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At least 865 records · Page 48Linked to original sources
PubMed1997

Eighteen novel human genes regionally mapped on chromosome 11.

Expression sequence tags (EST) obtained by sequencing a randomly primed cDNA library and gene signatures (GS) obtained by sequencing a 3'-directed cDNA library can identify genes that are active in the source cells. Eight ESTs and ten GSs which represent novel human genes, except for one GS, and which have been assigned to human chromosome 11 were used to select cosmids from a chromosome 11-specific cosmid library. These cosmids were regionally mapped using the fluorescence in situ hybridization technique.

Animals↗
PubMed1996

Physical map of the malaria vector Anopheles gambiae.

Random cDNA clones, cosmid clones and RAPD polymorphic fragments have been localized by in situ hybridization to the ovarian nurse cell polytene chromosomes of the malaria vector Anopheles gambiae. We thus established 85 molecular markers for 110 sites within the whole A. gambiae polytene chromosome complement. The cDNA clones analyzed were isolated at random, and their exact localizations were determined by in situ hybridization. For 15 of the cDNA clones, a partial nucleotide sequence has been obtained; for nine of them sequence searches in the GenBank database revealed high degrees of similarity with published sequences. The cosmid clones analyzed were obtained as the result of screening with a few of the aforementioned cDNA clones of particular interest, or taken from a small set of randomly isolated cosmid clones. The RAPD clones are polymorphic fragments, potentially diagnostic for the various chromosomal forms of A. gambiae that are currently being analyzed.

Animals↗
PubMed2001

Integration of the Aedes aegypti mosquito genetic linkage and physical maps.

Two approaches were used to correlate the Aedes aegypti genetic linkage map to the physical map. STS markers were developed for previously mapped RFLP-based genetic markers so that large genomic clones from cosmid libraries could be found and placed to the metaphase chromosome physical maps using standard FISH methods. Eight cosmids were identified that contained eight RFLP marker sequences, and these cosmids were located on the metaphase chromosomes. Twenty-one cDNAs were mapped directly to metaphase chromosomes using a FISH amplification procedure. The chromosome numbering schemes of the genetic linkage and physical maps corresponded directly and the orientations of the genetic linkage maps for chromosomes 2 and 3 were inverted relative to the physical maps. While the chromosome 2 linkage map represented essentially 100% of chromosome 2, approximately 65% of the chromosome 1 linkage map mapped to only 36% of the short p-arm and 83% of the chromosome 3 physical map contained the complete genetic linkage map. Since the genetic linkage map is a RFLP cDNA-based map, these data also provide a minimal estimate for the size of the euchromatic regions. The implications of these findings on positional cloning in A. aegypti are discussed.

Aedes↗
PubMed1992

Identification and characterization of a new gene in the human Xq28 region.

A human Xqter chromosome cosmid library was screened with a mixed probe derived from porcine kidney mRNA. A new expressed gene was identified in a cosmid clone known to be part of a G6PD cosmid contig. This gene is most likely a housekeeping gene because the cDNA clone recognizes a 1 kb mRNA transcript in all cell lines and tissues tested. Hybridizing genomic DNA of several species with a cDNA probe indicated that the gene is highly conserved during evolution and that it belongs to a gene family. The genomic sequence shows a 100% homology with the recently identified QM cDNA sequence.

Adrenoleukodystrophy↗
PubMed1992

High-resolution in situ hybridization using DNA halo preparations.

To improve DNA resolution of fluorescence in situ hybridization we have adapted a nuclear extraction technique, resulting in highly extended DNA loops arranged around the nuclear matrix in a halo-like structure. In situ hybridization signals from alphoid and cosmid DNAs appear as beads-on-a-string, which, according to preliminary experiments, results from the association of individual probe fragments. By multicolor hybridizations we have been able to determine relative map position and to easily detect 10 kb overlap between individual cosmid clones, each of which shows linear beaded signals of ca. 10 microns, suggesting that the DNA is essentially linearized in our protocol. The map configuration can be typically derived from analysis of 5-10 cells only. The resolution range of the technique is at least 10-200 kb, and probably as little as a few kb, thus greatly extending the abilities of the existing FISH methodologies. This novel technique is much more efficient and practicable than pronuclei hybridizations, another method for high resolution FISH, and readily produces results with probes of a variety of genomic origin. In conclusion the DNA halo technique should be able to contribute significantly to the assessment of cosmid and YAC overlaps as well as to the sizing of gaps between adjacent contigs generated in genome projects.

Cells, Cultured↗
PubMed1993

Identification of human chromosome 9 specific genes using exon amplification.

We have recently developed a method, exon amplification, that is designed for isolation of exon sequences from genomic DNA. To assess the efficacy of this method we have analyzed cosmid genomic clones derived from human chromosome 9, and have cloned several products from this analysis. Approximately 63% of cosmids produced at least one product derived from functioning splice sites within the target genomic fragment, and in many cases multiple products were isolated. In addition, an easily identifiable class of false positives was produced from 56% of cosmids analyzed; these are readily eliminated from subsequent study. Sequence analysis and database searches revealed that the majority (87%) of the putative exon clones were unique, the remainder being derived from repetitive sequences. Analysis of sequence conservation by Southern blotting in addition to cDNA screening experiments suggested that most, if not all, of these unique sequences represent true exons. The results of these studies indicate that exon amplification is a rapid and reliable approach for isolation of exon sequences from mammalian genomic DNA.

Animals↗
PubMed1993

Isolation of the human Xp21 glycerol kinase gene by positional cloning.

The gene for human glycerol kinase deficiency (GK) maps in Xp21.3 in a critical region of about 50-250 kb located distal to the Duchenne muscular dystrophy gene (DMD) by analysis of patient deletions and YAC contigs. We have used a genomic exon amplification strategy to isolate potential exons from two cosmids which mapped to this interval. The genomic exons were used to isolate six overlapping cDNA clones from human fetal liver which encode the X-linked glycerol kinase gene. The cDNA clones map to cosmids, YAC clones and deletions in patients which define the GK critical region and also hybridize to several autosomal fragments and one Xq fragment in genomic DNA. The GK gene is expressed most in human liver with three transcript sizes of 1.85, 2.7, and 3.7 kb. Sequence analysis of 1.5 kb of several overlapping liver cDNA clones predicted a protein with approximately 63% similarity to the E. coli and B. subtilis glycerol kinase genes. The liver cDNA clones have sequence identity with four genomic exons and the 3' untranslated region from an Xp21.3 cosmid thus indicating that this is the expressed GK gene which when deleted in patients gives rises to GK deficiency.

Amino Acid Sequence↗
PubMed1986

Structure and nucleotide sequence of the 5' region of the human and feline c-sis proto-oncogenes.

Comparative analysis of cosmid clones containing the human and feline c-sis genetic regions revealed the similar structural organization of these areas in the two species. The areas shared seven different genetic regions in and around the c-sis locus and of these was related to v-sis. Another region, 1.9 kbp in size and located about 8 kbp upstream of the v-sis homologous region in the human genome, also hybridized to the main c-sis transcriptional product of 3.5 kb. Comparison with a recently described c-sis cDNA clone (Collins et al., Nature 316, 748-750 (1985)) revealed that the 1.9 kbp DNA region contained a large 5' c-sis exon of at least 1050 bp. In this exon, the presumed initiation site of the predicted PDGF-2 containing precursor protein was located and appeared to be preceded by a large untranslated region. In the region immediately upstream of this exon, a TATA box and a consensus sequence for a potential Sp1 binding site were found at similar positions in both species. This region also exhibited promoter activity when tested in an assay in which coding sequences of bacterial chloramphenicol acetyltransferase (CAT; acetyl-CoA: chloramphenicol 3-O-acetyltransferase, EC 2.3.1.28) were placed under its control. The five other DNA regions were found upstream and downstream of the human c-sis transcription unit and also in an intron. Four of them contained repetitive sequences. Hybridization analysis of human and feline c-sis containing cosmid clones with a mixed synthetic nucleotide probe, which corresponded to sequences encoding amino acid residues 2-7 of chain 1 of platelet-derived growth factor (PDGF-1), suggested that the c-sis cosmid clones did not include PDGF-1-specific genetic sequences.

Acetyltransferases↗
PubMed1987

Overabundance of rare-cutting restriction endonuclease sites in the human genome.

A human chromosome 3-specific cosmid library was constructed from a somatic cell hybrid containing human chromosome 3 as its only human component. This library was screened to identify 230 human recombinants which contained an average insert size of 37 kilobases. DNA prepared from 54 of these cosmids, representing 2000 kilobases of human DNA, was then tested for restriction endonuclease sites for EcoRI, HindIII, KpnI, XhoI, and DraI, as well as those of the rare-cutting restriction endonucleases NotI, SfiI, NruI, MluI, SacII, and BssHII. Sites for the latter enzymes were much more abundant than would be expected from theoretical calculations, reflecting non-random clustering of these sites. This has important implications for the use of these enzymes in the construction of physical maps of chromosomes. Some individual cosmids contained large numbers of rare sites, offering an alternative means of physically mapping chromosomes based upon identifying clusters of rare restriction sites. These clusters appear to be spaced an average of 1000 kb apart.

Animals↗
PubMed1997

RAPD-based screening of genomic libraries for positional cloning.

RAPD markers are frequently used for positional cloning. However, RAPD markers often contain repeated sequences which prevent genomic library screening by hybridisation. We have developed a simple RAPD analysis of genomic libraries based on the identification of cosmid pools and clones amplifying the RAPD marker of interest. Our method does not require the cloning or characterisation of the RAPD marker as it relies on the analysis of cosmid pools or clones using a simple RAPD protocol. We applied this strategy using four RAPD markers composed of single copy or repeated sequences linked to avirulence genes of the rice blast fungus Magnaporthe grisea . Cosmids containing these RAPD markers were easily and rapidly identified allowing the construction of physical contigs at these loci.

Ascomycota↗
PubMed2000

A rapid method for efficient gene replacement in the filamentous fungus Aspergillus nidulans.

The construction of mutant fungal strains is often limited by the poor efficiency of homologous recombination in these organisms. Higher recombination efficiencies can be obtained by increasing the length of homologous DNA flanking the transformation marker, although this is a tedious process when standard molecular biology techniques are used for the construction of gene replacement cassettes. Here, we present a two-step technology which takes advantage of an Escherichia coli strain expressing the phage lambda Red(gam, bet, exo) functions and involves (i) the construction in this strain of a recombinant cosmid by in vivo recombination between a cosmid carrying a genomic region of interest and a PCR-generated transformation marker flanked by 50 bp regions of homology with the target DNA and (ii) genetic exchange in the fungus itself between the chromosomal locus and the circular or linearized recombinant cosmid. This strategy enables the rapid establishment of mutant strains carrying gene knock-outs with efficiencies >50%. It should also be appropriate for the construction of fungal strains with gene fusions or promoter replacements.

Aspergillus nidulans↗
PubMedDate not supplied

Isolation and characterization of Rhizobium (IC3342) genes that determine leaf curl induction in pigeon pea.

Nodulation by the Rhizobium strain IC3342 causes a leaf curl syndrome in certain tropical legumes such as pigeon pea (Cajanus cajan) (N.M. Upadhyaya, J.V.D.K. Kumar Rao, D.S. Letham, and P.J. Dart, Physiological and Molecular Plant Pathology 39:357-373, 1991). Transposon (Tn5) mutagenesis of this leaf curl-inducing (Curl+) Rhizobium strain yielded two Curl- Fix- and three Curl- Fix+ mutants. Plasmid visualization and subsequent Southern blot hybridization analyses with Tn5, nif and nod gene probes showed that the Tn5 element had inserted into the symbiotic (Sym) plasmid in three of the mutants. Restriction endonuclease analyses indicated that none of the Tn5 insertions were closely linked. Tn5-containing EcoRI fragments were cloned from each mutant and used as probes to isolate the corresponding wild-type DNA fragments from a cosmid (pLAFR3) genomic library. Fix+ and/or Curl+ phenotypes were restored in each mutant by the introduction of cosmids containing the corresponding wild-type DNA. A closely related but Curl- Rhizobium strain ANU240 was shown, by Southern hybridization, to contain conserved DNA sequences of all but one of the identified genetic regions of the Curl+ Rhizobium strain IC3342. Cosmids containing the genetic region unique to the strain IC3342, designated lcr1, conferred a Curl+ phenotype on the strain ANU240. DNA sequence analysis of the cloned lcr1 region revealed five open reading frames (ORFs). The ORF2 showed homology with the Escherichia coli regulatory gene ompR, and ORF4 showed homology with E. coli and Rhizobium meliloti regulatory genes fnr and fixK, respectively.

Amino Acid Sequence↗
PubMed1997

Cloning and characterization of four genes of Rhizobium leguminosarum bv. trifolii involved in exopolysaccharide production and nodulation.

Four different genes of Rhizobium leguminosarum bv. trifolii strain RBL5599 involved in exopolysaccharide (EPS) production were identified by complementation of Tn5-induced EPS-deficient mutants (Exo mutants) with a cosmid bank. On one cosmid pssA was located, which was found to be almost identical to the pss4 gene from R. leguminosarum bv. viciae VF39 and highly homologous to a family of glycosyl transferases. Two pssA mutants, exo2 and exo4, were characterized and found to produce 19 and 1% of the wild-type amount of EPS, respectively. The three other genes were found to be closely linked on a different complementing cosmid. pssC revealed similarity to exoM and exoW of R. meliloti, both encoding glucosyl transferases involved in the synthesis of succinoglycan. A mutation in this gene (mutant exo50) did reduce EPS synthesis to 27% of the wild-type amount. We found an operon closely linked to pssC, consisting of two overlapping genes, pssD and pssE, that is essential for EPS production. Homology of pssD and pssE was found with cps14F and cps14G of Streptococcus pneumoniae, respectively: two genes responsible for the second step in capsule polysaccharide synthesis. Furthermore, pssD and pssE were homologous to the 5' and 3' parts, respectively, of spsK of Sphingomonas S88, which encodes a putative glycosyl transferase. Structural analysis of EPS produced by Exo mutants exo2, exo4, and exo50 showed it to be identical to that of the parental strain RBL5599, with the exception of acetyl groups esterified to one of the glucose residues being absent.

Amino Acid Sequence↗
PubMed2002

The presence of diverse IS elements and an avrPphD homologue that acts as a virulence factor on the pathogenicity plasmid of Erwinia herbicola pv. gypsophilae.

The pathogenicity of Erwinia herbicola pv. gypsophilae (Ehg) and Erwinia herbicola pv. betae (Ehb) is dependent on a native plasmid (pPATH(Ehg) or pPATH(Ehb)) that harbors the hrp gene cluster, genes encoding type III effectors, phytohormones, biosynthetic genes, and several copies of IS1327. Sequence analysis of the hrp-flanking region in pPATH(Ehg) (cosmid pLA150) revealed a cluster of four additional IS elements designated as ISEhel, ISEhe2, ISEhe3, and ISEhe4. Two copies of another IS element (ISEhe5) were identified on the upstream region of the indole-3-acetic acid operon located on the same cosmid. Based on homology of amino acids and genetic organization, ISEhe1 belongs to the IS630 family, ISEhe2 to the IS5 family, ISEhe3 and ISEhe4 to different groups of the IS3 family, and ISEhe5 to the IS1 family. With the exception of ISEhe4, one to three copies of all the other IS elements were identified only in pathogenic strains of Erwinia herbicola pv. gypsophilae and Erwinia herbicola pv. betae whereas ISEhe4 was present in both pathogenic and nonpathogenic strains. An open reading frame that exhibited high identity (89% in amino acids) to AvrPphD of Pseudomonas syringae pv. phaseolicola was present within the cluster of IS elements. An insertional mutation in the AvrPphDEh, reduced gall size in gypsophila by approximately 85%. In addition, remnants of known genes from four different bacteria were detected on the same cosmid.

Bacterial Proteins↗
PubMed1990

Infertility in male transgenic mice: disruption of sperm development by HSV-tk expression in postmeiotic germ cells.

Previous experiments revealed that male transgenic mice bearing a cosmid that included the Class II E alpha gene, about 35 kb of 5' flanking DNA, and the cosmid vector sequences were sterile. To ascertain the cause of the sterility, various subfragments of the cosmid were tested in transgenic mice. Only those pieces of DNA that included some of the E alpha flanking chromosomal DNA and the herpes simplex virus (HSV)-thymidine kinase (tk) gene that was in the vector resulted in male sterility. Histological analysis revealed abnormalities in nuclear morphology of elongating spermatids and retention of mature spermatids within the seminiferous epithelium. Immunocytochemical studies showed that the HSV-tk gene was expressed at low levels in postmeiotic round spermatids and at higher levels in more mature elongating spermatids. To determine whether expression of HSV-tk in spermatids might be responsible for the sterility, the protamine gene promoter was used to direct the expression of HSV-tk to postmeiotic germ cells. Since the mice so treated were also sterile, the data suggest that expression of this enzyme in spermatids is responsible for the sterility phenotype.

Animals↗
PubMed1994

Absence of varicella-zoster virus (VZV) glycoprotein V does not alter growth of VZV in vitro or sensitivity to heparin.

Varicella-zoster virus (VZV) encodes at least five glycoproteins, gpI to gpV. VZV gpV, M(r) 100K to 110K, is the product of VZV open reading frame (ORF) 14. VZV gpV is homologous to herpes simplex virus gC and pseudorabies virus gIII. To determine whether gpV is required for viral replication, we inserted a stop codon after the fifteenth codon of the ORF14 gene in a cosmid containing the gene. Transfection of human melanoma cells with the cosmid containing the mutant ORF14 gene and three other cosmids resulted in the production of infectious VZV. Immunoprecipitation indicated that the mutant virus did not express gpV. VZV that did not express gpV grew at the same rate as parental virus and was inhibited by heparin to a similar extent. The pattern of inhibition by heparin of the gpV mutant was similar to that reported for a herpes simplex virus mutant that does not contain gC, but different from that described for a pseudorabies virus mutant devoid of gIII. These results indicate that VZV gpV is not required for viral replication in vitro.

Base Sequence↗
PubMed1990

Cloning of two genes from Bacillus circulans WL-12 which encode 1,3-beta-glucanase activity.

Two genes encoding distinct 1,3-beta-glucanases have been cloned from Bacillus circulans and expressed in Escherichia coli. A cosmid library of B. circulans WL-12 DNA was constructed in the broad-host-range cosmid pLAFR1 and screened in E. coli for clones which exhibited 1,3-beta-glucanase activity. Two 1,3-beta-glucanase-positive clones were identified which contained genes encoding two independent 1,3-beta-glucanases as shown by biochemical, physical and molecular analyses. The cosmids, designated pMON5401 (27.1 kb insert) and pMON5402 (24.7 kb insert), encoded 68 kDa and 40 kDa 1,3-beta-glucanases, respectively. Both 1,3-beta-glucanases were purified from their respective E. coli strains, characterized biochemically, and were shown to exhibit lytic activity against purified yeast cell wall preparations.

Amino Acid Sequence↗
PubMed1992

Physical and genetic mapping of the catA region of Pseudomonas aeruginosa.

A prime plasmid has been used as the basis for the construction of a physical and genetic map of a 125 kb segment of the Pseudomonas aeruginosa PAO chromosome. Using pMO1811, a prime plasmid selected for the catA region, a series of Tn5 insertions were obtained which identified two new markers gcu (glycine utilization) and oap (organic acids and alcohols permeability) in the 125 kb region and located them in relation to other known markers of this region. A cosmid bank was constructed from the prime plasmid and an ordered array of cosmid clones for this region identified by restriction endonuclease mapping with EcoRI, HindIII and KpnI, as well as complementation mapping and chromosome walking. By Southern hybridization analyses, it was confirmed that the chromosomal insert carried by pMO1811 was flanked by single, tandemly arranged copies of IS21 and the orientation of the insert on this prime was determined. This cosmid bank provides a resource for the further analysis of this region of the P. aeruginosa genome.

Alcohols↗