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At least 451 records · Page 25Linked to original sources
PubMed2004

Construction of a metastasis-associated gene subtracted cDNA library of human colorectal carcinoma by suppression subtraction hybridization.

AIM: To construct a differentially-expressed gene subtracted cDNA library from two colorectal carcinoma (CRC) cell lines with different metastatic phenotypes by suppression subtractive hybridization. METHODS: Two cell lines of human CRC from the same patient were used. SW620 cell line showing highly metastatic potential was regarded as tester in the forward subtractive hybridization, while SW480 cell line with lowly metastatic potential was treated as tester in the reverse hybridization. Suppression subtractive hybridization (SSH) was employed to obtain cDNA fragments of differentially expressed genes for the metastasis of CRC. These fragments were ligated with T vectors, screened through the blue-white screening system to establish cDNA library. RESULTS: After the blue-white screening, 235 white clones were picked out from the positive-going hybridization and 232 from the reverse. PCR results showed that 200-700 bp inserts were seen in 98% and 91% clones from the forward and reverse hybridizations, respectively. CONCLUSIONS: A subtractive cDNA library of differentially expressed genes specific for metastasis of CRC can be constructed with SSH and T/A cloning techniques.

Cell Line, Tumor↗
PubMed1987

Isolation and expression analysis of two yeast regulatory genes involved in the derepression of glucose-repressible enzymes.

Yeast strains carrying one of the two regulatory mutations cat1 and cat3 are defective in derepression of several glucose-repressible enzymes that are necessary for utilizing non-fermentable carbon sources. Hence, these strains fail to grow on ethanol, glycerol or acetate. The synthesis of isocitrate lyase, malate synthase, malate dehydrogenase and fructose-1,6-bisphosphatase is strongly affected in cat1 and cat3 strains. Genes CAT1 and CAT3 have been isolated by complementation of the cognate mutations after transformation with an episomal plasmid gene library. The restriction map of CAT1 proved its allelism to the earlier isolated SNF1 gene. Both genes appear to exist as single-copy genes per haploid genome as indicated by Southern hybridization. Northern analysis has shown that the 1.35 kb CAT3 mRNA is constitutively expressed, independent of the carbon source in the medium. Derepression studies with CAT3 transformants using a multi-copy plasmid showed over-expression of glyoxylate cycle enzymes. This result would be consistent with a direct effector function for the CAT3 gene product.

Crosses, Genetic↗
PubMed1996

Nitrobacter winogradskyi cytochrome c oxidase genes are organized in a repeated gene cluster.

Cytochrome c oxidase (EC 1.9.3.1) is one of the components of the electron transport chain by which Nitrobacter, a facultative lithoautotrophic bacterium, recovers energy from nitrite oxidation. The genes encoding the two catalytic core subunits of the enzyme were isolated from a Nitrobacter winogradskyi gene library. Sequencing of one of the 14 cloned DNA segments revealed that the subunit genes are side by side in an operon-like cluster. Remarkably the cluster appears to be present in at least two copies per genome. It extends over a 5-6 kb length including, besides the catalytic core subunit genes, other cytochrome oxidase related genes, especially a heme O synthase gene. Noteworthy is the new kind of gene order identified within the cluster. Deduced sequences for the cytochrome oxidase subunits and for the heme O synthase look closest to their counterparts in other alpha-subdivision Proteobacteria, particularly the Rhizobiaceae. This confirms the phylogenetic relationships established only upon 16S rRNA data. Furthermore, interesting similarities exist between N. winogradskyi and mitochondrial cytochrome oxidase subunits while the heme O synthase sequence gives some new insights about the other similar published alpha-subdivision proteobacterial sequences.

Amino Acid Sequence↗
PubMed1998

Cloning and sequence analysis of the poly (3-hydroxyalkanoic acid)-synthesis genes of Pseudomonas acidophila.

Pseudomonas acidophila can grow with CO2 as a sole carbon source by the possession of a recombinant plasmid that clones genes that confer chemolithoautotrophic growth ability derived from the H2-oxidizing bacterium Alcaligenes hydrogenophilus. H2-oxidizing bacteria produce poly(3-hydroxybutyric acid) (PHB) from CO2, but recombinant P. acidophila can produce the more useful biopolymer poly(3-hydroxyalkanoic acid) (PHA). In this study, the pha genes of P. acidophila were cloned and a sequence analysis was carried out. A gene library was constructed using the cosmid vector pVK102. A recombinant cosmid carrying the pha genes was selected by the complementation of a PHB-negative mutant of Alcaligenes eutrophus H16. The resulting recombinant cosmid pIK7 contained a 14.8-kb DNA insert. Subcloning was done. and the recombinant plasmid pEH74 was selected by hybridization with the A. eutrophus H16 pha genes. Escherichia coli possessing pEH74 produced PHB, indicating that pEH74 contained the pha genes of P. acidophila. The nucleotide sequences of the PHA-synthesis genes phaA (beta-ketothiolase), phaB (acetoacetyl-CoA reductase), and phaC (PHA synthase) in pEH74 were determined. The homologies of phaA, phaB, and phaC between P. acidophila and A. eutrophus H16 were 64.7, 76.1 and 56.6%, respectively.

Acetyl-CoA C-Acyltransferase↗
PubMed1987

Human chromosome-specific DNA libraries: use of an oligodeoxynucleotide probe to detect non-recombinants.

The frequencies of non-recombinants are directly determined in 21 human DNA libraries prepared from flow-purified chromosomes. A radiolabeled 18-base oligodeoxynucleotide, whose sequence spans the cloning site, stably hybridizes to non-recombinants. When the cloning site is interrupted by an insert, less stable hybridization occurs. Non-recombinant frequencies range from 1-34% with the frequency of false positives being less than 1%. Libraries are being prepared for each human chromosome as part of the National Laboratory Gene Library Project and the specifications of seven new libraries are presented.

Base Sequence↗
PubMed1988

Structure of an unusual sea urchin U1 RNA gene cluster.

Genomic clones containing multiple copies of the Lytechinus variegatus U1 gene have been isolated from a gene library in the phage lambda EMBL3. These clones contain both types of U1 RNA gene repeats interspersed in the same 15-kb fragment. In addition, about 1/3 of the repeat units contain a 260-bp insert 460 bp prior to the first nucleotide of the U1 RNA sequence. The inserted sequence is abundant in the sea urchin genome as judged by Southern blots of genomic DNA. There are no repeated sequences flanking the insert. The insert occurs at the same position in the highly conserved 5'-flanking region at which a deletion has previously been reported.

Animals↗
PubMed1995

A K-252a-resistance gene, sks1+, encodes a protein similar to the Caenorhabditis elegans F37 A4.5 gene product and confers multidrug resistance in Schizosaccharomyces pombe.

A gene named sks1+ was cloned as a suppressor of the K-252a-sensitivity phenotype of Schizosaccharomyces pombe (Sp) from a gene library of the parental Sp chromosomal DNA constructed with a multicopy vector pDB248'. The gene encoded a 308-amino-acid (aa) protein similar to the Caenorhabditis elegans F37 A4.5 gene product and to the mouse and Drosophila Mov34 gene products. The sks1+ null mutants obtained by gene disruption were non-viable, indicating that sks1+ is essential for vegetative growth. The parental Sp strain carrying multiple copies of sks1+ showed distinct cross-resistance to staurosporine, thiabendazole and vanadate in addition to K-252a, although Sks1 has no similarity in aa sequence to those of ATP-binding cassette (ABC)-type transporters. The multicopy plasmid containing sks1+ conferred multidrug resistance (MDR), even in a mutant cell defective in pmd1+ encoding an ABC-type transporter. It is therefore unlikely that the function of pmd1+ is involved in MDR conferred by sks1+. These results suggest that sks1+ is a functionally novel MDR gene.

Amino Acid Sequence↗
PubMed1996

Cloning and characterization of an Actinobacillus pleuropneumoniae outer membrane protein belonging to the family of PAL lipoproteins.

A 14-kDa outer membrane protein (OMP) was purified from Actinobacillus pleuro-pneumoniae serotype 2. The protein strongly reacts with sera from pigs experimentally or naturally infected with any of the 12 serotypes of A. pleuropneumoniae. The gene encoding this protein was isolated from a gene library of A. pleuropneumoniae serotype 2 reference strain by immunoscreening. Expression of the cloned gene in Escherichia coli revealed that the protein is also located in the outer membrane fraction of the recombinant host. DNA sequence analysis of the gene reveals high similarity of the protein's amino acid sequence to that of the E. coli peptidoglycan-associated lipoprotein PAL, to the Haemophilus influenzae OMP P6 and to related proteins of several other Gram-negative bacteria. We have therefore named the 14-kDa protein PalA, and its corresponding gene, palA. The 20 amino-terminal amino acid residues of PalA constitute a signal sequence characteristic of membrane lipoproteins of prokaryotes with a recognition site for the signal sequence peptidase II and a sorting signal for the final localization of the mature protein in the outer membrane. The DNA sequence upstream of palA contains an open reading frame which is highly similar to the E. coli tolB gene, indicating a gene cluster in A. pleuropneumoniae which is very similar to the E. coli tol locus. The palA gene is conserved and expressed in all A. pleuropneumoniae serotypes and in A. lignieresii. A very similar palA gene is present in A. suis and A. equuli.

Actinobacillus pleuropneumoniae↗
PubMed1983

Molecular cloning and characterization of a gene for rat U2 small nuclear RNA.

Six phage clones that contain sequences hybridizable with the small nuclear RNA U2 were isolated from a rat gene library. Of these clones, one which includes a candidate for a functional U2 RNA gene was selected and characterized. The sequence within the clone which hybridizes with rat U2 RNA was completely co-linear with that of the RNA. A T-A-T-A box was not found in the region of more than 400 base-pairs which lies upstream of the gene. However, several block homologies were found with the upstream sequences of a rat U1 RNA gene candidate cloned in our laboratory. An "identifier sequence", which was reported to be an element of gene regulation related to differentiation, was found downstream of the coding region at the same distance and with the same orientation as the identifier sequence located downstream of the U1 RNA gene candidate. We detected a presumed U2 RNA precursor elongated by about 11 nucleotides at the 3' end by S1 nuclease mapping using a fragment from the clone. A potential termination signal for transcription was found within the elongated region of the presumed precursor. Southern blot analysis suggests that families of U2 RNA genes that have conserved flanking sequences are present in the genomes of rat, mouse, man, calf and chicken.

Animals↗
PubMed1985

Characterization of a rat tRNA gene cluster: comparison of nucleotide sequences of the gene for tRNALeu newly found in repeating units.

In the rat, DNA carrying a cluster of the genes for tRNAAsp, tRNAGly, and tRNAGlu, aligned in that order, is repeated about 10 times. Seven DNA clones corresponding to the independent repeating units were isolated from a rat gene library. Nucleotide sequence analysis of these clones revealed the presence of a fourth tRNA gene, the gene for tRNALeu, in the cluster. The tRNALeu gene is located about 600 base pairs (bp) upstream from the tRNAAsp gene and its polarity differs from those of the other three tRNA genes. Among the repeating units, the nucleotide sequence of tRNALeu is conserved to a relatively high degree.

Animals↗
PubMed1998

A pheromone-independent CarR protein controls carbapenem antibiotic synthesis in the opportunistic human pathogen Serratia marcescens.

Strain ATCC 39006 of Serratia marcescens makes the same carbapenem, (5R)-carbapen-2-em-3-carboxylic acid (Car), as the Erwinia carotovora strain GS101. Unlike E. carotovora, where the onset of production occurs in the late-exponential phase of growth in response to the accumulation of the small diffusible pheromone N-(3-oxohexanoyl)-L-homoserine lactone (OHHL), in S. marcescens carbapenem is produced throughout the growth phase and does not appear to involve any diffusible pheromone molecule. Two cosmids capable of restoring antibiotic production in E. carotovora group I carbapenem mutants were isolated from an S. marcescens gene library. These cosmids were shown to contain a homologue of the E. carotovora carR gene, encoding a CarR protein with homology to the LuxR family of transcriptional regulators. The S. marcescens carR was subcloned and shown to be capable of complementing in trans, in the absence of OHHL, an E. carotovora carR carI double mutant, releasing the heterologous E. carotovora host from pheromone dependence for carbapenem production. The apparent OHHL-independence of the S. marcescens CarR explains the constitutive nature of carbapenem production in this strain of S. marcescens.

Amino Acid Sequence↗
PubMed1993

Presence of a Vanadium Nitrogenase in Azotobacter paspali.

There have been no previous studies on the genetics of Azotobacter paspali, an aerobic bacterium which forms a highly specific diazotrophic association with Bahia grass (Paspalum notatum). We constructed A. paspali strains defective in the molybdenum nitrogenase so that alternative N(2)ases could be studied. The cosmid vector pTBE and genomic DNA fragments ( approximately 50 kb) of A. paspali ATCC 23367 were used to construct a gene library in Escherichia coli. Recombinant cosmids containing sequences homologous to molybdenum nitrogenase nifDK structural genes were identified by hybridization. A 2.9-kb fragment bearing the putative nifDK genes of A. paspali was subcloned and mutagenized in vitro by the insertion of a kanamycin resistance gene cassette. The mutation was recombined into the chromosome of A. paspali with the suicide vector pCU101. One resultant mutant strain, AP2, was incapable of diazotrophic growth in a molybdenum-containing medium (Nif) without vanadium but grew well in a molybdenum-deficient medium with vanadium. The nitrogenase system in AP2 reduced acetylene to ethylene and produced ethane as 2.4% of the total products. Molybdenum levels as low as 10 nM prevented the diazotrophic growth of AP2, even in the presence of vanadium at levels up to 10 muM. These results are consistent with the existence of a vanadium nitrogenase system in A. paspali.

Journal Article↗
PubMed1995

Streptococcus salivarius ATCC 25975 possesses at least two genes coding for primer-independent glucosyltransferases.

Fractionation of the culture medium showed that Streptococcus salivarius ATCC 25975 secreted a glucosyltransferase (Gtf) that was primer independent. On the basis of this observation, a gene library of S. salivarius chromosomal DNA cloned into lambda L47.1 was screened for a gene(s) coding for such an activity. As a result of this screening process, two new gtf genes, gtfL and gtfM, both of which coded for primer-independent Gtf activities, were isolated. GtfL produced an insoluble glucan that was refractory to digestion by the endo-(1-->6)-alpha-D-glucanase. of Chaetonium gracile, while GtfM produced a soluble glucan that was readily degraded by the glucanase. Comparison of the deduced amino acid sequences of gtfL and gtfM with 10 other available Gtf sequences allowed the relatedness of the conserved catalytic regions to be assessed. This analysis showed that the 12 enzymes did not form clusters based on their primer dependencies or on their product solubilities. Further analysis of the YG repeats in the C-terminal glucan-binding domains of GtfJ, GtfK, GtfL, and GtfM from S. salivarius showed that there was strong homology between a block of contiguous triplet YG repeats present in the four alleles. These blocks of YG repeats were coded for by a region of each gene that appeared to have arisen as a result of a recent duplication event(s).

Amino Acid Sequence↗
PubMed2000

Cloning of the histidine biosynthetic genes from Corynebacterium glutamicum: organization and analysis of the hisG and hisE genes.

The physically linked hisG and hisE genes, encoding for ATP-phosphoribosyltransferase and phosphoribosyl-ATP-pyrophosphohydrolase were isolated from the Corynebacterium glutamicum gene library by complementation of Escherichia coli histidine auxotrophs. They are two of the nine genes that participate in the histidine biosynthetic pathway. Molecular genetics and sequencing analysis of the cloned 9-kb insert DNA showed that it carries the hisG and hisE genes. In combining this result with our previous report, we propose that all histidine biosynthetic genes are separated on the genome by three unlinked loci. The coding regions of the hisG and hisE genes are 279 and 87 amino acids in length with a predicted size of about 30 and 10 kDa, respectively. Computer analysis revealed that the amino acid sequences of the hisG and hisE gene products were similar to those of other bacteria.

ATP Phosphoribosyltransferase↗
PubMed1998

[Cloning and characterization of melanin biosynthesis genes from Streptomyces lincolnensis 78-11].

Using mel C1-C2 gene from pIJ702 as probe to hybridize wity chromosome DNA of S. lincolnensis 78-11, a series of positive bands were found, including a 3.2 kb BanHI fragment and a 2.6 kb SphI fragment. So a gene library of S. lincolnensis, containg 3.0-3.5 kb BamHI fragment, was constructed. Melanin biosynthesis genes, mel C1 and mel C2, were then cloned and the fragment, inserted in recombinant plasmid pRSB336 were sequenced. Among five ORFs of the 3152 bp BamHI fragment, mel C1 and mel C2 were highly homologous to the respective genes of the other three strains of Streptomyces, morever, the product of mel C2 gene from S. lincolnensis 78-11 was slightly homologous to tyrosinase from human-being by 17.3% and from rat by 24.5%. All of the above results indicated that mel C1, mel C2 and orf 3 constitute the melanin biosynthesis operon of S. lincolnensis. In the further study of the cloned melanin biosynthesis genes of S. lincolnensis 78-11, recombinant plasmids, pPZ518 and pPZ519, which contain promoter of neo gene and mel gene in different orientation respectively, were constructed, and then were used to transform S. lividans TK23. 12 transformants of pPZ518, selected at random, secreted light brown pigment in R2YE culture medium, but all transformants of pPZ519 and pES1 were white.

Amino Acid Sequence↗
PubMed1991

Increased yield of a lysozyme after self-cloning of the gene in Streptomyces coelicolor "Müller".

Streptomyces coelicolor "Müller" DSM3030 excretes a lysozyme comprising both beta-1,4-N-acetyl- and beta-1,4-N,6-O-diacetyl muramidase activities. The lysozyme is named Cellosyl. Gene libraries have been established using genomic DNA from the wild-type strain, S. coelicolor DSM3030, and from an overproducing mutant, S. coelicolor HP1, which exhibits about a twofold increase in lysozyme production. The lysozyme-encoding genes (cel) from both strains were detected by oligodeoxynucleotide hybridization. The nucleotide sequence of the cel genes isolated from both strains was shown to be identical. The different levels of lysozyme production could not be correlated with any mutations at the cel gene locus. The cel gene isolated from the wild-type strain could not be expressed in some other species of Streptomyces. However, self-cloning of the cel gene into S. coelicolor DSM3030 and HP1 resulted in a 2.5-fold increase in lysozyme production.

Amino Acid Sequence↗
PubMed1983

Tight linkage of retroviral-like sequences to a variant human c-mos gene in the human genome.

Chumakov et al. [Gene 17 (1982) 19-26] identified in the human gene library a number of recombinant phages that possess a homology to the v-mos gene. Here we report the unusual structure of one of these recombinants, lambda gp5. The 14.3-kb stretch of human DNA from this phage contains at least three regions of homology to the v-mos gene, together with multiple copies of Alu-family repeats. Moreover, we have shown the presence of retrovirus-related sequences in the close vicinity of the mos-homologous regions. These data point to the possibility of involvement of retrovirus in the process of c-mos gene amplification during the formation of a multigene family.

Gene Amplification↗
PubMed1997

The Deinococcus radiodurans uvr A gene: identification of mutation sites in two mitomycin-sensitive strains and the first discovery of insertion sequence element from deinobacteria.

Deinococcus radiodurans (Dr) possesses a prominent ability to repair the DNA injury induced by various DNA-damaging agents including mitomycin C (MC), ultraviolet light (UV) and ionizing radiation. DNA damage resistance was restored in MC sensitive (MC(S)) mutants 2621 and 3021 by transforming with DNAs of four cosmid clones derived from the gene library of strain KD8301, which showed wild type (wt) phenotype to DNA-damaging agents. Gene affected by mutation (mtcA or mtcB) in both mutants was cloned and its nucleotide (nt) sequence was determined. The deduced amino acid (aa) sequence of the gene product consists of 1016 aa and shares homology with many bacterial UvrA proteins. The mutation sites of both mutants were identified by analyzing the polymerase chain reaction (PCR) fragments derived from the genomic DNA of the mutants. A 144-base pair (bp) deletion including the start codon for the uvrA gene was observed in DNA of the mutant 3021, causing a defect in the gene. On the other hand, an insertion sequence (IS) element intervened in the uvrA gene of the mutant 2621, suggesting the insertional inactivation of the gene. The IS element comprises 1322-bp long, flanked by 19-bp inverted terminal repeats (ITR), and generated a 6-bp target duplication (TD). Two open reading frames (ORFs) were found in the IS element. The deduced aa sequences of large and small ORFs show homology to a putative transposase found in IS4 of Escherichia coli (Ec) and to a resolvase found in ISXc5 of Xanthomonas campestris (Xc), respectively. This is the first discovery of IS element in deinobacteria, and the IS element was designated IS2621.

Adenosine Triphosphatases↗