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Type 2 vasopressin receptor gene, the gene responsible nephrogenic diabetes insipidus, maps to Xq28 close to the LICAM gene.

Although long contigs have been assembled in Xq28, the interval between the anonymous probe St14 and the color vision locus is still incompletely defined. We report here that the recently cloned gene for type 2 vasopressin receptor (V2R) is physically linked to L1CAM using YACs and cosmids across about 180 kb of the region. Since it is known that L1CAM maps near the color pigment genes, this finding locates V2R in Xq28 in the area where nephrogenic diabetes insipidus (NDI) has been mapped by linkage analysis. The PFGE analysis of the clones positions V2R about 40 kb from the L1CAM gene in a region that appears to contain other unknown genes, since at least four putative CpG islands were identified by restriction analysis with rare cutter enzymes.

Animals↗

Cloning of salicylate hydroxylase gene and catechol 2,3-dioxygenase gene and sequencing of an intergenic sequence between the two genes of Pseudomonas putida KF715.

The salicylate hydroxylase can convert the salicylate to catechol, and catechol 2,3-dioxygenase catalizes the conversion of catechol to 2-hydroxymuconic semialdehyde. A salicylate hydroxylase gene and a catechol 2,3-dioxygenase have been cloned from chromosomal DNA of P. putida KF715. The two genes have different promoters. An open reading frame with 339 nucleotides preceded by a putative ribosome-binding sequence (GGAGG) was identified in the intergenic sequence between salicylate hydroxylase gene and catechol 2,3-dioxygenase gene of P. putida KF715 and its sequence analyzed. This open reading frame can encode a polypeptide of molecular weight 13 kDa containing 112 amino acids, whose sequence exhibited 87% homology with that of ferredoxin encoded in NAH7 of P. putida PpG7 and significant homology with those of redox components in phenol hydroxylase, benzoate 1,2-dioxygenase, toluate 1,2-dioxygenase, xylene monooxygenase, and toluene 4-monooxygenase.

Amino Acid Sequence↗

Chromosomal mapping of a family of human glutamine synthetase genes: functional gene (GLUL) on 1q25, pseudogene (GLULP) on 9p13, and three related genes (GLULL1, GLULL2, GLULL3) on 5q33, 11p15, and 11q24.

Glutamine synthetase (GS) is a ubiquitous enzyme that catalyzes the ATP-dependent conversion of glutamate to glutamine using ammonia as the nitrogen source. Using human GS cDNA as a probe, a bacterial artificial chromosome (BAC) library consisting of two-fold coverage of the human genome was screened, and 18 clones were obtained. The restriction analysis of the human insert DNAs provided a basis to divide these 18 BAC clones into five groups, suggesting the existence of a GS gene family in the human genome. PCR analysis using three sets of primers designed from the reported sequences for GS cDNA and a processed pseudogene identified the corresponding BAC clones. Fluorescence in situ hybridization analysis revealed the chromosomal localization of these five genes: the GS gene to 1q25 (GLUL), the processed pseudogene to 9p13 (GLULP), and three related gene to 5q33 (GLULL1), 11p15 (GLULL2), and 11q24 (GLULL3), respectively.

Chromosome Mapping↗

Water deficit modulates gene expression in growing zones of soybean seedlings. Analysis of differentially expressed cDNAs, a new beta-tubulin gene, and expression of genes encoding cell wall proteins.

Transfer of soybean seedlings to low-water-potential vermiculite (psi w = -0.3 MPa) results in a reversible decrease in hypocotyl growth and modulation of several polysomal mRNAs (Plant Physiol 92: 205-214). We report here the isolation of two cDNA clones (pGE16 and pGE95) which correspond to genes whose mRNA levels are increased, and one cDNA clone (pGE23) which corresponds to a gene whose mRNA level is decreased in the hypocotyl zone of cell elongation by water deficit. In well-watered seedlings mRNAs hybridizing to pGE16 and pGE95 are most abundant in mature regions of the seedling, but in water-deficient seedlings mRNA levels are reduced in mature regions and enhanced in elongating regions. RNA corresponding to soybean proline-rich protein 1 (sbPRP1) shows a similar tissue distribution and response to water deficit. In contrast, in well-watered seedlings, the gene corresponding to pGE23 was highly expressed in the hypocotyl and root growing zones. Transfer of seedlings to low-water-potential vermiculite caused a rapid decrease in mRNA hybridizing to pGE23. Sequence analysis revealed that pGE23 has high homology with beta-tubulin. Water deficit also reduced the level of mRNA hybridizing to JCW1, an auxin-modulated gene, although with different kinetics. Furthermore, mRNA encoding actin, glycine-rich proteins (GRPs), and hydroxyproline-rich glycoproteins (HRGPs) were down-regulated in the hypocotyl zone of elongation of seedlings exposed to water deficit. No effect of water deficit was observed on the expression of chalcone synthase. Decreased expression of beta-tubulin, actin, JCW1, HRGP and GRP and increased expression of sbPRP1, pGE95 and pGE16 in the hypocotyl zone of cell elongation could participate in the reversible growth inhibition observed in water-deficient soybean seedlings.

Actins↗

Molecular analysis of the dhp1+ gene of Schizosaccharomyces pombe: an essential gene that has homology to the DST2 and RAT1 genes of Saccharomyces cerevisiae.

The DST2 gene of Saccharomyces cerevisiae encodes a DNA strand exchange protein, STP beta, which is required for homologous recombination in both mitotic or meiotic cells. We have cloned a DST2-related gene from the fission yeast Schizosaccharomyces pombe and designated it dhp1+. The nucleotide sequence of dhp1+ revealed an open reading frame encoding a protein composed of 991 amino acids. The predicted amino acid sequence was significantly homologous to the S. cerevisiae STP beta, but lacked the carboxy-terminal sequence present in STP beta. Furthermore, dhp1+ shows greater homology to RAT1/HKE1, a gene which is involved in RNA trafficking and processing. Genetic experiments showed that dhp1+ on an S. cerevisiae expression vector could rescue both the defects of the S. cerevisiae DST2 disruptant, slow growth rate and a sporulation defect, and the lethality of the S. cerevisiae rat1ts mutation. This implies the functional similarity of dhp1+ to both DST2 and RAT1. However unlike DST2, dhp1+ is an essential gene for cell growth in S. pombe, suggesting that dhp1+ is not the true homologue of DST2 but rather of RAT1 in S. pombe. The possible roles of dhp1+ in recombination and cell growth in S. pombe are discussed.

Amino Acid Sequence↗

[Gene structure and gene expression. Chicken lysozyme gene].

In the chicken oviduct the synthesis of the egg-white proteins ovalbumin, conalbumin, ovomucoid and lysozyme is controlled by the female sex steroids. The steroid-controlled rate of synthesis of these proteins is closely correlated to the cellular concentration of the egg-white protein mRNAs. The accumulation of these mRNAs is achieved by a decreased rate of degradation and an increased rate of synthesis. To understand the molecular mechanism that regulates the expression of this group of genes we have isolated the lysozyme and ovomucoid gene by gene-cloning. The structure and expression of the lysozyme gene has been studied.

Animals↗

The yptV1 gene encodes a small G-protein in the green alga Volvox carteri: gene structure and properties of the gene product.

Small G-proteins encoded by ras-like genes are ubiquitous in eukaryotic cells. These G-proteins are believed to play a role in central processes, such as signal transduction, cell differentiation and membrane vesicle transport. By screening genomic and cDNA libraries of the colonial alga, Volvox carteri f. nagariensis, with ypt DNA probes from Zea mays, we have identified the first member of a ypt gene family, yptV1, within a green alga. The 1538-bp yptV1 gene of V. carteri consists of nine exons and eight introns and has three potential polyadenylation sites 210, 420 and 500 bp downstream from the UGA stop codon. The derived 203-amino-acid polypeptide, YptV1, exhibits 81% similarity with Ypt1 from mouse, with the corresponding genes sharing four identical intron positions. Recombinant YptV1 (reYptV1) produced in Escherichia coli retains the ability to bind GTP after SDS-PAGE and immobilization on nitrocellulose. Immunological studies using polyclonal antibodies against reYptV1 indicate that the protein is present in the membrane fraction of a V. carteri extract and is expressed throughout the whole life-cycle of the alga. Similar to other Ras-like proteins, YptV1 contains two conserved C-terminal cysteine residues suggesting post-translational modification(s), such as isoprenylation or palmitoylation, required for membrane anchoring. The presumptive role of YptV1 in cytoplasmic vesicle transport is briefly discussed.

Amino Acid Sequence↗

Over-expression of the Saccharomyces cerevisiae exo-beta-1,3-glucanase gene together with the Bacillus subtilis endo-beta-1,3-1,4-glucanase gene and the Butyrivibrio fibrisolvens endo-beta-1,4-glucanase gene in yeast.

The EXG1 gene encoding the main Saccharomyces cerevisiae exo-beta-1,3-glucanase was cloned and over-expressed in yeast. The Bacillus subtilis endo-1,3-1,4-beta-glucanase gene (beg1) and the Butyrivibrio fibrisolvens endo-beta-1,4-glucanase gene (end1) were fused to the secretion signal sequence of the yeast mating pheromone alpha-factor (MF alpha 1S) and inserted between the yeast alcohol dehydrogenase II gene promoter (ADH2P) and terminator (ADH2T). Constructs ADH2P-MF alpha 1S-beg1-ADH2T and ADH2P-MF alpha 1S-end 1-ADH2T designated BEG1 and END1, respectively, were expressed separately and jointly with EXG1 in S. cerevisiae. The construction of fur 1 ura3 S. cerevisiae strains allowed for the autoselection of these multicopy URA3-based plasmids in rich medium. Enzyme assays confirmed that co-expression of EXG1, BEG1 and END1 enhanced glucan degradation by S. cerevisiae.

Amino Acid Sequence↗

The left border of the genomic inversion of pseudorabies virus contains genes homologous to the UL46 and UL47 genes of herpes simplex virus type 1, but no UL45 gene.

The genome of pseudorabies virus (PrV) is collinear with the herpes simplex virus type 1 (HSV1) genome, except for an inversion in the unique long region, the right extremity of which resides within the BamHI fragment 9 and the left within the BamHI fragment 1. We previously sequenced the right border of the inversion which is situated next to the UL44-gC gene and found that it encodes the UL24, UL25, UL26 and UL26.5 gene counterparts of HSV1. We have now sequenced 5317 base pairs of the BamHI fragment 1, upstream of the UL27-gB gene. We found two open reading frames homologous to UL46 and UL47 of HSV1 yet UL45 was absent and replaced by a set of strictly repeated sequences. PrV UL46 and UL47 are transcribed into two 3' co-terminal messenger RNAs with early and late kinetics, respectively. Comparison of the PrV UL46 and UL47 protein sequences with their counterparts from alphaherpesviruses indicated a strong similarity. The genome is rearranged in this region with respect to HSV1 and the inversion must have taken place, on the left side, within the UL46-UL27 intergenic region. Thus, the inversion should include genes UL27 to UL44.

Amino Acid Sequence↗

Cloning of a novel murine gene Sfmbt, Scm-related gene containing four mbt domains, structurally belonging to the Polycomb group of genes.

A novel cDNA clone encoding a protein structurally related to the transcriptional repressor Polycomb group (PcG) proteins, which regulate homeotic genes and others, was isolated from mouse and rat brain. The coding protein contained the SPM domain and mbt repeats, both of which are characteristic of the PcG proteins, and showed significant similarity in amino acid sequence to the Drosophila Sex comb on midleg (Scm) protein. Since this novel protein contains the mbt repeats in four tandem copies, we designated this murine gene as Sfmbt for Scm-related gene containing four mbt domains. Cloning and characterization of the mouse Sfmbt gene revealed that the coding sequence comprised 20 exons, dispersed along approximately 40kb, and mapped to the proximal part of Chromosome 14. Northern blot analysis showed that the Sfmbt mRNAs were expressed most abundantly in the adult testis, and less intensively in all other tissues examined.

Amino Acid Sequence↗

Gene therapy for hepatocellular carcinoma based on tumour-selective suicide gene expression using the alpha-fetoprotein (AFP) enhancer and a housekeeping gene promoter.

The aim of this study was to examine whether the human alpha-fetoprotein (AFP) enhancer could be used to induce hepatocellular carcinoma (HCC)-selective expression of the herpes simplex virus thymidine kinase (HSV-tk) gene which is under the control of the phosphoglycerate kinase (pgk) promotor. The human AFP enhancer was linked with the non-tissue-specific, human housekeeping pgk promoter in a retroviral vector. AFP-producing HCC cells infected with retroviruses carrying the HSV-tk gene under the control of the AFP enhancer/pgk promoter were much more susceptible to the prodrug, ganciclovir (GCV), than those infected with the same retroviruses without the AFP enhancer. Non-HCC cells infected with retroviruses carrying the HSV-tk gene under the control of the AFP enhancer/pgk promoter exhibited profoundly increased resistance to GCV compared with those infected with the same retroviruses without the AFP enhancer. Northern blot analysis revealed that the AFP enhancer caused enhanced HSV-tk expression in AFP-producing HCC cells and suppressed HSV-tk expression in non-HCC cells. Our results indicate that the AFP enhancer could give HCC selectivity to the pgk promoter, and that this novel strategy may be useful for HCC-selective cancer gene therapy.

Blotting, Northern↗

Interaction between mendelian and non-mendelian genes. Regulation of the transmission of non-mendelian genes by a mendelian gene in Chlamydomonas reinhardtii.

The non-Mendelian genetic element of a given mating type of Chlamydomonas reinhardtii was first doubly marked by succesive mutageneses and then transferred into a number of offspring cells of the opposite mating type. In subsequent crosses of these offspring clones, the transmission pattern of the original non-Mendelian markers was analyzed. The results indicate that the mating-type gene of a given cell regulates the transmission and perhaps the recombination of the non-Mendelian genes regardless of whether the non-Mendelian markers were isolated originally in the same cell or obtained from another cell of the opposite mating type through genetic transmission. The regulation of the transmission of non-Mendelian genes in C. reinhardtii is thus apparently different from the autonomous control of the transmission of non-Mendelian mitochondrial genes in yeast by a genetic factor located on the mitochondrial genome.

Chlamydomonas↗

The promoter of the spinach PsaF gene for the subunit III of the photosystem I reaction center directs beta-glucuronidase gene expression in transgenic tobacco roots. Implication of the involvement of phospholipases and protein kinase C in PsaF gene expression.

PsaF is a nuclear encoded gene for the subunit III of photosystem I. It is located at the lumenal side of the thylakoid membrane and interacts with plastocyanin. Starting from a low-level expression in the cotyledons of etiolated seedlings the gene is upregulated by light. Light can be replaced by Ca2+ or phosphoinositides like phorbol myristate acetate, an analogue of diacylglycerol. We tested the effects of these components on PsaF promoter-driven gene expression in roots and found that the PsaF promoter includes a positive regulatory region [-220/-179] activated by cytokinin and a negative regulatory region [-687/-221] activated by abscisic acid. In addition, the promoter is activated by Ca2+, mastoparan and phorbol myristate acetate which suggests a role for phospholipases and protein kinase C in PsaF gene expression.

Abscisic Acid↗

Isolation and sequencing of glycosyltransferase gene and UDP-glucose dehydrogenase gene that are located on a gene cluster involved in a new exopolysaccharide biosynthesis in Streptomyces.

Streptomyces sp. 139 produces a new exopolysaccharide (EPS) that shows anti-rheumatic arthritis activity in vivo. To investigate the gene cluster involved in EPS biosynthesis, degenerate primers were designed to amplify an internal fragment of the priming glycosyltransferase gene that catalyzes the first step in EPS biosynthesis. Using this PCR product as probe, positive cosmid clones were selected from a genomic library of Streptomyces sp. 139, which led to the localization of ste (Streptomyces eps) gene cluster on the approximately 65-kb chromosomal region. A 4.0-kb Bam HI fragment from all positive cosmids that hybridized to this probe was sequenced, which revealed two genes encoding the priming glycosyltransferase and UDP-glucose dehydrogenase. The putative priming glycosyltransferase is suggested to catalyze the first step in the biosynthesis of EPS repeating unit and UDP-glucose dehydrogenase is suggested to convert UDP-glucose into UDP-glucuronic acid involved in nucleotide sugar precursor synthesis of EPS biosynthesis.

Amino Acid Sequence↗

A recombination repair gene of Schizosaccharomyces pombe, rhp57, is a functional homolog of the Saccharomyces cerevisiae RAD57 gene and is phylogenetically related to the human XRCC3 gene.

To identify Schizosaccharomyces pombe genes involved in recombination repair, we identified seven mutants that were hypersensitive to both methyl methanesulfonate (MMS) and gamma-rays and that contained mutations that caused synthetic lethality when combined with a rad2 mutation. One of the mutants was used to clone the corresponding gene from a genomic library by complementation of the MMS-sensitive phenotype. The gene obtained encodes a protein of 354 amino acids whose sequence is 32% identical to that of the Rad57 protein of Saccharomyces cerevisiae. An rhp57 (RAD57 homolog of S. pombe) deletion strain was more sensitive to MMS, UV, and gamma-rays than the wild-type strain and showed a reduction in the frequency of mitotic homologous recombination. The MMS sensitivity was more severe at lower temperature and was suppressed by the presence of a multicopy plasmid bearing the rhp51 gene. An rhp51 rhp57 double mutant was as sensitive to UV and gamma-rays as an rhp51 single mutant, indicating that rhp51 function is epistatic to that of rhp57. These characteristics of the rhp57 mutants are very similar to those of S. cerevisiae rad57 mutants. Phylogenetic analysis suggests that Rhp57 and Rad57 are evolutionarily closest to human Xrcc3 of the RecA/Rad51 family of proteins.

Adenosine Triphosphatases↗

Polymorphisms of the angiotensinogen gene, the endothelial nitric oxide synthase gene, and the interleukin-1beta gene promoter in women with idiopathic recurrent miscarriage.

Interleukin (IL)-1beta, angiotensinogen (Agt), and endothelium-derived nitric oxide synthase (eNOS) are thought to be involved in idiopathic recurrent miscarriage (IRM). We investigated the correlation between IRM and common polymorphisms in Agt, Nos3 and IL-1beta genes: one polymorphism in the promoter region of the IL-1beta gene, one in exon 2 of the Agt gene, and one in exon 7 of the Nos3 gene. A total of 130 women with a history of IRM and 67 healthy control women were included in the study. Genotyping for the C/T transition at position -511 in the promoter region of IL1B, for the single base M235T polymorphism of Agt, and for the missense Glu298Asp variant of Nos3 was performed using PCR, an allele-specific oligonucleotide hybridization assay, and pyrosequencing, respectively. Allele and genotype frequencies of all polymorphisms were similar among women with IRM and controls. Between women with primary and secondary recurrent miscarriages, no statistically significant differences between allele and genotype frequencies were observed. Despite promising experimental data, our data fall short of showing any significant association between a variant of the promoter region of IL1B, the M235T polymorphism of Agt, and the Glu298Asp missense variant of Nos3 and the occurrence of IRM.

Abortion, Habitual↗

Two additional 5' exons in the human Vigilin gene distinguish it from the chicken gene and provide the structural basis for differential routes of gene expression.

Vigilin, a 150-kDa protein, contains 14 tandemly arranged domains, each consisting of a KH RNA-binding motif and a spacer region. Here, we report on the physical structure of the human Vigilin gene with 29 exons, thereby outnumbering the chicken gene by two additional 5' exons. These additional exons, 1A and 1B, are alternatively though concurrently spliced to exon 1C which is homologous to the first exon in the chicken gene. None of the additional human exons code for an amino-terminal extension of Vigilin, due to in-frame stop codons. Structural features of exon 1A, however, would allow the translation of a 13-amino-acid peptide from an upstream open reading frame preceding the vigilin open reading frame. We suggest that exons 1A and 1B have been gained during evolution, allowing alternative routes of expression control of the human Vigilin gene.

Alternative Splicing↗

The gene overexpressed in polycythemia rubra vera, PRV-1, and the gene encoding a neutrophil alloantigen, NB1, are alleles of a single gene, CD177, in chromosome band 19q13.31.

BACKGROUND: PRV-1 mRNA is overexpressed by neutrophils from polycythemia vera patients and is homologous to NB1 a gene overexpressed in reactive neutrophilia. STUDY DESIGN AND METHODS: These investigations were designed to confirm searches of genome databases suggesting that PRV-1 and NB1 are alleles of the same gene, CD177, and confirm a pseudogene adjacent to CD177. Methods included polymerase chain reaction (PCR), cloning, sequencing, and fluorescent hybridization studies. RESULTS: The coding region of PRV-1 was PCR-amplified from human fetal RNA, cloned, and used to screen the RPCI-11 bacterial artificial chromosome (BAC) library. Five BACs were reactive with the PRV-1 probe. PCR analysis of the BACs with primers encompassing PRV-1 exons, containing four known single-nucleotide polymorphisms, followed by sequencing rendered amplicons identical to PRV-1 in all five BACs. Analysis of all five by restriction digestion yielded fragments possible only if both the gene and the pseudogene are present. End sequencing of the BACs localized them to the same chromosome region. G-banding and fluorescence in situ hybridization at the 400- and 850-band levels of resolution mapped one BAC to chromosome band 19q13.2 and sublocalized the BAC to band 19q13.31, respectively. CONCLUSION: PRV-1 and NB1 are alleles of the same gene now referred to as CD177. Changes in CD177 expression may be a marker of increased or decreased myelopoiesis and are therefore an effect of, rather than a cause of, myeloproliferative disorders.

Alleles↗