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At least 973 records · Page 54Linked to original sources
PubMed1998

Isolation, characterization, and expression of the gene encoding the beta subunit of the mitochondrial processing peptidase from Blastocladiella emersonii.

A 2.3-kb BamHI-KpnI fragment was isolated from a partial genomic library and shown by nucleotide sequence analysis to contain the entire coding region of the gene encoding the beta subunit of the Blastocladiella mitochondrial processing peptidase (beta-MPP). The predicted beta-MPP protein has 465 amino acids and a calculated molecular mass of 50.8 kDa. S1 nuclease protection assays revealed an intron, 209 bp in size, interrupting the coding region between the putative signal sequence and the mature protein. Northern blot analysis showed that beta-MPP mRNA levels decrease significantly during B. emersonii sporulation, reaching basal levels in the zoospore stage. The amount of beta-MPP protein, determined in Western blots, unlike its mRNA, does not vary significantly throughout the fungal life cycle.

Amino Acid Sequence↗
PubMed1995

Cloning and expression of Kluyveromyces fragilis LAC4 gene.

The genomic library of Kluyveromyces fragilis was constructed in E. coli TG1, and 5 beta-galactosidase gene (LAC4) clones have been obtained from the library by complementation of the Kluyveromyces lactis lac4-8 mutation. The studies on the structure and the function of the LAC4 gene revealed that (i) the gene can also complement E. coli lacZ mutation; (ii) the physical map of the K. fragilis LAC4 gene was very similar to that of K. lactis; (iii) the beta-galactosidase levels expressed by the clone strains were much higher than that expressed by the original strain; (iv) the variation of the beta-galactosidase level of different clone strains induced by lactose or galactose was related to the retained degree of the 5' flanking region of LAC4 gene, suggesting that there might be a lactose specific transcription activating element in the region.

Cloning, Molecular↗
PubMed1994

Characterization of the human gene (TBXAS1) encoding thromboxane synthase.

The gene encoding human thromboxane synthase (TBXAS1) was isolated from a human EMBL3 genomic library using human platelet thromboxane synthase cDNA as a probe. Nucleotide sequencing revealed that the human thromboxane synthase gene spans more than 75 kb and consists of 13 exons and 12 introns, of which the splice donor and acceptor sites conform to the GT/AG rule. The exon-intron boundaries of the thromboxane synthase gene were similar to those of the human cytochrome P450 nifedipine oxidase gene (CYP3A4) except for introns 9 and 10, although the primary sequences of these enzymes exhibited 35.8% identity each other. The 1.2-kb of the 5'-flanking region sequence contained potential binding sites for several transcription factors (AP-1, AP-2, GATA-1, CCAAT box, xenobiotic-response element, PEA-3, LF-A1, myb, basic transcription element and cAMP-response element). Primer-extension analysis indicated the multiple transcription-start sites, and the major start site was identified as an adenine residue located 142 bases upstream of the translation-initiation site. However, neither a typical TATA box nor a typical CAAT box is found within the 100-b upstream of the translation-initiation site. Southern-blot analysis revealed the presence of one copy of the thromboxane synthase gene per haploid genome. Furthermore, a fluorescence in situ hybridization study revealed that the human gene for thromboxane synthase is localized to band q33-q34 of the long arm of chromosome 7. A tissue-distribution study demonstrated that thromboxane synthase mRNA is widely expressed in human tissues and is particularly abundant in peripheral blood leukocyte, spleen, lung and liver. The low but significant levels of mRNA were observed in kidney, placenta and thymus.

Amino Acid Sequence↗
PubMed1997

Molecular characterization of the glyceraldehyde-3-phosphate dehydrogenase gene of Phaffia rhodozyma.

The glyceraldehyde-3-phosphate dehydrogenase (GPD; EC1.2.1.12)-encoding gene (gpd) was isolated from a genomic library of Phaffia rhodozyma CBS 6938. Unlike some other eukaryotic organisms the gpd gene is represented by a single copy in P. rhodozyma. The complete nucleotide sequence of the coding, as well as the flanking non-coding regions was determined. The nucleotide sequence of gpd predicted six introns and a polypeptide chain of 339 amino acids. The codon usage in the gpd gene of P. rhodozyma was highly biased and was significantly different from the codon usage in other yeasts. Phylogenetic analysis of different yeasts and filamentous asco- and basidiomycetes gpd sequences indicated that the gpd gene of P. rhodozyma forms a cluster with the corresponding genes of filamentous basidiomycetes.

Amino Acid Sequence↗
PubMed1991

A minor globin gene of the bivalve mollusc Anadara trapezia.

A minor haemoglobin gene was isolated from an Anadara trapezia genomic library using a synthetic oligonucleotide probe based on the identical amino acid sequence of the F-helical region of all the major Anadara globins previously sequenced. The amino acid sequence inferred from the coding region of the gene indicated that it is different from that of the three major chains alpha, beta and gamma, but most like the beta-chain. This beta-variant sequence shows 100% homology in the conserved F-helix region. The minor gene was found to contain two long intervening sequences, 1214 bp and 1435 bp, longer than those present in the genes for vertebrate globins or leghaemoglobins but shorter than those in myoglobin genes.

Amino Acid Sequence↗
PubMed1995

Comparative analyses reveal a highly conserved endoglucanase in the cellulolytic genus Fibrobacter.

An RNA probe complementary to the endoglucanase 3 gene (cel-3) of Fibrobacter succinogenes S85 hybridized to chromosomal DNAs from isolates representing the genetic diversity of the genus. The probe was subsequently used to identify putative cel-3-containing clones from genomic libraries of representative Fibrobacter isolates. Comparative sequence analyses of the cloned cel-3 genes confirmed that cel-3 is conserved among Fibrobacter isolates and that the ancestral cel-3 gene appears to have coevolved with the genus, since the same genealogy was inferred from sequence comparisons of 16S rRNAs and cel-3 genes. Hybridization comparisons using a xylanase gene probe suggested similar conservation of this gene. Together the data indicate that the cellulolytic apparatus is conserved among Fibrobacter isolates and that comparative analyses of homologous elements of the apparatus from different members, in relationship to the now established phylogeny of the genus, could serve to better define the enzymatic basis of fiber digestion in this genus.

Amino Acid Sequence↗
PubMedDate not supplied

PCR amplification of DNA sequences from the transcription factor IID and cation transporting ATPase genes in Pneumocystis carinii.

Oligonucleotide primers were used to amplify DNA sequences from a plasma membrane cation transporting ATPase gene and a transcription factor IID (TFIID) gene from Pneumocystis carinii genomic DNA. The entire P. carinii ATPase gene was cloned from a genomic library by hybridization to the PCR-amplified DNA product. The nucleotide sequence of the gene contained a 2,799 base-pair open reading frame that encoded a 102,274 dalton protein composed of 933 amino acids. The P. carinii ATPase protein was 69-74% identical to four fungal proton pumps but less than 35% identical to protozoan and mammalian cation transporting ATPase genes or the Ca++ ATPases of Saccharomyces. The nucleotide sequence of a portion of the TFIID gene could be translated to produce a peptide of 53 amino acids in two regions of the sequence, interrupted by a 45 bp intron. The predicted TFIID amino acid sequence was identical to yeast TFIID genes in this region.

Adenosine Triphosphatases↗
PubMed1991

Structure, expression, chromosomal location and product of the gene encoding ADH1 in Petunia.

A genomic clone for an alcohol dehydrogenase (Adh) gene has been isolated from Petunia hybrida cv. V30 by screening a Petunia genomic library with a maize Adh1 probe. A combination of RFLP and allozyme segregation data failed to demonstrate which of two Adh loci, both of which map to chromosome 4, was the source of the cloned gene. The product of the cloned genes has been identified unequivocally by a transient expression assay in Petunia protoplasts. We have designated this gene Petunia Adh1. The expression of this gene is tightly regulated in the developing anther, where its gene product is the predominant ADH isozyme. It is anaerobically inducible in roots, stems and leaves of seedlings. The induction of enzyme activity is correlated with induction of Adh1 mRNA.

Alcohol Dehydrogenase↗
PubMed2000

A genome-wide screening in Saccharomyces cerevisiae for genes that confer resistance to the anticancer agent cisplatin.

Cisplatin is a potent DNA-damaging agent that has demonstrated anticancer activities against several tumors. However, manifestation of cellular resistance is a major obstacle in anticancer therapy that severely limits the curative potential of cisplatin. Therefore, understanding the molecular basis of cisplatin resistance could significantly improve the clinical efficacy of this anticancer agent. Here, we employed Saccharomyces cerevisiae as a model organism to study cisplatin resistance mechanisms and describe a one-step cisplatin selection to identify and characterize novel cisplatin resistance genes. Screening a multicopy yeast genomic library enabled us to isolate several yeast clones for which we could confirm that the cisplatin resistance phenotype was linked to the introduced fragment. In a first attempt, a number of open reading frames could be identified. Among these genes, PDE2 and ZDS2 were repeatedly identified as genes whose overexpression confers cellular resistance to cisplatin. PDE2, encoding cAMP-phosphodiesterase 2, is of particular interest because the overexpression of this yeast gene is known to induce cisplatin resistance in mammalian cells as well, providing proof of the principle of our experimental approach. In addition, the identification of PDE2 shows that our yeast screening system can directly be informative for drug resistance in mammalian cells.

Chromosome Mapping↗
PubMed1991

Structure and expression of sunflower ubiquitin genes.

Two ubiquitin genes, designated UbB1 and UbB2, were isolated from a sunflower genomic library. They encode polyubiquitin transcripts corresponding to six repeats of the monomer. Northern blot analysis identified several different transcript size classes: both UbB1 and UbB2 transcripts are found in the most abundant 1.6 kb class. In contrast to the previously isolated UbF transcript which is present at high levels in flowers, UbB1 and UbB2 are expressed constitutively at low levels in different tissues. The levels of the two transcripts increase after heat stress. The two genes exhibit strong homology suggesting that they may result from duplication and conversion. Surprisingly, UbB1 gene shows structural similarities with the chicken ubiquitin heat shock gene, in particular the presence of an intron located just in front of the first ATG.

Amino Acid Sequence↗
PubMed2006

Tall fescue genomic SSR markers: development and transferability across multiple grass species.

Simple sequence repeat (SSR) markers are highly informative and widely used for genetic and breeding studies. Currently, a very limited number of SSR markers are available for tall fescue (Festuca arundinacea Schreb.) and other forage grass species. A tall fescue genomic library enriched in (GA/CT)( n ) repeats was used to develop primer pairs (PPs) flanking SSRs and assess PP functionality across different forage, cereal, and turf grass species. A total of 511 PPs were developed and assessed for their utility in six different grass species. The parents and a subset of a tall fescue mapping population were used to select PPs for mapping in tall fescue. Survey results revealed that 48% (in rice) to 66% (in tall fescue) of the PPs produced clean SSR-type amplification products in different grass species. Polymorphism rates were higher in tall fescue (68%) compared to other species (46% ryegrass, 39% wheat, and 34% rice). A set of 194 SSR loci (38%) were identified which amplified across all six species. Loci segregating in the tall fescue mapping population were grouped as loci segregating from the female parent (HD28-56, 37%), the male parent (R43-64, 37%), and both parents (26%). Three percent of the loci that were polymorphic between parents were monomorphic in the pseudo F1 mapping population and the remaining loci segregated. Sequencing of amplified products obtained from PP NFFAG428 revealed a very high level of sequence similarity among the grass species under study. Our results are the first report of genomic SSR marker development from tall fescue and they demonstrate the usefulness of these SSRs for genetic linkage mapping in tall fescue and cross-species amplification.

Base Sequence↗
PubMed1990

The complete nucleotide sequence of the growth-hormone gene from the common carp (Cyprinus carpio).

We have isolated and sequenced a phase clone from a common carp (Cyprinus carpio) genomic library that carries a gene encoding growth hormone (GH). This gene consists of five exons and four introns spanning a region of about 3 kilobase pairs. Its exons correspond with one of two reported cDNAs of carp GH except for nine differences in the nucleotide sequence, while the encoded amino-acid sequences are identical. The sequence upstream from the transcription start point contains two tandem repeats of AACTCTCATG (from -85 to -62) and the typical TATA box. All the introns start with a consensus GT dinucleotide and end with AG. The arrangement of exons and introns is very similar to that seen in mammalian GH, but quite different from the GH genes of rainbow trout and Atlantic salmon.

Amino Acid Sequence↗
PubMed1994

Cloning and characterization of the pepD gene of Aspergillus niger which codes for a subtilisin-like protease.

Serine proteases constitute an important group of extra- and intracellular proteases in fungi. These enzymes are characterized by conserved regions around the active site residues, Asp, His and Ser. Based on this amino acid (aa) sequence conservation, we have used degenerate primer PCR to isolate subtilisin-specific genomic probes from Aspergillus niger, and cloned a gene, pepD, by screening a lambda genomic library using a PCR probe. The pepD gene contains three putative introns, which are 51-, 47- and 55-bp long and has an open reading frame coding for a protein which consists of 416 aa. The deduced aa sequence shows similarity to subtilisin-like proteases, in particular to fungal alkaline proteases. Signal sequence cleavage prediction indicates that the first 20 aa are probably removed upon transfer to the endoplasmic reticulum. The conservation of the pro-enzyme cleavage site in fungal alkaline proteases suggests that the mature protein is derived from this polypeptide via the removal of an additional 101 aa, resulting in a mature 30,294-Da enzyme consisting of 295 aa.

Amino Acid Sequence↗
PubMed1995

Cloning, sequencing and expression of Pseudomonas testosteroni gene encoding 3 alpha-hydroxysteroid dehydrogenase.

We describe the cloning, sequencing and expression of the 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSD) gene of Pseudomonas testosteroni. A genomic library of P. testosteroni total DNA constructed from SauIIIA digests ligated to an lambda gt11 vector was probed with a polyclonal antibody raised against purified enzyme. Subclones derived from a recombinant phage containing a 1746 bp insert were sequenced and found to contain an open reading frame of 696 bp that corresponds to a protein of 231 amino acid residues. A search for homologous proteins was performed. No similarity was observed when comparing 3 alpha-HSD with known members of the short-chain dehydrogenase family. However a small proteic fragment (80 amino acids) shows homology with the N-terminal sequence of bacterial L7/L12 ribosomal proteins.

3-Hydroxysteroid Dehydrogenases↗
PubMed2001

Cloning and characterization of a gene encoding an immunosuppressive factor from Actinobacillus actinomycetemcomitans.

Actinobacillus actinomycetemcomitans is a pathogen of localized juvenile periodontitis and adult periodontitis. Immunomodulating activity is generally thought to be important in colonization by such pathogenic bacteria. Among the proteins possessing these activities, a 14 kDa immunosuppressive factor of A. actinomycetemcomitans has been reported by Kurita-Ochiai and Ochiai (Infect Immun 64: 50-54, 1996). To evaluate this factor, we cloned and characterized the gene encoding it. The immunosuppressive factor was screened from a genomic library of A. actinomycetemcomitans using an oligonucleotide probe based on the amino acid sequence of the factor. The clone obtained, pHI13, contained a 1.5 kbp fragment. The immunosuppressive factor located in its center. Southern blot analysis showed that this factor is common among A. actinomycetemcomitans strains. The open reading frame consisted of 324 bp coding for 107 amino acid residues. The relative molecular mass of the deduced amino acid sequence was calculated to be 11,595. BLAST analysis indicated that the amino acid sequence is highly homologous with those of thioredoxins from Haemophilus influenzae (76.6%), Neisseria meningitidis (67.3%), and Pseudomonas aeruginosa (59.3%). These results suggest that the 14 kDa immunosuppressive factor characterized in this study is a thioredoxin.

Aggregatibacter actinomycetemcomitans↗
PubMed1994

Nucleotide sequence of a gene for nitrite reductase from Arabidopsis thaliana.

A nitrite reductase (NiR) gene was recovered from Arabidopsis thaliana genomic library by the homology with a cDNA of spinach NiR and sequenced. Based on the comparison with the spinach cDNA, the Arabidopsis NiR gene was concluded to contain 4 exons [exon 1 of 376 bp (beginning with ATG start codon), exon 2 of 355 bp, exon 3 of 289 bp and exon 4 of 741 bp (ending at TGA stop codon)] and 3 introns (intron 1 of 196 bp, intron 2 of 81 bp and intron 3 of 77 bp). This conclusion was confirmed by the analysis using the RT-PCR method. The deduced amino acid sequence of the coding region of the Arabidopsis NiR gene had high similarities with those of NiR genes of other plants including spinach.

Arabidopsis↗
PubMed1992

Isolation and sequence analysis of carp gonadotropin beta-subunit gene.

Using the cDNA encoding the beta subunit of carp gonadotropin (cGTH-beta) as a probe, 14 clones containing cGTH-beta gene have been isolated from a carp genomic library. Nucleotide sequence analysis indicated that the transcriptional unit of the cGTH-beta gene is 1.2 Kb. Similar to mammalian GTH-beta genes, cGTH-beta gene contains three exons and two introns. The locations of the exon/intron junctions also correspond to those of mammalian GTH-beta gene. Using the primer extension assay, the start site of transcription was determined to be 35 or 37 bp upstream from the translation initiation codon. The TATAA box is present in the 5' flanking region of the gene, 21 bp upstream from the start site of transcription. Three polyadenylation signals, AATAAA, are located in the 3' noncoding region, 111, 430, and 442 bp downstream from the stop codon of translation, respectively.

Amino Acid Sequence↗
PubMed2006

Screening and its potential application of lipolytic activity from a marine environment: characterization of a novel esterase from Yarrowia lipolytica CL180.

To develop an enantioselective lipase/esterase hydrolyzing racemic ofloxacin ester to levofloxacin, samples were collected from a variety of marine environments such as cold sea, hydrothermal vent area, sediment, tidal flat area, arctic sea, marine organisms, and so on. Microorganisms were isolated by plating on an enrichment medium with simultaneous detection of lipolytic activities and screened for the hydrolysis of ofloxacin ester. Three candidates among isolates were selected, and one of them, identified as Yarrowia lipolytica CL180, hydrolyzed preferentially S-enantiomer of racemic ofloxacin ester. The lipase/esterase gene (yli180) was cloned by screening a genomic library. The sequence analysis revealed an open reading frame consisting of 1,431 bp that encoded a protein of 476 amino acids with a molecular mass of 53 kDa. The yli180 gene was expressed in Escherichia coli and purified to homogeneity. The optimum activity of the recombinant protein (rYli180) occurred at pH 7.5 and 35 degrees C, respectively. rYli180 preferentially hydrolyzed p-nitrophenyl esters of fatty acids with short chain lengths of < or =10 carbon atoms. This study represents a novel esterase of type B1 carboxylesterase/lipase family from a marine isolate, showing a potential usage as a biocatalyst because of enantioselectivity toward racemic ofloxacin ester.

Amino Acid Sequence↗