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At least 1,135 records · Page 63Linked to original sources
PubMed1996

Comparison of a vitellogenin gene between two distantly related rhabditid nematode species.

Three vitellogenin genes from the free-living nematode Caenorhabditis elegans have previously been characterized at the molecular level. In order to study evolutionary relationships within this poorly understood taxon, we have cloned a vitellogenin gene, CEW1-vit-6, from a distantly related species belonging to the same family as C. elegans. Screening of a genomic library with a probe to total poly(A+) RNA yielded three clones that hybridized more intensely than all others, and all three corresponded to a single gene homologous to C. elegans vit-6. Comparison of CEW1-vit-6 with Ce-vit-6 reveals both strong similarities and surprising differences. Life Ce-vit-6, the gene is about 5 kb long and contains four unusually small introns (38-41 nt), but only one interrupts the gene at the same location as a Ce-vit-6 intron. The promoter region contains five matches to Vitellogenin Promoter Element 1 (VPE1) and no matches to VPE2, both previously shown to be required for vit gene transcription in C. elegans. Codon usage is in general similar to that of the Ce-vit genes, but a few codon biases are quite different. Alignment of the CEW1-vit-6 protein with Ce-vit-6 and Ce-vit-2 products suggests the existence of two domains which have evolved at different rates. Sequence comparison shows that nematode vitellogenins are much more closely related to vertebrate than to insect vitellogenins.

Amino Acid Sequence↗
PubMed1997

An endoglucanase from the anaerobic fungus Orpinomyces joyonii: characterization of the gene and its product.

An endoglucanase gene (celA) was isolated from a genomic library of the ruminal fungus Orpinomyces joyonii. DNA sequence analysis of celA revealed an intronless gene encoding a typical signal sequence, an N-terminal catalytic domain, two repeated regions linked by a short Ser/Thr-rich linker and a domain of unknown function. The deduced amino acid sequence of the catalytic domain showed homology with the family 5 cellulases. While the catalytic domain of CelA was not homologous to the catalytic domain of the endoglucanase gene (EG3) from the ruminal bacterium Fibrobacter succinogenes, the repeated regions of CelA were very similar to the noncatalytic domain of EG3. This suggests that evolutionary shuffling of endoglucanase domains might occur among bacteria and fungi within the anaerobic ecosystem of the rumen. The celA gene was expressed in Escherichia coli, and the periplasmic endoglucanase was used for the characterization studies of the enzyme. CelA exhibited both endoglucanase and xylanase activities. Its pH optimum was 4 and the temperature optimum was 40 degrees C. Deletion analysis showed that the repeated sequences and C-terminal domain of CelA were not required for enzyme activity.

Amino Acid Sequence↗
PubMed1994

Structure of a gene and cDNA of a major constitutive form of testosterone 6 beta-hydroxylase (P450/6 beta A) encoding CYP3A2: comparison of the cDNA with P450PCN2.

P450/6 beta A gene was isolated from a rat genomic library and its encoding protein was characterized by the expression of the corresponding cDNA in COS-1 cells. Between exon regions of P450/6 beta A gene and P450PCN2 cDNA (F. J. Gonzalez, B.-J. Song, and J. P. Hardwick (1986) Mol. Cell. Biol. 6, 2969-2976), 12 nucleotide differences were observed, involving two amino acid changes, His-->Asp [429] and Asp-->Gly [445], respectively. A cDNA (6 beta-A cDNA), whose nucleotide sequence was completely identical with the corresponding exon of P450/6 beta A gene, was isolated from a rat cDNA library. Northern blotting using specific oligonucleotide probes showed that 6 beta-A mRNA, but not P450PCN2 mRNA, was a major form in livers of the male rats. 6 beta-A protein expressed in COS-1 cells (at about 0.1 to 0.3% of total microsomal protein) catalyzed testosterone 6 beta-hydroxylation. The reaction was enhanced by the addition of NADPH-P450 reductase (2.5-fold) and by the simultaneous addition of cytochrome b5 and NADPH-P450 reductase (13.7-fold). Catalytic properties of 6 beta-A for typical CYP3A substrates were consistent with those of purified rat P450(6 beta-1) and P450(6 beta-3), corresponding, respectively, to CYP3A2 or the variant form (K. Nagata, F. J. Gonzalez, Y. Yamazoe, and R. Kato (1990) J. Biochem. 107, 718-725). Apparent Michaelis constants (Km) of 6 beta-A for testosterone 6 beta-hydroxylation and a O-dealkylation ratio of propoxycoumarin to pentoxycoumarin, however, showed higher degrees of similarity to those of purified P450(6 beta-3) than those of P450(6 beta-1).

Amino Acid Sequence↗
PubMed1995

Molecular characterization of a Campylobacter jejuni lipoprotein with homology to periplasmic siderophore-binding proteins.

A genomic library of Campylobacter jejuni (NCTC 11351) was used to identify genes which could confer a hemolytic phenotype to Escherichia coli. Accordingly, when transformants were screened on blood plates, hemolytic colonies appeared at a frequency of 3 x 10(-4). The gene conferring the hemolytic activity was identified by subcloning and was found to be responsible for the phenotype of all hemolytic transformants isolated. The open reading frame conferring this activity encodes a protein of 36,244 Da with a typical endopeptidase type II leader sequence. The protein is modified with palmitic acid when it is processed in E. coli, confirming that it is a typical lipoprotein. The deduced gene product of 329 amino acids has significant homology to the group of solute binding proteins from periplasmic-binding-protein-dependent transport systems for ferric siderophores, including the FatB protein from Vibrio anguillarium and the FhuD protein from Bacillus subtilis. In particular, the protein contained the signature sequence for siderophore-binding proteins, suggesting that the protein may be the siderophore-binding protein component of an iron acquisition system of C. jejuni.

Amino Acid Sequence↗
PubMed2006

Molecular basis of ergosterol-induced protection of grape against botrytis cinerea: induction of type I LTP promoter activity, WRKY, and stilbene synthase gene expression.

Type I lipid transfer proteins (LTPs) are basic, 9-kDa cystein-rich proteins believed to be involved in plant defense mechanisms. A 2,100-bp fragment containing the coding region of Vitis vinifera lipid transfer protein 1 (VvLTP1) and 1,420-bp of its promoter region was isolated by screening a grape genomic library. In silico analysis revealed several putative, defense-related, cis-regulatory elements such as W- and MYB-boxes, involved in the binding of WRKY and MYB transcription factors, respectively. The 5'-truncated versions of the VvLTP1 promoter were generated, cloned in front of the beta-glucuronidase (GUS) reporter gene, and introduced in tobacco plants and grapevine cell suspensions using Agrobacterium spp. Single MYB- and the W-boxes identified on the 0.250-kbp fragment were sufficient to induce GUS activity in transgenic tobacco plants after transient expression of MYB and WRKY. Ergosterol, a nonspecific fungal elicitor, induced GUS activity in transgenic grapevine cell suspensions transformed with the 1,420- and 750-bp promoter containing a palindromic arrangement of two W-boxes but not the 650- or 250-bp fragment, where only one W-box was present. Moreover, ergosterol triggered WRKY, VvLTP1, and stilbene synthase gene expression in grape plantlets and enhanced protection against Botrytis cinerea. The molecular basis of ergosterol-induced protection is discussed.

Acyltransferases↗
PubMed1991

Characterisation of a genomic clone covering the structural mouse MyoD1 gene and its promoter region.

We have isolated the mouse MyoD1 gene flanked by its promoter region by screening a genomic library with synthetic oligonucleotides. The structural gene is interrupted by two G + C rich introns. Transfection of the cloned gene inserted into an expression vector converts fibroblasts to myoblasts. Sequence analysis of about 650 bp of the 5' upstream region revealed the presence of several potential regulatory elements such as a TATA-box, an AP2-box, two SP1-boxes and a CAAT-box. In addition, there are three half palindromic estrogen response elements, a potential cAMP response element and various muscle specific elements such as a muscle-specific CAAT-box (MCAT) and four potential binding sites for MyoD1. Using S1 protection analysis the major start site of transcription in muscle and myoblast cells was mapped 3 bp upstream of the published cDNA 5' end. Promoter activity of the 650 bp upstream fragment was tested by in vitro transcription and by transfection analysis of myoblasts and fibroblasts. In all promoter test systems used, MyoD1 promoter activity was detected in myoblasts as well as in fibroblasts. Furthermore, DNA methylation was found to turn off MyoD1 promoter activity both in myoblasts and in fibroblasts.

Amino Acid Sequence↗
PubMed1998

Partial characterization of the Streptomyces lividans xlnB promoter and its use for expression of a thermostable xylanase from Thermotoga maritima.

Xylanase activity assays were used to screen a Streptomyces coelicolor genomic library in Escherichia coli, and a xylanase gene that is 99% identical to the xylanase B gene (xlnB) of S. lividans (GenBank accession no. M64552) was identified. The promoter region of this gene was identified by using a transcriptional fusion between the upstream region of the S. coelicolor xlnB gene and the xylE reporter gene. Transcription from the xlnB promoter was found to be induced by xylan and repressed by glucose. A single apparent transcription start site was identified by both primer extension analysis and in vitro run off transcription assays. Analysis of deletions of the promoter identified a region required for glucose repression. By using the transcriptional and protein localization signals of the Streptomyces xlnB gene, an in-frame translational fusion between the end of the xlnB signal sequence and the ATG of the Thermotoga maritima xynA gene was constructed. The xynA gene encodes a thermostable xylanase that has been demonstrated to be useful in the bleaching of Kraft pulp. The xlnB-xynA gene fusion was expressed in Streptomyces, and the activity of the protein produced was thermostable and was localized to the supernatant fraction of harvested cells.

Amino Acid Sequence↗
PubMed1990

Nucleotide sequence of the HLA-A26 class I gene: identification of specific residues and molecular mapping of public HLA class I epitopes.

A cosmid clone bearing an HLA class I gene has been isolated from a human genomic library by hybridization to a class I-specific probe. This clone encodes the HLA-A26 molecule characterized by immunologic reagents on murine transfected L cells. Nucleotide sequencing of the A26 allele has been performed, and the deduced amino acid sequence was compared with previously published HLA class I sequences. Amino acid sequence homologies between HLA-A26 molecules and members of the HLA-AW19 cross-reactive group were observed and allowed us to demonstrate that residue Q144 is the only critical residue involved in the binding of the 4E monoclonal antibody defining an epitope common to all HLA-B, -C, and -Aw19 alleles. This study also permitted designation of a V residue at position 189 in the third domain as possibly involved in the binding of the B1-23-2 monoclonal antibody. Furthermore, we located clusters of variability in reference to the three-dimensional structure of the HLA-A molecules, i.e., the ninth residue of the first beta-strand domain, the upper surface of the first helical region, and both beta and alpha structures of the alpha 2 domain.

Amino Acid Sequence↗
PubMed1999

A group of alpha-1,4-glucan lyase genes from the fungi Morchella costata, M. vulgaris and Peziza ostracoderma. Cloning, complete sequencing and heterologous expression.

We here report genes encoding a newly discovered class of starch- and glycogen-degrading enzyme, alpha-1,4-glucan lyase (EC 4.2.2.13), which degrades starch and glycogen to 1,5-anhydro-D-fructose. Two lyases were purified and partially sequenced from the macrofungi Morchella costata and M. vulgaris. The obtained lyase amino acid sequences were used to generate PCR primers, which were further used to probe the fungal genomic libraries. Two lyase genes (Agll1;Mo.cos and Agll1;Mo.vul) from the two fungi were fully sequenced and found to contain a coding region of 3201 bp and 3213 bp, respectively. A total of 13 small introns were found in each of the two genes with identical positions. The two lyase genes share 86% identity at the amino acid level. They encode mature lyases with 1066 and 1070 amino acids, respectively. The deduced molecular masses of 121,530 and 121,971 Da agree with the values found for the two purified lyases. A structure analysis of the promoter regions of the lyase genes revealed a number of putative regulatory DNA elements, such as the AREA and CREA sites, which are related to nitrogen and carbon metabolism, respectively, and the CCAAT/CAAT boxes, which are related to basal expression of genes. A third lyase gene (Agll1;Pe.ost) from the fungus Peziza ostracoderma was partially sequenced to 557 bp. The amino acid sequence deduced from this nucleotide fragment shares 76% identity with the M. costata lyase. Heterologous expression of the M. costata lyase gene was achieved intracellularly in Pichia pastoris and Aspergillus niger.

Amino Acid Sequence↗
PubMed1997

Partial characterization of the 5'-flanking region of trout IP: a Po-like gene containing a PLP-like promoter.

The IP gene of trout encodes two Po-like glycoproteins which are expressed by oligodendrocytes in the fish CNS. A 679 bp fragment of its 5'-flanking region was isolated from a genomic library and sequenced. The transcription start point was determined 124 bp upstream the ATG initiator codon by primer extension analysis. Apart from a modified TATA-box and an inverted CCAAT-box located at canonical distances from the transcription start site several eucaryotic cis-acting regulatory elements were identified in the 679 bp upstream region, including an AP-1 binding site, a brain specific Sp1 motif, a cyclic AMP responsive element and a consensus sequence for POU homeodomain protein binding. The occurence of respective DNA-binding proteins for Sp1, AP-1 and POU in the nuclei of trout oligodendrocyte progenitor cells was verified by gel retardation experiments. Functional activity of various subfragments of the 679 bp upstream region was demonstrated by CAT reporter gene analysis. A computer-assisted sequence alignment of the trout IP 5'-flanking end with the corresponding region of the mammalian PLP gene promoter revealed four sites of high homology, while similarity with the mammalian Po gene promotor was low. The results are discussed with respect to the phylogenetic shift from Po-like proteins to PLP during evolution of the vertebrate CNS myelin sheath.

Animals↗
PubMed1998

Equus caballus gelsolin--cDNA sequence and protein structural implications.

We have generated and characterized the cDNA from equine smooth muscle that encodes gelsolin, an actin-modulating protein. Overlapping cDNA clones synthesized by the reverse transcriptase/polymerase chain reaction and clones isolated from a horse genomic library provided the complete primary structure for the intracellular isoform of gelsolin, while cDNA complemented with protein sequence data produced the full-length primary transcript of the gelsolin isoform found circulating in equine plasma. The deduced amino acid sequences of the intracellular and secreted versions of equine gelsolin infer polypeptides of 731 and 755 residues with apparent molecular masses of 80.7 kDa and 83.2 kDa, respectively. Multiple sequence alignment analysis of equine, human, porcine, and murine orthologs of gelsolin demonstrates prominent similarities among all of these proteins, with the horse and human molecules exhibiting the largest degree of likeness with respect to polypeptide length and overall sequence composition. Both horse and human plasma gelsolins are comprised of 755 amino acids with 94% of the residues identical, while the degree of sequence identity in the shorter (731 residues) cytoplasmic gelsolins is 95%. Analysis of the sequences and structures of the six related domains that comprise gelsolin emphasizes the strong correlation that exists between primary structural conservation among mammalian gelsolins and maintenance of the three-dimensional domain fold characteristic of members of this protein family.

Amino Acid Sequence↗
PubMed2003

Identification and characterization of Lactobacillus helveticus PepO2, an endopeptidase with post-proline specificity.

A post-proline endopeptidase (PepO2) was detected in cell extracts from a genomic library of Lactobacillus helveticus CNRZ32 by using the synthetic substrate N-acetyl-beta-casein-(f203-209)-rho-nitroanilide in a coupled reaction with aminopeptidase N. Isolates with activity for this substrate contained plasmids with visually indistinguishable restriction profiles. Nucleotide sequence analysis revealed a 1,947-bp open reading frame, designated pepO2, encoding a putative 71.4-kDa protein. Analysis of the predicted peptide sequence revealed that L. helveticus PepO2 contained the zinc-dependent metalloprotease motif HEXXH and exhibited levels of amino acid sequence similarity of 72, 61, 59, and 53% to L. helveticus PepO, Lactococcus lactis PepO2, L. lactis PepO, and Lactobacillus rhamnosus PepO, respectively. Northern hybridization results indicated that the transcript containing pepO2 was monocistronic. Despite the high degrees of amino acid similarity to PepO proteins from other lactic acid bacteria, the specificity of the L. helveticus PepO2 for post-proline bonds distinguishes it from other PepO-type endopeptidases characterized to date. The specificity for post-proline bonds also suggests that this enzyme may play a central role in the hydrolysis of casein-derived bitter peptides, such as beta-casein(f193-209).

Amino Acid Sequence↗
PubMed1989

Three separate mitochondrial DNA sequences are contiguous in human genomic DNA.

We isolated from a HeLa genomic library 38 plaques that hybridized to total mitochondrial (mt) DNA isolated from human placenta. One clone (HLmt-17.8) hybridized to a 740 base-pair (12 S ribosomal RNA gene and displacement loop) mtDNA probe and was characterized in more detail. Within its 17.8 x 10(3) base-pair insert a 1.6 x 10(3) base-pair mtDNA fragment was similar to three non-sequential coding genes of human mtDNA, including a part of the 12 S ribosomal RNA (684-971), the cytochrome oxidase I (6553-7302), and two NADH dehydrogenase [ND4L/ND4] (10,606-11,159). The similarity to human mtDNA sequences was 92.0%, 92.3% and 92.4%, respectively, the highest degree of similarity to human mtDNA so far reported. This is also the first report of several adjacent mtDNA-like sequences in cellular chromosomes. The mtDNA-like sequences in HLmt-17.8 was found in the DNAs of human placenta, freshly isolated human leukocytes, foreskin and several human cell lines; but it was not present in other primates or lower organisms. The HLmt-17.8 mtDNA-like region appears to be a pseudogene that transferred into the nucleus in humans more recently than nine million years ago.

Base Sequence↗
PubMed1997

Cloning, nucleotide sequence and expression of a mannitol dehydrogenase gene from Pseudomonas fluorescens DSM 50106 in Escherichia coli.

A NAD-dependent mannitol dehydrogenase (MtlD) was purified to homogeneity from P. fluorescens DSM50106 and the N-terminal amino acid sequence was determined. An oligonucleotide deduced from this peptide sequence was used as a probe to isolate the mannitol dehydrogenase gene (mtlD) from a genomic library of P. fluorescens. Nucleotide sequence analysis of a 1.8 kb NruI fragment containing the entire mtlD gene revealed an open reading frame of 1482 bp encoding a protein with a calculated molecular weight of 54.49 kDa. The enzyme shared a high similarity with a mannitol dehydrogenase from Rhodobacter sphaeroides and a putative mannitol dehydrogenase of Saccharomyces cerevisae with an overall identity in amino acid sequence of 44% and 42%, respectively, whereas the similarity to mannitol-1-phosphate dehydrogenases of Escherichia coli or Enterococcus faecalis was only about 23% of identical amino acids. By construction of inducible expression plasmids the specific activity of the mannitol dehydrogenase synthesized in E. coli was increased from 0.02 U (mg protein)(-1) to 10 U (mg protein)(-1). After fusion of six histidine codons to the 3' end of mtlD gene and expression in E. coli active mannitol dehydrogenase could be purified in a two-step procedure by affinity chromatography using a Ni2+ matrix column. The purified enzyme exhibited a specific activity of 46 U (mg protein)(-1) and was shown to be a polyol dehydrogenase with a broad substrate spectrum oxidizing efficiently mannitol, sorbitol and arabitol.

Amino Acid Sequence↗
PubMed1996

Two chitin synthase genes from Ustilago maydis.

PCR was used to amplify fragments corresponding to CHS genes from Ustilago maydis, utilizing as primers oligonucleotides devised according to the conserved regions of fungal CHS genes. The PCR product was employed as a probe to screen a genomic library of the fungus. Two different CHS genes (Umchs1 and Umchs2) were thus identified in the positive clones recovered. Their sequence revealed high similarity with the CHS genes previously cloned from other fungi, especially in their central region. Alignment with the deduced protein sequences of all CHS genes reported up to date showed the existence of seven conserved domains. Transcripts from both genes were detected in the yeast and mycelial forms. In general, the transcripts from the Umchs1 gene appeared to be present at a higher level than the transcripts from the Umchs2 gene; the transcripts from both genes appeared to be more abundant in the mycelial form. Gene replacement of either gene and analysis of the resulting phenotype demonstrated that they are non-essential. Nevertheless, growth, chitin synthase activity levels, and chitin content of mycelial cells induced by cultivation in acidic media were all reduced in chs1 and chs2 mutants. However, mating, virulence and dimorphic behaviour were unaffected. Overall, the results indicate that the CHS1 and CHS2 genes encode products with redundant functions in U. maydis.

Amino Acid Sequence↗
PubMed1992

The gene for a novel human lamin maps at a highly transcribed locus of chromosome 19 which replicates at the onset of S-phase.

A previously described human DNA fragment which is replicated early in S-phase of HL-60 cell DNA (C. Tribioli, G. Biamonti, M. Giacca, M. Colonna, S. Riva, and A. Falaschi, Nucleic Acids Res. 15:10211-10232, 1987) was used to screen a genomic library in lambda Ch28. A clone which contained a 13.7-kb insert (L30E) found to code for several transcripts was isolated. The transcription of L30E DNA exhibited a complex pattern and a tissue-specific and proliferation-dependent type of regulation. The data were consistent with two tandemly arranged transcription units, the 3' end of one separated from the 5' end of the other by a sequence of about 600 bp containing an active promoter. The isolation and sequencing of L30E-specific cDNAs permitted identification of two genes, one of which encoded a B-type human lamin (analogous to mouse lamin B2). L30E DNA was mapped by in situ hybridization at the G-negative subtelomeric band p13.3 of chromosome 19. Interestingly, in synchronized HL-60 cells, L30E DNA is replicated in the first minute of S-phase. Replication of the lamin gene early in S-phase may reflect a coupling between early replication and transcription of genes for S-phase-specific proteins such as lamins.

Amino Acid Sequence↗
PubMed1994

Two murine GSTpi genes are arranged in tandem and are differentially expressed.

An 8-kilobase mouse genomic fragment containing two intact glutathione S-transferase (GST) genes has been isolated from a mouse lambda genomic library. Each of the genes (designated mGSTpiA and mGSTpiB) is less than 3 kilobases in size and is comprised of seven exons that give rise to a 630-base pair open reading frame encoding 209 amino acids. The deduced amino acid sequences of the gene products differ in only 6 amino acids at positions 10, 11, 89, 104, 106, and 109. These two genes are highly homologous to rat GST-P and to a lesser extent to human GST-pi. Northern blot analysis of mRNAs from a variety of mouse tissues demonstrated that mGSTpiB is ubiquitously expressed, whereas mGST-piA is more selectively expressed in gallbladder, colon, heart, and skeletal muscle. Primer extension analysis revealed four potential transcription start sites in mGST-piB and one in mGSTpiA. Although both genes were expressed in vitro and in vivo only mGSTpiB product metabolized 1-chloro-2,4-dinitrobenzene, a common GST substrate. Further in vitro expression studies of three chimeric mGSTpi genes suggested that one or both of the amino acids at positions 10 and 11 of mouse GSTpi enzymes are important for the enzyme's ability to metabolize 1-chloro-2,4-dinitrobenzene.

Amino Acid Sequence↗
PubMed1998

Structural analysis of Arabidopsis thaliana chromosome 5. VIII. Sequence features of the regions of 1,081,958 bp covered by seventeen physically assigned P1 and TAC clones.

A total of 17 Pl and TAC clones each representing an assigned region of chromosome 5 were isolated from P1 and TAC genomic libraries of Arabidopsis thaliana Columbia, and their nucleotide sequences were determined. The length of the clones sequenced in this study summed up to 1,081,958 bp. As we have previously reported the sequence of 9,072,622 bp by analysis of 125 P1 and TAC clones, the total length of the sequences of chromosome 5 determined so far is now 10,154,580 bp. The sequences were subjected to similarity search against protein and EST databases and analysis with computer programs for gene modeling. As a consequence, a total of 253 potential protein-coding genes with known or predicted functions were identified. The positions of exons which do not show apparent similarity to known genes were also assigned using computer programs for exon prediction. The average density of the genes identified in this study was 1 gene per 4277 bp. Introns were observed in 74% of the potential protein genes, and the average number per gene and the average length of the introns were 4.3 and 168 bp, respectively. The sequence data and gene information are available on the World Wide Web database KAOS (Kazusa Arabidopsis data Opening Site) at http://www.kazusa.or.jp/arabi/.

Arabidopsis↗