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At least 469 records · Page 26Linked to original sources
PubMed2001

The characterization of prepro-insulin-like growth factor-1 Ea-2 expression and insulin-like growth factor-1 genes (devoid 81 bp) in the zebrafish (Danio rerio).

In this study, we cloned zebrafish (Danio rerio) IGF-1 cDNA and gene from zebrafish brain cDNA library and adult zebrafish genomic library, respectively. Based on two cDNAs sequence with different length of 5'- and 3'-untranslated region (5UTR and 3UTR) and one nucleotide difference at glutamine (A9, CAG) of A domain represented at IGF-1 sequence. One of zebrafish IGF-1 genes named as IGF-1a gene. The zebrafish IGF-1a gene spanned approximately 15 kb and is divided into five exons. The results of IGF-1 cDNA and genomic Southern blotting, all indicated that the zebrafish have more than one IGF-1 gene. The genomic organization of zebrafish IGF-1a gene in an exon is devoid of 81 bp segment which is located at 3' end of exon 3 encoded 27 amino acid of E domain. The segment of 27 amino acid exists in known teleost IGF-1 genes but is absent in zebrafish IGF-1 gene. The E domain of zebrafish IGF-1 Ea-2 is encoded by 3' end of exon 3 (16 amino acid), full of exon 4 (12 amino acid) and exon 5 (19 amino acid). The sequence data revealed the zebrafish IGF-1a gene encoded IGF-1a Ea-2 mRNA. In combination RT-PCR with Southern blotting, zebrafish IGF-1 genes abundantly expressed IGF-1 Ea-2 mRNA in all tested adult tissues and developmental stages of embryo. The IGF-1 Ea-2 mRNA was first detected during embryo development from blastula stage to hatching, during yolk absorption and at feeding. All these findings suggest that the expression of pro-IGF-1 Ea-2 is not controlled by alternative splicing but alternative gene usage in the zebrafish.

3' Untranslated Regions↗
PubMed2001

Generation of a P1 artificial chromosome library of the Southern pufferfish.

We describe the generation of a P1 artificial chromosome genomic library from the Southern pufferfish, Spheroides nephelus. The arrayed library consists of approximately 30,000 clones and has an average insert size of 125-150 kb. The coverage is estimated to encompass seven to eight genome equivalents. The library has been used for isolating numerous genomic clones and for establishing contigs of several multigene families. Analysis of several of the clones from this library suggests a preponderance of CA repeat tracts relative to their abundance in humans. The library and high-density filters have been made available to the scientific public through genomics distribution companies.

Animals↗
PubMed1991

Isolation and characterization of a species-specific DNA probe for Candida albicans.

As a model for the isolation of species-specific sequences of DNA, we isolated and characterized a species-specific DNA fragment from the Candida albicans genome. We used a series of differential colony hybridization experiments, in which a C. albicans genomic library was hybridized with genomic DNA probes from related organisms to minimize the number of potentially specific C. albicans DNA fragments to be tested. Six clones were tested by dot blot analysis, and one of them, a 1348 bp EcoRI DNA fragment, was confirmed as specific for C. albicans. This species-specific fragment could be utilized as a DNA probe for rapid, sensitive, and specific diagnosis of C. albicans DNA in clinical specimens.

Base Sequence↗
PubMed2006

A framework linkage map of perennial ryegrass based on SSR markers.

A moderate-density linkage map for Lolium perenne L. has been constructed based on 376 simple sequence repeat (SSR) markers. Approximately one third (124) of the SSR markers were developed from GeneThresher libraries that preferentially select genomic DNA clones from the gene-rich unmethylated portion of the genome. The remaining SSR marker loci were generated from either SSR-enriched genomic libraries (247) or ESTs (5). Forty-five percent of the GeneThresher SSRs were associated with an expressed gene. Unlike EST-derived SSR markers, GeneThresher SSRs were often associated with genes expressed at a low level, such as transcription factors. The map constructed here fulfills 2 definitions of a "framework map". Firstly, it is composed of codominant markers to ensure map transferability either within or among species. Secondly, it was constructed to achieve a level of statistical confidence in the support-for-order of marker loci. The map consists of 81 framework SSR markers spread over 7 linkage groups, the same as the haploid chromosome number. Most of the remaining 295 SSR markers have been placed into their most likely interval on the framework map. Nine RFLP markers and 1 SSR marker from another map constructed using the same pedigree were also incorporated to extend genome coverage at the terminal ends of 5 linkage groups. The final map provides a robust framework with which to conduct investigations into the genetic architecture of trait variation in this commercially important grass species.

Chromosome Mapping↗
PubMed2002

CART gene promoter transcription is regulated by a cyclic adenosine monophosphate response element.

OBJECTIVE: To define the regulatory DNA sequence controlling the expression of the cocaine-amphetamine-regulated transcript (CART) gene expression. RESEARCH METHODS AND PROCEDURES: A rat genomic library was screened for genomic clones containing the CART gene and upstream DNA sequence. A clone containing exons 1 and 2 of the rat CART gene plus 1.2 kb of upstream sequence was isolated. The 1.2-kb upstream sequence and truncated segments of this sequence were cloned into a luciferase reporter vector for analysis of transcriptional activity in the CART expressing pituitary GH3 cell line. Luciferase reporter assays, gel shift assays, and site-directed mutagenesis were used to analyze potential regulatory regions in the 1.2-kb 5' DNA sequence. RESULTS: Sequence analysis of the 1.2-kb upstream sequence reveals several potential regulatory elements. Luciferase reporter assays demonstrate that within the first 162 bp of the start codon is a functional cyclic adenosine monophosphate (cAMP) response element that can induce cAMP-regulated gene expression of the CART promoter in GH3 cells. Mutation of this cAMP response element abolishes cAMP-stimulated transcription [corrected]. Furthermore, the promoter is active in a neuronal cell line where it also demonstrates cAMP responsiveness. DISCUSSION: cAMP-mediated transcription of the CART promoter may be an important aspect of the regulation of the CART gene. However, the cellular context of expression is also important because the CART promoter is not responsive to cAMP stimulation in all cell types. Other transcription factor binding sequences are likely to play a key role in CART promoter regulation.

Animals↗
PubMed1985

An extracellular matrix protein in plants: characterization of a genomic clone for carrot extensin.

Extensins are hydroxyproline-rich glycoproteins found in many plant cell walls as a major protein component. The peptide Ser-Hyp-Hyp-Hyp-Hyp is abundant in the extensins. Using extensin cDNA clones as probes, we isolated six different clones from carrot genomic libraries. One of the genomic clones, pDC5A1, was characterized and found to contain an open reading frame encoding extensin and a single intron in the 3'-non-coding region. The derived amino acid sequence contains a signal peptide sequence and 25 Ser-Pro-Pro-Pro-Pro repetitive sequences. Two extensin transcripts were found corresponding to pDC5A1 with different 5' start sites. These transcripts increased in abundance after wounding. This is consistent with the reported extensin accumulation in the cell wall upon wounding.

Base Sequence↗
PubMed1995

Molecular cloning of the GTP-cyclohydrolase structural gene RIB1 of Pichia guilliermondii involved in riboflavin biosynthesis.

The structural gene of GTP-cyclohydrolase, involved in riboflavin biosynthesis, was cloned from a Pichia guilliermondii genomic library. A 1855 bp genomic DNA fragment complementing the riboflavin auxotrophies of an Escherichia coli ribA mutant, defective in GTP-cyclohydrolase II, and a P. guilliermondii rib1 mutant was isolated and sequenced. An open reading frame with the potential to encode a protein of 344 amino acids with a predicted molecular mass of 38,711 Da was detected. The P. guilliermondii enzyme shows a high degree of homology to GTP-cyclohydrolases type II from E. coli and Baccillus subtilis and to GTP-cyclohydrolase from Saccharomyces cerevisiae. Functional GTP-cyclohydrolase from P. guilliermondii may consist of four identical subunits.

Amino Acid Sequence↗
PubMed1993

Cloning of the rat insulin- like growth factor binding protein-5 gene and DNA sequence analysis of its promoter region.

To understand the regulation of the insulin-like growth factor binding protein-5 (IGFBP-5) gene expression, we have cloned the IGFBP-5 gene from rat genomic libraries and determined its genomic organization as well as the DNA sequence at the 5' flanking region of the gene. The rat IGFBP-5 gene spans at least 17 kilobases (kb) of the genome and contains 4 exons interrupted by 3 introns of approximately 10, 0.6 and 0.7 kb, respectively. Southern blot analysis of the rat chromosome DNA revealed a single copy gene for IGFBP-5 in the haploid genome. Primer extension experiments demonstrated a single transcriptional start site located at 772 nucleotides 5' of the ATG translational start codon. In addition to a TATA box and a CAAT box, multiple putative cis-regulatory elements, including an AP-1, an AP-2 and a binding site for progesterone receptor are present in the promoter region. This finding suggests that the IGFBP-5 gene is one of those that are expressed in a tissue-specific manner.

Amino Acid Sequence↗
PubMed1994

Sequence homologies and linkage group conservation of the human and mouse Cenpc genes.

Using a previously identified human CENPC cDNA fragment, we have isolated cDNA clones corresponding to the complete mouse Cenpc coding sequence. Using these cDNAs as probes to genomic libraries, we have isolated genomic clones corresponding to the mouse and human genes and also to a mouse pseudogene. In situ hybridization mapping of these genes reveals that the human gene maps to 4q12-q13.3 and the mouse gene to 5E2-E5. These sites are in a region of linkage group conservation between the two species. Secondary sites are present in man on chromosome 12q21.2-q21.33 and in mouse on chromosome 2B. This mouse secondary site is a pseudogene on the basis of DNA sequence. These secondary sites are not syntenic in the two species.

Amino Acid Sequence↗
PubMed1994

Meiosis-dependent mRNA splicing of the fission yeast Schizosaccharomyces pombe mes1+ gene.

The mes1+ gene of the fission yeast Schizosaccharomyces pombe is essential for the second meiotic division. We have cloned a 1.1-kb HindIII fragment containing mes1+ by complementation from an S. pombe genomic library. Sequencing of the genomic and cDNA fragments indicates the existence of one small intron of 75 nucleotides, although both the 5'(G/GTTAGT) and 3'(CAG/T) intron-exon junctions deviate from the consensus sequences proposed for S. pombe. The putative translation product of the mature mes1+ mRNA is a 11-kDa protein of 101 amino acids which has no significant homology to any previously-reported proteins. Disruption of mes1 has no effect on cell growth but causes an arrest of meiosis before the second meiotic division. Northern-blot analysis revealed that mes1+ was preferentially transcribed under conditions of nitrogen starvation. When a h90 homothallic strain was shifted to a nitrogen-deficient medium, a pre-mRNA accumulated and then was gradually processed to generate a mature mRNA. This splicing did not occur in either a heterothallic haploid strain or in a homothallic mei2 mutant strain which was defective in the initiation of meiosis. Expression of the first exon alone was not able to suppress the mes1 null allele. These results indicate that mes1+ is required for the completion of meiosis, that splicing is required for the function of the mes1+ gene, and that this splicing requires the function of the mei2+ product.

Amino Acid Sequence↗
PubMed1982

The establishment of genomic DNA libraries for the human malaria parasite Plasmodium falciparum and identification of individual clones by hybridisation.

The DNA of Plasmodium falciparum has been purified and fragmented with the restriction endonucleases EcoRI and HindIII. The fragments have been incorporated in vitro into derivatives of bacteriophage lambda to make libraries in which most of the parasite DNA is represented. By Southern hybridisation we have been able to recover from these libraries specific clones containing (a) repetitive DNA sequences, (b) rRNA gene(s) and (c) sequences homologous to an actin gene probe. Parasite DNA from two independent sources differs markedly in the pattern of its repetitive DNA visualised by hybridisation to our repetitive clone. By contrast, the rRNA genes of the two isolates prove to be carried on identically sized fragments.

Bacteriophage lambda↗
PubMed1991

Isolation of the human cytochrome P-450 IIC8 gene: multiple glucocorticoid responsive elements in the 5' region.

The 5' portion of the cytochrome P-450 IIC8 gene has been isolated from a human genomic library. A 3.5 kb genomic fragment including 2477 bp of 5' flanking region and exon 1 of the gene was sequenced. S1 nuclease mapping and specific primer extension experiments localized the transcription start site 23 bp upstream from the ATG. Two and three putative TATA and CAAT boxes were identified together with seven core motifs for glucocorticoid responsive elements, an interesting feature in this constitutive P-450 subfamily.

Base Sequence↗
PubMed1993

Molecular cloning and structure of the human (GABATHG) GABA transporter gene.

A cDNA molecule encoding the human GABA transporter was synthesized by means of polymerase chain reaction (PCR) technique and used as probe for selecting a human genomic DNA fragment encoding GABA transporter. A positive clone harboring the whole gene was obtained from a human lymphocyte genomic library through utilizing the genomic 'walking' technique. The clone, designated as pHGAT, harbours a DNA fragment of about 39 kb in length inserted into the BamHI site in cosmid pWE15. The gene covers about 25 kb in length and is constituted by four EcoRI restricted fragments which are 13.7 kb, 3.1 kb, 4.2 kb and 7.2 kb long, respectively. The genomic clone contains 15 introns, including two introns prior to the initiator methionine (i.e., the translation start site is in exon 3). Eleven exons encode the twelve transmembrane regions in the transporter protein. Thus as in the case for a number of other membrane proteins, there appears to be a strong tendency for the putative transmembrane domains to be encoded by separate exons. It is noted that the structure of the human GABA transporter gene reported here differs from the mouse gene which is contains 12 introns.

Base Sequence↗
PubMed1992

Cloning of Campylobacter jejuni genes required for leucine biosynthesis, and construction of leu-negative mutant of C. jejuni by shuttle transposon mutagenesis.

Campylobacter jejuni is a Gram-negative pathogen responsible for diarrhoeal diseases in humans. To date, very little is known about the genetic organization and molecular biology of this microorganism. The cosmid vector pHC79 was used to construct a genomic library from the total genomic DNA of C. jejuni strain C31 in Escherichia coli and recombinant cosmids capable of complementing the auxotrophic defect in leucine biosynthesis of E. coli HB101 were identified. Three of 400 clones tested were found to be capable of complementing the nutritional defect of E. coli HB101 as well as those of independent leuB mutants of E. coli strains. These results indicated that the cloned genes responsible for leucine complementation encoded an enzyme analogous to the beta-isopropylmalate dehydrogenase specified by the leuB gene in E. coli strains. The sizes of the recombinant cosmids which became stabilized in E. coli cells ranged from 12.9 to 15.4 kb compared to the expected, originally packaged, 45- to 50-kb molecules, attesting to major rearrangements occurring in this background. The recombinant plasmid pILL547 was shown to carry genes that were analogous to the leuB gene and also to the leuC and leuD genes of E. coli. The gene required for leuB complementation was subcloned on a 1.6-kb restriction fragment and was mapped more precisely by insertional mutagenesis using as transposon a newly constructed (MiniTn3-Km) element engineered to mutagenize Campylobacter genes. The leuB gene of C. jejuni was shown to be expressed from its own promoter in E. coli cells. In E. coli minicells, the cloned insert encoded a polypeptide with an apparent molecular weight of 40 kDa. A leucine auxotrophic mutant of C. jejuni strain C31 was constructed in vitro by allelic exchange, replacing the original copy of the leucine gene by an allele mutated by the insertion of the kanamycin transposable element.

Campylobacter jejuni↗