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At least 415 records · Page 23Linked to original sources
PubMed1997

Gene knockout reveals a novel gene cluster for the synthesis of a class of cell wall lipids unique to pathogenic mycobacteria.

Surface-exposed unusual lipids containing phthiocerol and phenolphthiocerol are found only in the cell wall of slow-growing pathogenic mycobacteria and are thought to play important roles in host-pathogen interaction. The enzymology and molecular genetics of biosynthesis of phthiocerol and phenolphthiocerol are unknown. We postulate the domain organization of a set of multifunctional enzymes and a cluster of genes (pps) that would encode these enzymes for the biosynthesis of phthiocerol and phenolphthiocerol. A cosmid containing the postulated pps gene cluster was identified by screening a genomic library of Mycobacterium bovis BCG with the postulated homologous domains from mycocerosic acid synthase and fatty acid synthase genes as probes. Homologous cosmids were also identified in the genomic libraries of Mycobacterium tuberculosis and Mycobacterium leprae. M. bovis BCG was transformed with a pps disruption construct, made from the BCG cosmid by introducing the hygromycin resistance gene as the positive-selectable marker and the sacB gene as the counter-selectable marker. Gene disruption by homologous recombination with double crossover was confirmed by polymerase chain reaction and Southern hybridization. Chromatographic analysis showed that the phenolphthiocerol derivative, mycoside B, and phthiocerol dimycocerosates were not produced by the gene knockout mutants. This result confirms the identity of the pps genes. With the identification of the pps gene clusters in both M. tuberculosis and M. leprae, it should be possible to test the postulated roles of these unique lipids in tuberculosis and leprosy.

Acyl Coenzyme A↗
PubMed1986

Molecular cloning of the murine adenosine deaminase gene from a genetically enriched source: identification and characterization of the promoter region.

A genomic library was prepared with DNA from a genetically enriched mouse cell line in which amplified copies of the adenosine deaminase (ADA) gene account for over 5% of the genome. Overlapping cosmid clones encompassing the entire ADA structural gene were isolated from this genomic library and used for subsequent structural and functional analyses. Nuclease protection and primer extension analyses served to identify the location of multiple transcription initiation sites at the 5' end of the structural gene. Promoter activity was found by functional analyses to reside within a 240-base-pair fragment which contains the transcription initiation sites. Sequences upstream of the transcription initiation sites are very G + C rich (77%) and include a 22 nucleotide stretch of deoxyguanylate residues and two potential Sp1 transcription factor-binding sites. Comparison of the mouse and human ADA gene promoters revealed the presence of several regions that are highly conserved with regard to both sequence content and location and may represent genetic elements which are involved in ADA gene expression.

Acetyltransferases↗
PubMed1991

A novel approach to the rapid isolation and nucleotide sequencing of genomic clones.

In this study, a genomic library subdivided into fractions was rapidly screened by a Southern detection technique. Deletion libraries were obtained from recovered genomic clones by single random cuts with nuclease S1. These deletion libraries proved useful for localizing genes in the inserts and yielded, after size fractionation, nested deletions suitable for nucleotide sequencing. An heterologous vector (pDB21) carried the insert used as probe for all hybridizations involved in the process of genomic clones isolation and characterization.

Base Sequence↗
PubMed1997

Functional differences among the six Saccharomyces cerevisiae tRNATrp genes.

The Saccharomyces cerevisiae haploid genome includes six copies of the gene encoding tRNATrp which are scattered on five chromosomes. Other, non-functional tDNATrp fragments also occur in the genome. The segments of all six genes which encode the 72-nucleotide mature tRNATrp, as well as a 34-nucleotide intervening sequence, are identical. However, the 5' and 3' flanking sequences diverge virtually at the boundaries of the coding region. We have used an assay based on suppression of UGA mutations by multi-copy clones of tDNATrp to search for functional differences among these genes. Previous studies with one tDNATrp had demonstrated that moderate suppression of a UGA mutation, leu2-2, resulted from introduction of a multi-copy clone of the gene. Attempts to use this assay to select tDNATrp clones from a yeast genomic library yielded only four of the six different clones. The other two genes were amplified by PCR and cloned in pRS202, a 2 mu vector also used for the genomic library. Plasmids bearing the six tRNA genes were transformed into S. cerevisiae strain JG369.3B and scored for their ability to suppress the leu2-2 mutation as well as his4-260, another UGA marker. Two of the six tRNATrp clones were unable to suppress either marker, two evidenced weak suppression of the Leu auxotrophy, and two were able to suppress both markers. Growth rates in liquid media requiring suppression were measured for cell lines carrying each of the clones. Differences greater than 50-fold were observed in media lacking histidine. An evolutionary tree based on 5'-flanking sequence corresponds reasonably well with suppressor activity, while a similar analysis of 3'-flanking sequence does not. This suggests that the functional differences are based on divergence in the 5'-flanking sequences of the tRNATrp genes.

Base Sequence↗
PubMed1998

Discovery of three novel G-protein-coupled receptor genes.

We report here the molecular cloning, tissue distribution, and chromosomal localization of novel genes encoding G-protein-coupled receptors (GPCRs). A search of a mouse database of expressed sequence tags revealed an EST partially encoding a GPCR, which was used to screen a mouse genomic library to obtain the translational open reading frame (ORF). The resultant clone, GPR27, contained an intronless ORF, encoding a receptor of 379 amino acids. In an alternate strategy, human genomic DNA was subjected to polymerase chain reaction (PCR) amplification, using degenerate oligonucleotides based on GPR1. Two PCR products partially encoding GPCRs were isolated and used to screen a genomic library to obtain the translational ORF. One of the resultant clones, GPR30, contained an intronless ORF encoding a receptor of 375 amino acids. The other clone, GPR35, also contained an intronless ORF encoding a receptor of 309 amino acids. Transcripts corresponding to GPR27 and GPR30 were detected in several areas of human and rat CNS, While GPR35 expression was detected only in the rat intestine. Through fluorescence in situ hybridization analysis the gene encoding GPR30 was localized to chromosome 7p22 and GPR35 to chromosome 2q37.3.

Amino Acid Sequence↗
PubMed2005

Construction of a cytogenetically anchored microsatellite map in rabbit.

Rabbit (Oryctolagus cuniculus) represents a valuable source of biomedical models and corresponds to a small but active economic sector in Europe for meat and fur. The rabbit genome has not been thoroughly studied until recently, and high-resolution maps necessary for identification of genes and quantitative trait loci (QTL) are not yet available. Our aim was to isolate over 300 new and regularly distributed (TG)n or (TC)n rabbit microsatellites. To achieve this purpose, 164 microsatellite sequences were isolated from gene-containing bacterial artificial chromosome (BAC) clones previously localized by fluorescence in situ hybridization (FISH) on all the rabbit chromosomes. In addition, 141 microsatellite sequences were subcloned from a plasmid genomic library, and for 41 of these sequences, BAC clones were identified and FISH-mapped. TC repeats were present in 62% of the microsatellites derived from gene-containing BAC clones and in 22% of those from the plasmid genomic library, with an average of 42.9% irrespective of the microsatellite origin. These results suggest a higher proportion of (TC)n repeats and a nonhomogeneous distribution of (TG)n and (TC)n repeats in the rabbit genome compared to those in man. Among the 305 isolated microsatellites, 177 were assigned to 139 different cytogenetic positions on all the chromosomes except rabbit Chromosome 21. Sequence similarity searches provided hit locations on the Human Build 35a and hypothetical assignments on rabbit chromosomes for ten additional microsatellites. Taken together, these results report a reservoir of 305 new rabbit microsatellites of which 60% have a cytogenetic position. This is the first step toward the construction of an integrated cytogenetic and genetic map based on microsatellites homogeneously anchored to the rabbit genome.

Animals↗
PubMed1998

Rhizobium leguminosarum contains a group of genes that appear to code for methyl-accepting chemotaxis proteins.

Methyl-accepting chemotaxis proteins (MCPs) play important roles in the chemotactic response of many bacteria. Oligonucleotide primers designed to amplify the conserved signalling domain of MCPs by PCR were used to identify potential MCP-encoding genes in Rhizobium leguminosarum. Using a PCR-derived probe created from these primers a genomic library of R. leguminosarum VF39SM was screened; at least five putative MCP-encoding genes (termed mcpB to mcpF) were identified and isolated from the library. One of these putative genes (mcpC) is located on one of the indigenous plasmids of VF39SM. Fifteen different cosmids showing homology to an mcpD probe were also isolated from a genomic library. The complete DNA sequences of mcpB, mcpC and mcpD were obtained. All three genes code for proteins with characteristics typical of MCPs. However, the protein encoded by mcpB has a relatively large periplasmic domain compared to that in other MCPs. Partial DNA sequences of mcpE and mcpF had strong similarity to sequences from the methylation domains of known MCPs. Mutants defective in mcpB, mcpC, mcpD or mcpE were created using insertional mutagenesis strategies. Mutation of mcpB resulted in impairment of chemotaxis to a wide range of carbon sources on swarm plates; phenotypes for the other three mutants have yet to be elucidated. The mcpB, mcpC and mcpD mutants were tested for loss of nodulation competitiveness. When co-inoculated with the wild-type, the mcpB and mcpC mutants formed fewer nodules than the wild-type, whereas the mcpD mutant was just as competitive as the wild-type. The results overall suggest that R. leguminosarum possesses mcp-like genes, and that at least some of these play a role in early steps in the plant-microbe interaction.

Amino Acid Sequence↗
PubMed1997

Cloning, analysis, and overexpression of the gene encoding isobutylamine N-hydroxylase from the valanimycin producer, Streptomyces viridifaciens.

The flavoprotein isobutylamine N-hydroxylase (IBAH) catalyzes the oxidation of isobutylamine to isobutylhydroxylamine, a key step in the biosynthesis of the azoxy antibiotic valanimycin. By using oligonucleotide primers designed from peptide sequence information derived from native IBAH, a fragment of the gene (vlmH) encoding IBAH was amplified by PCR from a genomic library of the valanimycin-producing organism, Streptomyces viridifaciens MG456-hF10. The gene fragment was then employed as a probe to clone the entire vlmH gene from an S. viridifaciens genomic library. Overexpression of the vlmH gene in Escherichia coli gave a soluble protein that was purified to homogeneity. The purified protein exhibited the catalytic activity expected for IBAH. The deduced amino acid sequence of IBAH exhibited the greatest similarity to the Sox/DszC protein from Rhodococcus sp. strain IGT38, a flavoprotein involved in the oxidation of dibenzothiophene to the corresponding sulfone. Significant similarities were also found between the amino acid sequence of IBAH and those of the acyl coenzyme A dehydrogenases.

Amino Acid Sequence↗
PubMed1997

Cloning and chromosomal mapping of four putative novel human G-protein-coupled receptor genes.

We report the discovery of four novel human putative G-protein-coupled receptor (GPCR) genes. Gene GPR20 was isolated by amplifying genomic DNA with oligos based on the opioid and somatostatin related receptor genes and subsequent screening of a genomic library. Also, using our customized search procedure of a database of expressed sequence tags (dbEST), cDNA sequences that partially encoded novel GPCRs were identified. These cDNA fragments were obtained and used to screen a genomic library to isolate the full-length coding region of the genes. This resulted in the isolation of genes GPR21, GPR22 and GPR23. The four encoded receptors share significant identity to each other and to other members of the receptor family. Northern blot analysis revealed expression of GPR20 and GPR22 in several human brain regions while GPR20 expression was detected also in liver. Fluorescence in situ hybridization (FISH) was used to map GPR20 to chromosome 8q, region 24.3-24.2, GPR21 to chromosome 9, region q33, GPR22 to chromosome 7, region q22-q31.1, and GPR23 to chromosome X, region q13-q21.1.

Amino Acid Sequence↗
PubMed1986

Characterization of a defective phage system for the analysis of bacteriophage T4 DNA replication origins.

We have developed a defective phage system for the isolation and analysis of phage T4 replication origins based on the T4-mediated transduction of plasmid pBR322. During the initial infection of a plasmid-containing cell, recombinant plasmids with T4 DNA inserts are converted into fully modified linear DNA concatamers that are packaged into T4 phage particles, to create defective phage (transducing particles). In order to select T4 replication origins from genomic libraries of T4 sequences cloned into the plasmid pBR322, we searched for recombinant plasmids that transduce with an unusually high efficiency, reasoning that this should select for T4 sequences that function as origins on plasmid DNA after phage infection. We also selected for defective phage that can propagate efficiently with the aid of a coinfecting helper phage during subsequent rounds of phage infection, which should select for T4 sequences that can function as origins on the linear DNA present in the defective phage. Several T4 inserts were isolated repeatedly in one or both of these selective procedures, and these were mapped to particular locations on the T4 genome. When plasmids were selected in this way from genomic libraries constructed using different restriction nucleases, they contained overlapping segments of the T4 genome, indicating that the same T4 sequences were selected. The inserts in two of the selected plasmids permit a very high frequency of transduction from circular plasmids; these have been shown to contain a special type of T4 replication origin.

DNA Replication↗
PubMed1994

Isolation of expressed sequences encoded by the human Xq terminal portion using microclone probes generated by laser microdissection.

The genes that cause a variety of neurologic and neuromuscular disorders have been mapped to the distal region of Xq. In an effort to isolate genes from this area, a regional genomic library of the distal 30% of Xq was constructed from a single metaphase spread by means of laser microdissection and single unique primer-polymerase chain reaction. Using pooled probes of 1000 clones from the genomic library, human brain cDNA libraries were screened for expressed sequences encoded by this region. From the 250,000 cDNA clones screened so far, 10 nonoverlapping sequences that mapped back to the target portion were isolated. The complete nucleotide sequences of these cDNA clones have been determined. Analysis of the sequences indicates that none has significant similarity to previously characterized primate genes. One sequence mapping to Xq27.3-qter contained an open reading frame of 281 amino acids and was expressed in every tissue tested. This gene, as well as others isolated in this manner, may prove to be a candidate gene for heritable disorders mapping to this region.

Amino Acid Sequence↗
PubMed2000

Small-scale isolation of high molecular weight DNA from Leishmania braziliensis.

In this paper, we report a method for isolation of high molecular weight DNA from Leishmania promastigotes. This technique is especially indicated for small-scale purification of DNA suitable for the construction of highly representative genomic libraries. In our protocol, lysis buffer is compatible with RNase treatment, avoiding an additional precipitation step and consequent shearing of DNA. In order to prove the quality of the DNA isolated by this method, a Leishmania braziliensis genomic library was constructed, and an L. braziliensis KMP-11 gene was cloned after screening the library with a heterologous probe.

Animals↗
PubMed1984

Rearrangements of host and viral DNA in mouse cells transformed by simian virus 40.

We have determined the structure of host DNA and viral DNA at the site of integration of Simian virus 40 (SV40) in a line of transformed Balb/c-3T3 cells (SVB400) isolated by single cell cloning after virus infection. Recombinant phage containing integrated viral DNA and flanking host DNA were purified from a genomic library and, in conjunction with restriction endonuclease cleavage analysis of the transformed cell DNA, were used to determine the organization of the integrated viral sequences. There is heterogeneity in the arrangement of the viral sequences resulting from tandem duplications of all or part of the SV40 genome with preservation of the viral-host junctions. The predominant arrangement is the result of tandem duplication of 41% of the SV40 genome from 0.64 to 0.23. Analysis of the structure of integrated viral DNA in SVB400 at different passage numbers and in single cell clones derived from the 20th passage indicated that rearrangements of viral DNA occur after the integration event and continue with passage of the cells. The organization of host sequences before and after the integration of SV40 was determined by restriction endonuclease cleavage analysis of parental 3T3 DNA and SVB400 DNA, and by analysis of recombinant phage isolated from genomic libraries. A deletion of at least 15 X 10(3) bases of host DNA occurred at the site of integration, which indicates that viral integration was not a result of a simple insertion of SV40. Nucleotide sequence analysis of the virus-host junctions showed that retained SV40 sequences were colinear with the viral genome, and that the junctions with SV40 DNA occurred at nucleotide numbers 1377 and 3610. There was no evidence of duplications of viral or host sequences at the junctions, and a comparison of the flanking mouse sequences with the deleted SV40 sequences revealed no significant homology at the point of joining of the two genomes.

Animals↗
PubMed1987

Genomic clones encoding chicken myosin heavy-chain genes.

A chicken genomic library was screened with a cDNA probe containing the 3' coding and noncoding portions of quail fast-twitch skeletal muscle myosin heavy chain (MHC). This probe hybridized to seven to nine bands on Southern blots of chicken genomic DNA, and 17 clones that hybridized to this probe were obtained from the genomic library. Partial restriction maps were constructed and probable orientation of transcription was determined for each of the 17 clones. These maps indicate the presence of at least 14 unique MHC genes or pseudogenes. Dot-blot hybridization analysis using DNA complementary to RNA from a variety of chicken tissues demonstrated that these genes are all related to the gene for sarcomeric MHC, and permitted tentative assignment of the tissue of expression for several of the MHC isoforms. To substantiate further the dot-blot data, a subclone of one of the genes (4b1), which showed significant homology with adult breast muscle RNA but which also showed weaker hybridization to RNA from other tissues, was sequenced. The sequence data verified that the clone contains a portion of a MHC gene, that it contains both 3' coding and noncoding regions, and that its predicted amino acid sequence is identical (with 96% nucleotide homology) to that of the 75-bp quail fast MHC cDNA clone published by Hastings and Emerson (1982). Thus, clone 4b1 contains a portion of one of the genes that is expressed in adult chicken breast skeletal muscle tissue.

Amino Acid Sequence↗
PubMed1999

Discovery of three novel orphan G-protein-coupled receptors.

We have discovered three novel human genes, GPR34, GPR44, and GPR45, encoding family A G-protein-coupled receptors (GPCRs). The receptor encoded by GPR34 is most similar to the P2Y receptor subfamily, while the receptor encoded by GPR44 is most similar to chemoattractant receptors. The receptor encoded by GPR45 is the mammalian orthologue of a putative lysophosphatidic acid receptor from Xenopus laevis. Partial sequence of GPR34 was discovered during a search of the GenBank database of expressed sequence tags (ESTs). This sequence information was used both to isolate the full-length translational open reading frame from a human genomic library and to assemble a contig from additional GPR34 EST cDNAs. Northern blot and in situ hybridization analyses revealed GPR34 mRNA transcripts in several human and rat brain regions. Also, we used polymerase chain reaction (PCR) to amplify human genomic DNA using degenerate oligonucleotides designed from sequences encoding transmembrane domains 3 and 7 of opioid and somatostatin receptors. Two PCR products partially encoding novel GPCRs, named GPR44 and GPR45, were discovered and used to isolate the full-length translational open reading frames from a human genomic library. Both GPR44 and GPR45 are expressed in the central nervous system and periphery. For chromosomal localization, fluorescence in situ hybridization analysis was performed to assign GPR34 to chromosomes 4p12 and Xp11. 3, GPR44 to chromosome 11q12-q13.3, and GPR45 to chromosome 2q11. 1-q12.

Amino Acid Sequence↗
PubMed1990

Cloning and expression of a second Aspergillus niger pectin lyase gene (pelA): indications of a pectin lyase gene family in A. niger.

Using the previously cloned Aspergillus niger N756 pectin lyase D gene as a probe, the corresponding pelD gene has been isolated from a genomic library of the laboratory strain A. niger N400. This gene encodes PLD, previously described as PLI, which is one of the two major pectin lyases isolated from the commercial pectinase preparation Ultrazym. Heterologous hybridization of the A. niger N400 genomic library with the pelD gene led to the isolation of another five genes: pelA, B, C, E, and F. These genes differ in their hybridization patterns with probes containing either the entire pelD gene, or 5' or 3' parts thereof. By partial sequencing, and expression in an A. niger transformant containing multiple copies of the pelA gene, we show that this gene, which hybridizes strongest with the pelD gene, encodes the other major pectin lyase from Ultrazym, PLII.

Amino Acid Sequence↗
PubMed1996

Characterization and genetic mapping of simple repeat sequences in the tomato genome.

Tomato genomic libraries were screened for the presence of simple sequence repeats (SSRs) with seventeen synthetic oligonucleotide probes, consisting of 2- to 5-basepair motifs repeated in tandem. GAn and GTn sequences were found to occur most frequently in the tomato genome (every 1.2 Mb), followed by ATTn and GCCn (every 1.4 Mb and 1.5 Mb, respectively). In contrast, only ATn and GAn microsatellites (n > 7) were found to be frequent in the GenBank database, suggesting that other motifs may be preferentially located away from genes. Polymorphism of microstellites was measured by PCR amplification of individual loci of by Southern hybridization, using a set of ten tomato cultivars. Surprisingly, only two of the nine microsatellite clones surveyed (five GTn, three GAn and one ATTn), showed length variation among these accessions. Polymorphism was also very limited between Lycopersicon esculentum and L. pennelli, two distant species. Southern analysis using the seventeen oligonucleotide probes identified GATAn and GAAAn as useful motifs for the detection of multiple polymorphic fragments among tomato cultivars. To determine the structure of microsatellite loci, a GAn probe was used for hybridization at low stringency on a small insert genomic library, and randomly selected clones were analyzed. GAn based motifs of increasing complexity were found, indicating that simple dinucleotide sequences may have evolved into larger tandem repeats such as minisatellites as a result of basepair substitution, replication slippage, and possibly unequal crossing-over. Finally, we genetically mapped loci corresponding to two amplified microsatellites, as well as nine large hypervariable fragments detected by Southern hybridization with a GATA8 probe. All loci are located around putative tomato centromeres. This may contribute to understanding of the structure of centromeric regions in tomato.

Base Sequence↗
PubMed2003

Brachyspira hyodysenteriae contains eight linked gene copies related to an expressed 39-kDa surface protein.

A tandemly linked set of four open reading frames (ORFs), identified as vspA-D (variable surface protein) had been identified from previous cloning and sequencing of clones from a genomic library constructed from Brachyspira hyodysenteriae strain B204. The predicted translation products of these closely related genes were homologous to (but not identical with) a characterized 39-kDa surface-exposed membrane protein from this animal pathogen. Additional screening of the genomic library has been performed to retrieve what are believed to be additional vsp genes including the one expected to encode this 39-kDa protein. Four new vsp genes have been identified and found to be associated in a second set of four tandemly linked alleles. This new gene cluster of 7481 nucleotides is not adjacent to the original vspA-D gene cluster described but does appear to have arisen from a gene (region) duplication event. The new vsp genes (identified as vspE-H) are oriented parallel to one another and appear to have a set of similar but distinct regulatory elements that may control separate expression of their ORFs. The four adjacent ORFs are of similar size (361-390 codons) and share from 83% to 90% identity in their amino acid sequence. The organization and homologies of these highly conserved multiple gene copies are discussed.

Alleles↗