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At least 937 records · Page 52Linked to original sources
PubMed1992

Molecular and functional characterization of the murine glucocerebrosidase gene.

A genomic clone of glucocerebrosidase (D-glucosyl-N-acyl-sphingosine glucohydrolase; E.C. 3.2.1.45) purified from a genomic library derived from a Balb/c mouse was analyzed by restriction mapping and nucleotide sequencing of its promoter and protein coding regions. Promoter activity was functionally assessed by ligation of a 2 kb glucocerebrosidase fragment to the protein coding segment of a bacterial neomycin resistance gene. Smaller segments of the 5' flanking sequence were then analyzed for their ability to initiate transcription of the chloramphenicol acetyltransferase reporter gene. A 319 bp Eco RI-Bgl II fragment (containing 259 bp upstream of the cDNA 5' limit) ligated to the chloramphenicol acetyltransferase open reading frame produced considerable activity.

3T3 Cells↗
PubMed1994

Analysis of integrated hepatitis B virus DNA and flanking cellular sequences in the hepatocellular carcinoma cell line HCC36.

A hepatocellular carcinoma cell line, HCC36, was established from an adult HBV carrier in Taiwan. From Southern blot analysis, there were at least four sites of integration of HBV DNA, and no viral replicative intermediates were detected. A genomic library was constructed from HCC36 DNA, and two phage clones, designated lambda 36A and lambda 36B, were shown to contain HBV DNA and flanking cellular sequences. In lambda 36A, HBV DNA sequences were quite conserved, and 7.4% base variation was detected. The viral sequences in lambda 36A and lambda 36B differed in only four bases, in addition to the microdeletion and -insertion observed in lambda 36B. The flanking cellular sequences identified in lambda 36A were human Alu sequences and in lambda 36B satellite sequences.

Base Sequence↗
PubMed1992

Methylation and sequence analysis around EagI sites: identification of 28 new CpG islands in XQ24-XQ28.

Thirty-two probes for CpG islands of the distal long arm of the human X chromosome have been identified. From a genomic library of DNA of the hamster-human cell hybrid X3000.1 digested with the rare cutter restriction enzyme EagI, 53 different human clones have been isolated and characterized by methylation and sequence analysis. The characteristic pattern of DNA methylation of CpG islands at the 5' end of genes of the X chromosome has been used to distinguish between EagI sites in CpG islands versus isolated EagI sites. The sequence analysis has confirmed and completed the characterization showing that sequences at the 5' end of known genes were among the clones defined CpG islands and that the non-CpG islands clones were mostly repetitive sequences with a non-methylated or variably methylated EagI site. Thus, since clones corresponding to repetitive sequences can be easily identified by sequencing, such libraries are a very good source of CpG islands. The methylation analysis of 28 different new probes allows to state that demethylation of CpG islands of the active X and methylation of those on the inactive X chromosome are the general rule. Moreover, the finding, in all instances, of methylation differences between male and female DNA is in very strong support of the notion that most genes of the distal long arm of the X chromosome are subject to X inactivation.

Amino Acid Sequence↗
PubMed1997

Characterization of the genomic structure and promoter of the mouse NAD+-dependent 15-hydroxyprostaglandin dehydrogenase gene.

The mouse NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH) gene and its 5'-flanking region was cloned from a 129 mouse ES bacteriophage P1 genomic library. The gene contains 7 exons and 6 introns and is 11.3 kb in length. The transcription initiation site was mapped at 35 bases upstream from the ATG start codon. The nucleotide sequence of the 1.6 kb promoter region contains two TATA boxes and a number of potential regulatory elements including Sp1, CRE, GRE, AP1, AP2, NF-IL6 and estrogen receptor binding site. Studies of the promoter's activity showed that the first 400 nucleotides of 5'-flanking region efficiently drove the transcription of the luciferase reporter gene in U936 cells upon stimulation with a phorbol ester.

Animals↗
PubMed1996

A novel myb-related gene from Arabidopsis thaliana.

A novel myb-like gene (Atmyb5) has been isolated from a genomic library of Arabidopsis thaliana. The gene contains a single intron in the region coding for the Myb domains. The Myb domains are highly homologous to other animal and plant Myb proteins. Arabidopsis plants transgenic for a chimeric Atmyb5 promoter/GUS gene expressed the enzyme in a developmentally controlled and tissue specific manner. The GUS activity was detected in developing leaf trichomes, stipules, epidermal cells on the margins of young rosette and cauline leaves, and in immature seeds. Atmyb5 mRNA appears between fertilization and the 16 cell stage of embryo development and persists beyond the heart stage.

Amino Acid Sequence↗
PubMed1993

Six members of the mouse forkhead gene family are developmentally regulated.

The 110-aa forkhead domain defines a class of transcription factors that have been shown to be developmentally regulated in Drosophila melanogaster and Xenopus laevis. The forkhead domain is necessary and sufficient for target DNA binding as shown for the rat hepatic nuclear factor 3 (HNF3) gene family. We have cloned six forkhead gene family members from a mouse genomic library in addition to the mouse equivalents of the genes for HNF3 alpha, -beta, and -gamma. The six genes, termed fkh-1 to fkh-6, share a high degree of similarity with the Drosophila forkhead gene, having 57-67% amino acid identity within the forkhead domain. fkh-1 seems to be the mammalian homologue of the Drosophila FD1 gene, as the sequences are 86% identical. fkh-1 to fkh-6 show distinct spatial patterns of expression in adult tissues and are expressed during embryogenesis.

Amino Acid Sequence↗
PubMed1994

Structure of genes that encode isozymes of aspartate aminotransferase in Panicum miliaceum L., a C4 plant.

The cytosolic and mitochondrial isozymes of aspartate aminotransferase (AspAT) function in the C4 photosynthetic cycle in NAD-malic enzyme-type C4 plants and are expressed at high levels in mesophyll cells and bundle sheath cells, respectively. We constructed a genomic library from Panicum miliaceum, a NAD-malic enzyme-type C4 plant, and cloned the genes for these isozymes. The sequence of the cloned gene for cytosolic AspAT spans 7800 bp and consists of 12 exons. The sequence of the cloned gene for mitochondrial AspAT spans 9000 bp and consists of 10 exons. The results of primer-extension analysis suggest that transcription may be initiated from multiple adjacent sites. Both genes have significant GC-rich regions around the site of initiation of transcription, and these regions showed no CpG suppression. The 5'- flanking regions of both genes include several short sequences similar to the regulatory elements found in other genes for components of the photosynthetic machinery. In particular, the cytosolic AspAT gene contains sequences similar to nuclear protein-binding sites in other mesophyll-expressed C4 photosynthetic genes and the mitochondrial AspAT gene contains elements for light-sensitive and constitutive expression of a bundle sheath-expressed gene. The results of Southern analysis indicated that there are at least two genes that encode each isozyme in the genome of P. miliaceum. A comparison of intron-insertion positions between AspAT genes of plants and animals revealed that several introns are located at identical positions. On the basis of a phylogenetic tree among AspATs and tyrosine aminotransferase, we have shown that the introns of aminotransferase genes antedate the divergence of eubacteria, archaebacteria, and eukaryotes.

Animals↗
PubMed1999

Cloning of a centromere binding factor 3d (CBF3D) gene from Candida glabrata.

The gene encoding centromere binding factor 3d (CBF3D) of the human pathogenic yeast Candida glabrata has been isolated by hybridization of Saccharomyces cerevisiae CBF3D (ScCBF3D) DNA to a C. glabrata partial genomic library. Sequence analysis revealed a 540 bp open reading frame encoding a protein of 179 amino acids with a calculated molecular mass of 20.9 kDa. The amino acid sequence is highly homologous (78.6% identity) to ScCbf3d and 48.3% identical to the human homologue p19 (SKP1). Southern blot analysis indicates that CgCbf3d is encoded by an unique nuclear gene. The cloned CgCBF3D gene can functionally substitute the S. cerevisiae homologue in a S. cerevisiae CBF3D-deletion mutant. The GenBank Accession No. for this gene is AF 072472.

Amino Acid Sequence↗
PubMed1991

Isolation of Escherichia coli mutants defective in uptake of molybdate.

For the study of molybdenum uptake by Escherichia coli, we generated Tn5lac transposition mutants, which were screened for the pleiotropic loss of molybdoenzyme activities. Three mutants A1, A4, and M22 were finally selected for further analysis. Even in the presence of 100 microM molybdate in the growth medium, no active nitrate reductase, formate dehydrogenase, and trimethylamine-N-oxide reductase were detected in these mutants, indicating that the intracellular supply of molybdenum was not sufficient. This was also supported by the observation that introduction of plasmid pWK225 carrying the complete nif regulon of Klebsiella pneumoniae did not lead to a functional expression of nitrogenase. Finally, molybdenum determination by induced coupled plasma mass spectroscopy confirmed a significant reduction of cell-bound molybdenum in the mutants compared with that in wild-type E. coli, even at high molybdate concentrations in the medium. A genomic library established with the plasmid mini-F-derived cop(ts) vector pJE258 allowed the isolation of cosmid pBK229 complementing the molybdate uptake deficiency of the chlD mutant and the Tn5lac-induced mutants. Certain subfragments of pBK229 which do not contain the chlD gene are still able to complement the Tn5lac mutants. Mapping experiments showed that the Tn5lac insertions did not occur within the chromosomal region present in pBK229 but did occur very close to that region. We assume that the Tn5lac insertions have a polar effect, thus preventing the expression of transport genes, or that a positively acting regulatory element was inactivated.

Blotting, Southern↗
PubMed1997

Molecular cloning of bovine CD14 gene.

Genomic DNA encoding bovine CD14 was isolated from a bovine (Holstein) genomic library. Utilizing PCR fragment of mouse CD14 gene as a probe, we screened 9 x 10(5) plaques and obtained 3 clones containing the bovine CD14 gene. DNA sequencing showed that bovine CD14 gene encodes 373 amino acids, and the coding sequence was separated by a 90 nt intron. The identity of the deduced amino acid sequence of bovine CD14 was 61-73% to those of mouse, rabbit and human. Northern blot analysis revealed that CD14 mRNA (1.5 kb) was expressed in the lung. Expression of CD14 mRNA was stimulated about 2-fold in bovine peripheral blood macrophage activated with LPS in vitro.

Amino Acid Sequence↗
PubMed1999

Isolation and expression of the gene encoding mitochondrial ADP/ATP carrier (AAC) from the pathogenic yeast Candida parapsilosis.

A gene homologous to Saccharomyces cerevisiae AAC genes coding for mitochondrial ADP/ATP carriers has been cloned from the pathogenic yeast Candida parapsilosis. A probe obtained by PCR amplification from C. parapsilosis DNA, using primers derived from the conserved transmembrane region of yeast ADP/ATP carriers, was used for screening of the C. parapsilosis genomic library. The cloned gene was sequenced and found to encode a polypeptide of 303 amino acids that shows homology with other yeast and fungal mitochondrial ADP/ATP carriers. The gene was designated CpAAC1 and was able to complement the growth phenotypes of S. cerevisiae double deletion mutant (Deltaaac2; Deltaaac3). The expression of the CpAAC1 gene was reduced under semi-anaerobic conditions and it was affected at normal aerobic conditions by the nature of carbon sources used for growth. Hybridization experiments indicate that C. parapsilosis possesses a single gene encoding a mitochondrial ADP/ATP carrier.

Aerobiosis↗
PubMed1994

Characterization of the genomic structure, chromosomal location and promoter of human prostaglandin H synthase-2 gene.

Prostaglandin H synthase (PGHS) is the rate-limiting enzyme in the conversion of arachidonic acid to prostanoids. The human PGHS has two isoforms. PGHS-1 is a house keeping gene whereas PGHS-2 is an inducible gene. We reported here the isolation of the entire PGHS-2 gene and its 5'-flanking region from a human bacteriophage P1 genomic library. The gene containing 10 exons is 7.5 kb in length and located at chromosome 1. The transcriptional start site was mapped at 134 bases upstream from the ATG start codon. Nucleotide sequence of 1.8 kb promoter region contains a TATA box and a number of potential regulatory elements including CRE, NF-kappa B, Sp1 and AP2 sites. Studies of the promoter activity showed that the first 460 nucleotides of 5'-flanking region efficiently drove transcription of the luciferase reporter gene in human umbilical vein endothelial cells upon stimulation with phorbor ester.

Animals↗
PubMed1990

Centromeric DNA of Kluyveromyces lactis.

A direct selection method was used to isolate centromeres from a genomic library of the yeast Kluyveromyces lactis. The method is based on the lethality at high copy number of the ochre-suppressing tRNA gene SUP11. Five different chromosomal fragments were found that confer mitotic stability to plasmids containing a replication origin of K. lactis (KARS). In addition, KARS plasmids containing these fragments have a copy number of approximately one, and each of the five fragments hybridizes to a different chromosome of K. lactis. From these results we conclude that five of the six centromeres of K. lactis have been isolated. These centromeres do not function in S. cerevisiae.

Centromere↗
PubMed1994

Protection of mice against experimental murine mycoplasmosis by a Mycoplasma pulmonis immunogen in lysogenized Escherichia coli.

A construct of the Mycoplasma pulmonis (MP) genomic library, using randomly sheared DNA, was cloned in lambda gt11 and transfected into C600 Escherichia coli organisms. Clones of E. coli expressing a fusion protein reactive with anti-MP and monospecific serum were transferred orally or intravenously into Balb/c mice. Expression of the fusion protein was induced by adding isopropyl-beta-D-thiogalactopyranoside to the drinking water. This vaccination protocol led to local and systemic antibody formation, to generation of immune lymphocytes and to protection against large numbers of virulent MP organisms. This approach might be generally successful in preventing infectious disease.

Administration, Oral↗
PubMed1992

Human interferon regulatory factor 1: intron-exon organization.

Interferon (IFN) regulatory factor 1 (IRF-1) is a transcriptional regulatory protein that mediates the transcriptional activation of the IFN-alpha and IFN-beta genes by viruses and IFNs. To characterize the mechanisms that govern the level of IRF-1 in cells, we isolated the IRF-1 gene and characterized the structure of its intronic and exonic domains and of its regulatory promoter region. A human placental genomic library was screened with an IRF-1 cDNA probe, and two clones that contained the IRF-1 gene and its 5' regulatory region were obtained. We used these clones to determine the complete nucleotide sequence for the IRF-1 gene, finding that the IRF-1 gene spanned 7.72 kb of DNA and included 10 exons and 9 introns. When the deduced amino acid sequences were compared among different species, the most conserved exons were exons 2, 3, and 4, in which the putative DNA binding domain for the IRF-1 protein is located.

Amino Acid Sequence↗
PubMed1996

Structure of the phenylalanine hydroxylase gene in Drosophila melanogaster and evidence of alternative promoter usage.

The complete Drosophila melanogaster phenylalanine hydroxylase gene isolated from a genomic library was sequenced. Gene structure consisted of five exons covering a region of around 3 kb. Position of introns in the C-terminal domain was conserved with mammalian aromatic amino acid hydroxylase genes. Putative promoter sequences in the 5'UTR and intron 1 were identified. A novel transcript was detected differing from that previously reported by the inclusion of a part of the intron 1 sequence. It could be produced using an alternative promoter. The deduced open reading frame would code a protein with a small difference at the N-terminus. Expression of the alternative transcripts was examined throughout development.

Amino Acid Sequence↗
PubMed1994

Stereochemical course of hydrolysis catalysed by Cellulomonas fimi CenE, a member of a new family of beta-1,4-glucanases.

The gene for a previously identified, extracellular, 120 kDa cellulose-binding protein (Cbp120) was isolated from a Cellulomonas fimi genomic library and expressed in Escherichia coli. Qualitative analysis of CM-cellulose hydrolysis shows that Cbp120 is an endo-beta-1,4-glucanase. Cbp120, now renamed CenE, catalyzes hydrolysis of cellohexaose with inversion of anomeric carbon configuration, characteristic of a single displacement reaction. Partial sequencing of its gene shows that CenE has significant sequence similarity with the catalytic domains of five enzymes from cellulolytic bacteria. It is proposed that the six enzymes form a new family of beta-1,4-glucanases. CenE is the first enzyme from this family to be characterized stereochemically.

Amino Acid Sequence↗
PubMed1994

Tandem lignin peroxidase genes of the fungus Trametes versicolor.

A DNA fragment containing two lignin peroxidase genes (LPG I and LPG II) has been isolated from a genomic library of the white-rot fungus Trametes versicolor. The genes are separated by 2.2 kbp and have the same direction of transcription. Conserved elements preceding the translation start have been identified. In addition to the TATA box, a stretch of 11 identical nucleotides is found 23-24 bp downstream of the TATA box. The putative mature peroxidases encoded by LPG I and LPG II are 87% identical in amino acid sequence. Both are preceded by two basic residues, also observed in lignin peroxidases from Phanerochaete chrysosporium. Strong evidence suggests that LPG I encodes a quantitatively dominant lignin peroxidase isozyme (TvLP12), whereas the product of LPF II has not been identified so far.

Amino Acid Sequence↗