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At least 361 records · Page 20Linked to original sources
PubMed1994

The human gonadotropin-releasing hormone (GnRH) receptor gene: cloning, genomic organization and chromosomal assignment.

The cDNA encoding the gonadotropin-releasing hormone (GnRH) receptor has recently been cloned and characterized in several species, including human. To determine the structure of the gene encoding the human GnRH receptor, we have screened a human genomic library and isolated seven positive clones, using cDNA probes derived from a human pituitary cDNA library. The isolated genomic clone contains the entire protein coding region of the GnRH receptor which is distributed between three exons and spans over 18.9 kb. Sequence analysis and restriction endonuclease mapping revealed the presence of two introns of 4.2 and 5.0 kb, respectively, both located within the open reading frame, designating the human GnRH receptor gene to the intron-containing class of the G-protein coupled receptor superfamily. Genomic Southern blot analysis indicated the presence of a single copy of the gene encoding for the GnRH receptor within the human genome. Using DNA from human-hamster somatic hybrid cell lines, the GnRH receptor gene was assigned to human chromosome 4, by means of PCR. The present study represents the first report on the GnRH receptor gene and its partial characterization should facilitate further investigation of the mechanisms by which expression of this gene is regulated.

Base Sequence↗
PubMed1998

Organization, 5'-flanking sequence and promoter activity of the rat GPC1 gene.

Glypicans are a member of a family of glycosylphosphatidylinositol anchored heparan sulfate proteoglycans that are expressed in cell and development specific patterns. Rat GPC1 cDNA probes were used to screen rat genomic libraries. Three overlapping genomic clones that contained the entire rat GPC1 gene were isolated. The rat GPC1 gene is approximately 15kb in length and consists of eight exons interrupted by introns of varying lengths. Two of the introns are quite short, with lengths of 41 and 43 base pairs. Each exon-intron splice junction exhibited the consensus splice site sequence. Exon 1 encodes the putative signal peptide and the serine residue of the first putative heparan sulfate attachment site. The last exon encodes the cluster of three potential COOH-terminal heparan sulfate attachment sites, the putative GPI anchor and polypeptide cleavage site, and the 3'-untranslated region including the polyadenylation signal. One of the genomic clones extended approximately 2.8 kb 5' of the exon 1 coding sequence, and is thus likely to contain sequences that regulate GPC1 gene expression. Sequence analysis of the 5'-flanking sequence revealed a lack of consensus TATA and CAAT boxes. A search for potential transcription factor binding sites revealed a number of such motifs, including Sp1 (GC box), NF-kappaB, and MyoD (E-box). This region of the rat GPC1 gene shows significant sequence homology to the 5'-flanking region of the human GPC3 gene. Functional promoter activity of the rat GPC1 sequence was demonstrated by its ability to drive the expression of a luciferase reporter gene in several cell types.

Animals↗
PubMed2000

The mouse N-acetylgalactosamine-6-sulfate sulfatase (Galns) gene: cDNA isolation, genomic characterization, chromosomal assignment and analysis of the 5'-flanking region.

Deficiency of lysosomal enzyme N-acetylgalactosamine-6-sulfate sulfatase (GALNS) leads to mucopolysaccharidosis IV A (MPS IV A), for which there is no definitive treatment so far. Although a number of mutations of the GALNS gene of MPS IV A patients have been described, pathogenesis of the disorder still remains elusive. In order to facilitate in vivo studies using model animals for MPS IV A, we isolated and performed molecular characterization of the mouse homolog of human GALNS. The 2.3-kb cDNA contains a 1560-bp open reading frame encoding 520 amino acid residues. The coding region has 84% similarity to the human GALNS cDNA at amino acid level. The mouse Galns gene was mapped by interspecific backcross analysis to the distal region of chromosome 8 where it co-segregates with Aprt. Northern blot analysis showed a wide expression of a single-copy gene, being higher especially in liver and kidney. The Galns gene was isolated from S129vJ genomic library and its genomic organization was characterized. The mouse Galns gene was about 50-kb long and organized into 14 exons and 13 introns. All intron-exon splice junctions conformed to the GT/AG consensus sequence except exon 8/intron 8 junction. Primer extension shows multiple transcription initiation sites between -44 and -75 although major transcription initiation site was observed at -90 bp from the ATG codon. The 5'-flanking region lacks canonical TATA and CAAT box sequences, but is G+C rich with 10 GC boxes (potential Sp1 binding sites), characteristic of a housekeeping gene promoter.

Amino Acid Sequence↗
PubMed1989

Characterization of AMD, the AMP deaminase gene in yeast. Production of amd strain, cloning, nucleotide sequence, and properties of the protein.

The structural gene for AMP deaminase (AMD) from Saccharomyces cerevisiae has been cloned and characterized. A yeast strain deficient in AMP deaminase activity was produced and shown to be deficient in AMP deaminase protein by Western blot analysis. The gene for AMP deaminase was located in a lambda gt11 library of yeast genomic DNA, and a DNA fragment from the lambda gt11 clone was used to locate homologous DNA in a yeast genomic library in the centromeric plasmid YCp50, a yeast-Escherichia coli shuttle vector. One plasmid was selected for its ability to restore AMP catalytic activity to the deficient strain. Yeast deficient in AMP deaminase or those overproducing the enzyme grow at near normal rates. The open reading frame corresponding to AMD codes for a protein of 810 amino acids, molecular weight 93,286. The yeast AMD transcript is 3.0 +/- 0.2 kb, and the transcriptional initiation sites have been identified. Western blot analysis of extracts prepared from actively growing yeast indicates a major band at approximately 96,000 molecular weight with several bands at lower molecular weight, including 83,000. When the AMD gene is expressed in E. coli, the large Mr form of AMP deaminase is produced. These results show that the purified enzyme (Mr = 83,000) is a truncated form of the full-length translation product. No adenine nucleotide binding sites were located based on the consensus sequence from other nucleotide binding proteins. No overall homology was found between yeast AMP deaminase and E. coli AMP nucleosidase. Although their metabolic roles and regulatory mechanisms are similar, these enzymes have arisen from separate ancestral proteins.

AMP Deaminase↗
PubMed1990

Genomic subtraction for cloning DNA corresponding to deletion mutations.

We have developed a technique, called genomic subtraction, for isolating the DNA that is absent in deletion mutants. The method removes from wild-type DNA the sequences that are present in both the wild-type and the deletion mutant genomes. The DNA that corresponds to the deleted region remains. Enrichment for the deleted sequences is achieved by allowing a mixture of denatured wild-type and biotinylated mutant DNA to reassociate. After reassociation, the biotinylated sequences are removed by binding to avidin-coated beads. This subtraction process is then repeated several times. In each cycle we hybridize the unbound wild-type DNA from the previous round with fresh biotinylated deletion mutant DNA. The unbound DNA from the final cycle is ligated to adaptors and amplified by using one strand of the adaptor as a primer in the polymerase chain reaction. The amplified sequences can then be used to probe a genomic library. We applied genomic subtraction to a yeast strain that has a 5-kilobase deletion, corresponding to 1/4000th of the genome. In the experiment reported here, three rounds of subtraction were sufficient to accurately identify genomic clones containing sequences that are missing in the deletion mutant. We discuss the limitations and some potential applications of the method.

Affinity Labels↗
PubMed1995

Structure and expression of a rat kappa opioid receptor gene.

A gene encoding the rat kappa opioid receptor (KOR) was cloned and characterized. Results of rat genomic library screening and genomic Southern blot analysis show the gene represented at one copy/haploid genome. Three introns are present within the gene; however, polymerase chain reaction using different sets of primers specifying neighboring exons indicates that alternative splicing does not occur. Using reverse transcription-polymerase chain reaction and primer extension techniques, we are able to demonstrate that two species of transcripts are differentially produced from the KOR gene in a tissue-specific manner. The first transcript that we designate as KOR1 is equivalent to the cDNA sequence reported by other groups and is believed to correspond to KOR subtype 1. KOR1 begins with exon 1 just downstream of two TATA boxes, whereas the second transcript, which we refer to as KORx, begins in intron 1 and thereby retains this intronic sequence in the mature mRNA. Within this intronic sequence there are two potential translation initiation codons that are in-frame with the proposed initiation codon of KOR. The potential open reading frame that starts further upstream in KORx may lead to the translation of a variant KOR protein having a novel peptide sequence at its amino terminus.

Amino Acid Sequence↗
PubMed1984

Characterization of the bovine prolactin gene.

The gene coding for bovine prolactin was shown to exist as a single copy per haploid genome. Three restriction fragment polymorphisms were detected in the prolactin gene by Southern blot analysis of DNA obtained from the semen of pedigreed bulls representing eight breeds. The organization of the bovine prolactin gene was determined by restriction mapping of a clone isolated from a genomic library and by genomic blots. The 5'-flanking region and two exons were sequenced and the transcription start site mapped by primer extension. Comparison of the bovine prolactin sequence reported here with the published sequence of the rat prolactin gene revealed extensive homology (79%), extending 360 nucleotides upstream from the cap site, after which the sequences diverge. The homology exceeds that of the coding regions. A possible alternate intron-exon splice site was noted within the sequence coding for the signal peptide.

Amino Acid Sequence↗
PubMed2002

A genetic map of Gibberella zeae (Fusarium graminearum).

We constructed a genetic linkage map of Gibberella zeae (Fusarium graminearum) by crossing complementary nitrate-nonutilizing (nit) mutants of G. zeae strains R-5470 (from Japan) and Z-3639 (from Kansas). We selected 99 nitrate-utilizing (recombinant) progeny and analyzed them for amplified fragment length polymorphisms (AFLPs). We used 34 pairs of two-base selective AFLP primers and identified 1048 polymorphic markers that mapped to 468 unique loci on nine linkage groups. The total map length is approximately 1300 cM with an average interval of 2.8 map units between loci. Three of the nine linkage groups contain regions in which there are high levels of segregation distortion. Selection for the nitrate-utilizing recombinant progeny can explain two of the three skewed regions. Two linkage groups have recombination patterns that are consistent with the presence of intercalary inversions. Loci governing trichothecene toxin amount and type (deoxynivalenol or nivalenol) map on linkage groups IV and I, respectively. The locus governing the type of trichothecene produced (nivalenol or deoxynivalenol) cosegregated with the TRI5 gene (which encodes trichodiene synthase) and probably maps in the trichothecene gene cluster. This linkage map will be useful in population genetic studies, in map-based cloning, for QTL (quantitative trait loci) analysis, for ordering genomic libraries, and for genomic comparisons of related species.

Chromosome Mapping↗
PubMed1982

Isolation and characterisation of the Xenopus laevis albumin genes: loss of 74K albumin gene sequences by library amplification.

The blood of the frog X.laevis contains 2 albumins of 68,000 and 74,000 daltons which are encoded in the liver by two related mRNAs. When an amplified X.laevis DNA library was screened with cloned albumin cDNA only 68,000 dalton albumin gene sequences were isolated. Hybridisation of the albumin cDNA to Southern-blots of Eco R1 digested X.laevis DNA showed that the sequences present in the recombinants did not account for all the fragments which hybridised on the Southern-blots. This indicated that 74K albumin gene sequences exist but that they are not present in the amplified DNA library. A X.laevis genomic library was therefore constructed and screened for albumin genes without amplification. Both 68K and 74K albumin gene sequences were isolated. Recombinants containing 74K albumin gene sequences grew extremely poorly and this probably explains why the 74K albumin sequences were ot isolated from the amplified library. Characterisation of the cloned DNA indicates that there is one sequence coding for the 68K albumin but two different sequences coding for the 75K albumin.

Animals↗
PubMed1996

Isolation and characterization of the human chromosomal gene for prostacyclin-stimulating factor.

Prostacyclin-stimulating factor (PSF) is a protein which acts on vascular endothelial cells and stimulates the production of prostacyclin. Recently, we were able to purify PSF from the conditioned medium of cultured human diploid fibroblasts and clone PSF cDNA. In this study, we screened a human genomic library and isolated genomic clones to determine the structure of the human chromosomal PSF gene. By determining the nucleotide sequence and transcription initiation site of this gene, we found that it comprises 5 exons and 4 introns. Southern hybridization analysis indicated the presence of a single copy of the PSF gene per haploid set of chromosomes. The 300 bp upstream of the transcription initiation site had a very high GC content, and 7 binding sites for the transcription regulating factor Sp1 were present.

Base Sequence↗
PubMed1986

Isolation of chicken cellular DNA sequences with homology to the region of viral oncogenes that encodes the tyrosine kinase domain.

A library of chicken genomic DNA was screened for sequences that could hybridize to a cloned DNA fragment containing the transforming gene (v-fps) of Fujinami sarcoma virus. In addition to c-fps, two unique chicken cellular DNA sequences were isolated that hybridized weakly to v-fps. These sequences hybridized with many other viral oncogenes encoding tyrosine kinases. Sequence analysis of the region where homology was detected revealed a region that is highly conserved among the tyrosine kinases both at the nucleotide and amino acid levels. Although we were unable to detect expression of either chicken cellular DNA sequence in a variety of avian tissues, the data suggest the existence of additional members of the tyrosine kinase gene family. Screening genomic libraries for sequences that hybridize weakly to functional regions of other genes may prove useful for the isolation and characterization of additional members of other gene families.

Animals↗
PubMed1995

Cloning of the porcine D1A dopamine receptor gene expressed in renal epithelial LLC-PK1 cells.

The porcine renal epithelial cell line LLC-PK1 expresses a D1 dopamine receptor coupled to stimulation of adenylyl cyclase. The molecular identity of this receptor is unknown. We isolated a partial cDNA from LLC-PK1 poly(A)+ RNA by the reverse transcription-polymerase chain reaction procedure with degenerate D1 receptor oligonucleotide primers and used the partial cDNA to screen a porcine genomic library. One such genomic clone (lambda PGD1A.1) contained an open-reading frame that encoded a 446-amino-acid protein that is 95% identical to the human D1A receptor. The functional properties of the genomic clone transiently transfected into COS-7 cells were consistent with expression of a D1 receptor. RNA hybridization analyses with LLC-PK1 poly(A)+ RNA were positive. Primer extension analysis indicated that the primary transcription initiation site of the porcine D1A gene expressed in LLC-PK1 cells is 1,033 nucleotides upstream from the translation start site. The 5'-flanking region of the gene lacks a TATA and CAAT box but is high in GC content (68%) and contains multiple Sp1 binding sites. There is a 97-bp intron within the 5'-noncoding region, separating exons 1 and 2. These results add support to the view that the D1A receptor is the major D1 receptor expressed in kidney and further suggest that LLC-PK1 cells may be a useful model for study of the regulation of the renal D1A receptor gene.

Amino Acid Sequence↗
PubMed1989

The androgen-dependent rat prostatic binding protein: comparison of the sequences in the 5' part and upstream region of the C1 and C2 genes and analysis of their transcripts.

The complete gene encoding the polypeptide C1 of the complex androgen-controlled prostatic binding protein was isolated from a rat genomic library. A new genomic fragment (C2B) containing only the 5' part of a C2-related gene was also purified. The segments containing exon 1 and a large part of the adjacent sequences were analysed and compared with the corresponding region of the C2A gene which has been completely sequenced previously. The high structural similarity extending over a large part of all three genomic fragments suggests the duplication of a common ancestral gene, followed by a more recent duplication of the C2-coding region. However, since the structural similarity upstream of position -150 between C2A and C2B abruptly disappears and no transcripts specific for the C2B region can be detected in prostate RNA, we propose that at a later stage in evolution the C2B region was disrupted and inactivated. Despite the common origin and the similar regulation of the two active genes, C1 and C2A, the only obvious conserved structural element is the homopurine stretch located at position -400, although sequence motifs resembling steroid hormone response elements are present at several locations.

Androgen-Binding Protein↗
PubMed1988

Molecular cloning and characterization of an antigen associated with early stages of melanoma tumor progression.

The melanoma-associated antigen ME491 is expressed strongly during the early stages of tumor progression. The ME491 gene was molecularly cloned by means of DNA-mediated gene transfer followed by screening a lambda genomic library with human repetitive Alu sequences as a probe. The cloned DNA, after transfection into mouse L-cells, generated a protein with characteristics that were indistinguishable in Western blot analysis from the ME491 antigen expressed by human melanoma cells. Repeat-free subfragments of the cloned DNA were used for further studies. By Northern blot analysis, the subfragments detected a single 1.2-kilobase mRNA in the transformants and various human melanoma cell lines. ME491 complementary DNA clones were then obtained by probing a melanoma complementary DNA library with the genomic subfragments. Nucleotide sequence analysis of the cloned complementary DNA indicated that the ME491 antigen consists of 237 amino acids (Mr 25,475) with four transmembrane regions and three putative N-glycosylation sites. No significant structural homology was observed with other proteins thus far reported. We observed that the amounts of mRNA varied greatly with different melanoma cell lines. Southern blot analysis revealed no amplification or rearrangement of the ME491 gene in the human melanoma cell lines tested, including both high and low expressors of this antigen. The ME491 gene has been mapped to chromosome region 12p12----12q13 by somatic cell hybrid analysis and more narrowly localized to 12q12----12q14 by in situ hybridization.

Amino Acid Sequence↗
PubMed2000

[Cloning and expression of metallothionein gene of Bombyx mori].

Yeast MTI gene from vector pCMI-1 was used as a probe. It appeared strong hybridization signals when the total DNA of Bombyx mori Huishu eggs hybridizes with the probe. The 1-6 kb DNA fragments were isolated from the EcoR I digested total DNA of Bombyx mori Huishu eggs and ligated with M13- vector digested by restriction EcoR I. The ligation mixtures were used to transform E. coli DH5 alpha. blue/White colonies selection was used to identify colonies with insert. Approximately 4000 white colonies were selected, so the part genomic library of Bombyx mori was constructed. Three positive colonies were gained from the genomic library by southern blotting analysis, designated T1 (pZHC-1), T5 (pZHC-5), T7 (pZHC-7). Digesting the recombinant plasmid pZHC-5 with 12 restriction enzymes, the results suggested that the inserted fragment was about 1.2 kb and there was only a Hind III site. The experiment of resistant to CuSO4 proved that the DH5 alpha cells contain recombinant plasmids were more resistance than the recepient DH5 alpha cells. According to these results, the inserted fragment possibly contains the gene encoding Metallothionein of Bombyx mori. The sequence analysis of the inserted fragment and its high-expressions in E. coli are in progress.

Animals↗
PubMed1997

Identification of homologs for thioredoxin, peptidyl prolyl cis-trans isomerase, and glycerophosphodiester phosphodiesterase in outer membrane fractions from Treponema pallidum, the syphilis spirochete.

In this study, we characterized candidate rare outer membrane (OM) proteins with apparent molecular masses of 19, 27, 38, and 38.5 kDa, which had been identified previously in OM fractions from Treponema pallidum (J. D. Radolf et al., Infect. Immun. 63:4244-4252, 1995). Using N-terminal and internal amino acid sequences, a probe for the 19-kDa candidate was PCR amplified and used to screen a T. pallidum genomic library in Lambda Zap II. The corresponding gene (tlp) encoded a homolog for periplasmic thioredoxin-like proteins (Tlp), which reduce c-type cytochromes. A degenerate oligonucleotide derived from the N terminus of the 27-kDa protein was used to PCR amplify a duplex probe from a T. pallidum genomic library in pBluescript II SK+. With this probe, the corresponding gene (ppiB) was identified and found to code for a presumptive periplasmic cyclophilin B-type peptidyl prolyl cis-trans isomerase (PpiB). We postulate that PpiB assists the folding of proteins within the T. pallidum periplasmic space. The N terminus of the 38-kDa candidate was blocked to Edman degradation. However, internal sequence data revealed that it was basic membrane protein (Bmp), a previously characterized, signal peptidase I-processed protein. Triton X-114 phase partitioning revealed that despite its name, Bmp is hydrophilic and therefore likely to be periplasmic. The final candidate was also blocked to Edman degradation; as before, a duplex probe was PCR amplified with degenerate primers derived from internal sequences. The corresponding gene (glpQ) coded for a presumptively lipid-modified homolog of glycerophosphodiester phosphodiesterase (GlpQ). Based upon findings with other treponemal lipoproteins, the hydrophilic GlpQ polypeptide is thought to be anchored by N-terminal lipids to the periplasmic leaflet(s) of the cytoplasmic membrane and/or OM. The discovery of T. pallidum periplasmic proteins with potentially defined functions provides fresh insights into a poorly understood aspect of treponemal physiology. At the same time, however, these findings also raise important issues regarding the use of OM preparations for identifying rare OM proteins of T. pallidum.

Amino Acid Sequence↗
PubMed1997

Cloning and characterization of BS-cadherin, a novel cadherin from the colonial urochordate Botryllus schlosseri.

The genomic DNA for a novel member of the cadherin family (BS-cadherin) was cloned and characterized from the colonial marine invertebrate, Botryllus schlosseri. Using a differential display of mRNA by means of PCR, a small cDNA fragment of 380 nucleotides was found to be specifically expressed in a colony undergoing allogeneic rejection processes, as compared with naive parts of the same genotype. This cDNA fragment was used as a probe to screen a genomic library of Botryllus schlosseri. A genomic fragment containing an ORF of 2718 nucleotides, with no introns, was isolated. The encoded protein exhibits a typical structure of cadherins; an extracellular domain with conserved repeated sequences (cadherin signatures), a single transmembrane domain and a conserved cytoplasmic tail region. The BS-cadherin amino-acid sequence shows 32-35% identity to mature classical cadherins type I, e.g., N-, P- and E-cadherin as well as mature classical cadherins type II, e.g., human cadherin-6, -8 and OB-cadherin. This cadherin represents a new cadherin gene family, evolutionarily distant to all other known classical cadherins.

Amino Acid Sequence↗