeval forms. Nowadays, fungal MTs of diverse lengths and sequence features are known, following the huge heterogeneity of the Kingdom of Fungi. At the opposite end of N. crassa MT, the recently reported Cryptococcus neoformans CnMT1 and CnMT2 (122 and 186 aa) constitute the longest reported fungal MTs, having been identified as virulence factors of this pathogen. CnMTs are high-capacity Cu-thioneins that appear to be built by tandem amplification of a basic unit, a 7-Cys segment homologous to N. crassa MT. Here, we report the in silico, in vivo and in vitro study of a still longer fungal MT, belonging to Tremella mesenterica (TmMT), a saprophytic ascomycete. The TmMT gene has 10 exons, and it yields a 779-bp mature transcript that encodes a 257 residue-long protein. This MT is also built by repeated fragments, but of variable number of Cys: six units of the 7-Cys building blocks--CXCX3CSCPPGXCXCAXCP-, two fragments of six Cys, plus three Cys at the N-terminus. TmMT metal binding abilities have been analyzed through the spectrophotometric and spectrometric characterization of its recombinant Zn-, Cd- and Cu-complexes. Results allow it to be unambiguous classified as a Cu-thionein, also of extraordinary coordinating capacity. According to this feature, when the TmMT cDNA is expressed in MT-devoid yeast cells, it is capable of restoring a high Cu tolerance level. Since it is not obvious that T. mesenterica shares the same physiological needs for a high capacity Cu-binding protein with C. neoformans, the existence of this peculiar MT might be better explained on the basis of a possible role in Cu-handling for the Cu-enzymes responsible in lignin degradation pathways.

  5. The Streptomyces peucetius drrC gene encodes a UvrA-like protein involved in daunorubicin resistance and production.

    PubMed Central

    Lomovskaya, N; Hong, S K; Kim, S U; Fonstein, L; Furuya, K; Hutchinson, R C

    1996-01-01

    The drrC gene, cloned from the daunorubicin (DNR)- and doxorubicin-producing strain of Streptomyces peucetius ATCC 29050, encodes a 764-amino-acid protein with a strong sequence similarity to the Escherichia coli and Micrococcus luteus UvrA proteins involved in excision repair of DNA. Expression of drrC was correlated with the timing of DNR production in the growth medium tested and was not dependent on the presence of DNR. Since introduction of drrC into Streptomyces lividans imparted a DNR resistance phenotype, this gene is believed to be a DNR resistance gene. The drrC gene could be disrupted in the non-DNR-producing S. peucetius dnrJ mutant but not in the wild-type strain, and the resulting dnrJ drrC double mutant was significantly more sensitive to DNR in efficiency-of-plating experiments. Expression of drrC in an E. coli uvrA strain conferred significant DNR resistance to this highly DNR-sensitive mutant. However, the DrrC protein did not complement the uvrA mutation to protect the mutant from the lethal effects of UV or mitomycin even though it enhanced the UV resistance of a uvrA+ strain. We speculate that the DrrC protein mediates a novel type of DNR resistance, possibly different from the mechanism of DNR resistance governed by the S. peucetius drrAB genes, which are believed to encode a DNR antiporter. PMID:8655504

  6. A human homolog of the Saccharomyces cerevisiae REV3 gene, which encodes the catalytic subunit of DNA polymerase ζ

    PubMed Central

    Gibbs, Peter E. M.; McGregor, W. Glenn; Maher, Veronica M.; Nisson, Paul; Lawrence, Christopher W.

    1998-01-01

    To get a better understanding of mutagenic mechanisms in humans, we have cloned and sequenced the human homolog of the Saccharomyces cerevisiae REV3 gene. The yeast gene encodes the catalytic subunit of DNA polymerase ζ, a nonessential enzyme that is thought to carry out translesion replication and is responsible for virtually all DNA damage-induced mutagenesis and the majority of spontaneous mutagenesis. The human gene encodes an expected protein of 3,130 residues, about twice the size of the yeast protein (1,504 aa). The two proteins are 29% identical in an amino-terminal region of ≈340 residues, 39% identical in a carboxyl-terminal region of ≈850 residues, and 29% identical in a 55-residue region in the middle of the two genes. The sequence of the expected protein strongly predicts that it is the catalytic subunit of a DNA polymerase of the pol ζ type; the carboxyl-terminal domain possesses, in the right order, the six motifs characteristic of eukaryotic DNA polymerases, most closely resembles yeast pol ζ among all polymerases in the GenBank database, and is different from the human α, δ, and ɛ enzymes. Human cells expressing high levels of an hsREV3 antisense RNA fragment grow normally, but show little or no UV-induced mutagenesis and are slightly more sensitive to killing by UV. The human gene therefore appears to carry out a function similar to that of its yeast counterpart. PMID:9618506

  7. Saccharomyces cerevisiae protein phosphatase 2A performs an essential cellular function and is encoded by two genes.

    PubMed Central

    Sneddon, A A; Cohen, P T; Stark, M J

    1990-01-01

    Two genes (PPH21 and PPH22) encoding the yeast homologues of protein serine-threonine phosphatase 2A have been cloned from a Saccharomyces cerevisiae genomic library using a rabbit protein phosphatase 2A cDNA as a hybridization probe. The PPH genes are genetically linked on chromosome IV and are predicted to encode polypeptides each with 74% amino acid sequence identity to rabbit type 2A protein phosphatase, indicating once again the extraordinarily high degree of sequence conservation shown by protein-phosphatases from different species. The two PPH genes show less than 10% amino acid sequence divergence from each other and while disruption of either PPH gene alone is without any major effect, the double disruption is lethal. This indicates that protein phosphatase 2A activity is an essential cellular function in yeast. Measurement of type 2A protein phosphatase activity in yeast strains lacking one or other of the genes indicates that they account for most, if not all, protein phosphatase 2A activity in the cell. Images Fig. 5. PMID:2176150

  8. The Rb97D gene encodes a potential RNA-binding protein required for spermatogenesis in Drosophila.

    PubMed Central

    Karsch-Mizrachi, I; Haynes, S R

    1993-01-01

    Many proteins that bind RNA contain a common RNA-binding domain, the RNP motif. We have been studying two Drosophila RNP motif proteins, Hrb98DE and Hrb87F, which are hnRNA-binding proteins. We report here the characterization of the Rb97D gene, which encodes a protein that is closely related to the Hrb proteins in the RNP motif domain, but has a distinctive proline-rich C-terminal domain. The gene is located at 97D on the right arm of the third chromosome, near the rough gene. Multiple transcripts from the Rb97D gene are present at varying levels throughout development. The transcripts are generated by alternative processing in the coding and 3' untranslated regions, and can encode two protein isoforms. Analysis of a mutant containing a P element inserted into the 5' untranslated region of the gene demonstrates that Rb97D is required for male fertility. Possible models for the function of Rb97D in testes are discussed. Images PMID:8502565