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At least 1,153 records · Page 64Linked to original sources
PubMed1992

Structure of the human cellular retinoic acid-binding protein II gene. Early transcriptional regulation by retinoic acid.

The gene for human cellular retinoic acid-binding protein II (CRABP-II) has been cloned. It was isolated from a human placenta genomic library and is contained within one bacteriophage clone. The gene spans 6 kilobases and consists of 4 exons. One major transcription initiation site was mapped to an A residue 137 nucleotides upstream of the ATG initiation codon. The upstream region contains a TATA box and potential AP2, Sp1, and Krox-24 binding sites, as well as a direct repeat with homology to a retinoic acid-responsive element. The CRABP-II mRNA was rapidly induced within 2-6 h in cultured human skin fibroblasts by retinoic acid, reaching a plateau after 6 h of treatment. The rapid increase of CRABP-II message was mainly due to an increased rate of transcription as determined by nuclear run-on experiments. Increased transcription could be detected as early as 1 h after addition of retinoic acid, peaked at 2 h, and returned to basal levels within 6 h. On-going protein synthesis was required for this transient increase of transcription, since the induction was blocked by cycloheximide. These data suggest that the human CRABP-II gene is transcriptionally regulated by a newly synthesized regulatory protein.

Amino Acid Sequence↗
PubMed2001

Identification of a novel low-temperature-response element in the promoter of the barley (Hordeum vulgare L) gene blt101.1.

Two winter barley (Hordeum vulgare L. cv. Igri) genomic clones, lambda gblt101.1 and lambda gblt101.2, encoding the blt101 gene family, were isolated from a genomic library. Deletion analysis of the blt101.1 promoter, using transient beta-glucuronidase (GUS) reporter expression assays, indicated that it contains at least three regulatory regions. A 107-bp region between nucleotides -168 and -275 with respect to the translation initiation codon, confers high-level GUS reporter expression at low temperature and contains a sequence (designated CR1) that is highly conserved in equivalent positions within the promoters of both members of the blt101 gene family. A 10-bp motif contained within CR1 binds proteins present in nuclear extracts from both control and low-temperature-treated barley tissue. Loss-of-function experiments, using transient-expression analysis, confirmed that this motif acts as a previously unreported low-temperature-responsive element. Nuclease sensitivity analysis of intact chromatin indicated that the blt101.1 promoter becomes more susceptible to DNase and micrococcal nuclease at low temperature, consistent with chromatin reorganisation upon transcriptional induction. It is proposed that both the 10-bp motif and chromatin reorganisation are involved in the regulation of blt101.1 at low temperature. This is the first detailed analysis of a low-temperature-specific plant promoter and identifies a novel low-temperature-response element.

Adaptation, Physiological↗
PubMed1993

The complete sequence of the gene encoding bovine alpha s2-casein.

From a bovine genomic library, five overlapping clones, spanning some 50 kb, have been isolated. These clones contain the complete alpha s2-casein-encoding gene (alpha s2ca) and its 5' and 3' flanking regions. The nucleotide (nt) sequence of the complete gene including 2510 bp of the 5' flanking region and 276 bp of the 3' region has been determined. The total length of alpha s2ca appears to be 18483 bp and, therefore, it is the longest of the four bovine casein-encoding genes. The alpha s2ca gene is comprised of 18 exons ranging in size from 21 to 266 nt. There are 16 Alu-like artiodactyla retroposons inserted at ten different locations within the gene. About 14% of the gene is composed of these repetitive sequences. Although the organization of alpha s2ca appears to be similar to that of the alpha s1-casein-encoding gene (alpha s1ca), sequence comparisons and the length of the exons indicate that it is more closely related to the beta-casein-encoding gene. Furthermore, it is shown that both genes could have evolved from a common ancestor by means of internal duplications.

Animals↗
PubMed1998

Cloning and characterization of the equine F18 gene, which has a novel exon.

A genomic clone isolated from an equine genomic library probed with an oligonucleotide (CAG)10 showed high sequence similarity to the human F18 gene and was tentatively named equine F18 gene. Because the human F18 gene is expressed in many tissues, we examined whether this equine clone was also expressed in equine tissues. The cDNA encoding equine F18 was obtained by the reverse transcriptase-polymerase chain reaction (RT-PCR) from equine thymus. The nucleotide sequence of the equine F18 cDNA (1940 bp) was determined and contained both the ATG initiation codon and a poly(A) sequence. The cDNA sequence contained sequence homologous to exon 3 of human F18 gene and a new exon that is not found in the human F18 gene. The equine F18 gene contains a CAG repetitive sequence (CRS) that is translated into a polyglutamine tract. The CRS was analyzed for polymorphism by PCR: four and two different alleles were observed with unrelated east-Asian and thoroughbred horses, respectively. The equine F18 gene was mapped to Xq29.1 by fluorescence in situ hybridization. The human F18 gene is also X-linked. These data strongly supported the conclusion that the clone contains the equine homologue of the human F18 gene.

Animals↗
PubMed1997

Characterization of cell cultures derived from Fugu, the Japanese pufferfish.

The Japanese pufferfish (genus Fugu), which possesses a highly compact genome, is becoming a popular model among those interested in sequencing and mapping the genomes of higher vertebrates. Although genomic libraries have been derived and used to study the molecular biology of Fugu, biological material derived from the living organism is difficult to obtain for laboratories distant from the Asian Pacific. We have established cell cultures from two Fugu species: kusafugu, Fugu niphobles, and torafugu, F. rubripes. Cultures derived from F. niphobles fry and F. rubripes eye have been passaged more than 60 times over the course of one year, representing approximately 180 population doublings. Proliferating cultures were also initiated from F. rubripes brain, liver, fin, spleen, kidney, swimbladder, and muscle. Karyotype analyses indicated that F. rubripes eye-derived cells possessed a chromosome number in the diploid range; F. niphobles fry cells were slightly hyperploid. Flow cytometry confirmed that the relative amounts of DNA present in cultured cells from both Fugu species were similar to that measured in blood cells collected from F. rubripes, and approximately one-seventh of that measured in diploid human cells. Telomerase activity was easily detectable in lysates prepared from F. niphobles fry cells and F. rubripes eye cells, consistent with the notion that these cultures are capable of indefinite proliferation.

Animals↗
PubMed1994

Cloning and characterisation of genes for tetrapyrrole biosynthesis from the cyanobacterium Anacystis nidulans R2.

The genes for 5-aminolevulinic acid dehydratase (ALAD) and uroporphyrinogen III synthase (UROS), two enzymes in the biosynthetic pathway for tetrapyrroles, were independently isolated from a plasmid-based genomic library of Anacystis nidulans R2 (also called Synechococcus sp. PCC7942), by their ability to complement Escherichia coli strains carrying mutations in the equivalent genes (hemB and hemD respectively). The identity of the genes was confirmed by comparing the appropriate enzyme activities in complemented and mutant strains. Subclones of the original plasmids that were also capable of complementing the mutants were sequenced. The inferred amino acid sequence of the cyanobacterial HemB protein indicates a significant difference in the metal cofactor requirement from the higher-plant enzymes, which was confirmed by overexpression and biochemical analysis. The organisation of the cyanobacterial hemD locus differs markedly from other prokaryotes. Two open reading frames were found immediately upstream of hemD. The product of one shows considerable similarity to published sequences from other organisms for uroporphyrinogen III methylase (UROM), an enzyme involved in the production of sirohaem and cobalamins (including vitamin B-12). The product of the other shows motifs which are similar to those found in proteins responsible for metabolic regulation in yeast and indicates that this family of transcription control proteins, which has previously been reported only from eukaryotes, is also represented in prokaryotes.

Amino Acid Sequence↗
PubMed1992

Genomic organization of the mouse OSF-1 gene.

The mouse OSF-1 protein (also known as pleiotrophin, HB-GAM, HBGF-8, or HBNF) gene was isolated from a mouse genomic library and sequenced. OSF-1 is a 15-kD secreted protein specifically expressed in bone and brain, and is believed to play a role in brain development and osteogenesis. The mouse OSF-1 gene consists of at least 5 exons and 4 introns and spans > 32 kb. Computer analysis of approximately 4 kb of 5'-flanking sequence of the OSF-1 gene revealed two candidate promoter regions. One candidate promoter contains a thyroid hormone/retinoic acid-responsive element and the other contains two glucocorticoid-responsive elements. DNA sequence analysis of novel OSF-1 cDNA clones indicates that two promoters can be utilized in MC3T3-E1 osteoblastic cells. The overall organization of the mouse OSF-1 gene is similar and the locations of the three exon-intron junctions within the coding region are identical to the mouse gene encoding the differentiation-related factor midkine (MK). Based on this similarity and on the high degree of nucleotide sequence homology (approximately 55%) of mouse OSF-1 and mouse MK, we conclude that OSF-1 and MK are generated from a common ancestral gene and are members of a family of structurally and probably functionally related proteins.

Amino Acid Sequence↗
PubMed1992

Cloning and chromosome mapping of the human interleukin-1 receptor antagonist gene.

By screening a human genomic library with an interleukin-1 receptor antagonist (IL-1ra) cDNA probe, we have isolated a 15 kb clone which contains the entire coding region of the gene as expressed in monocytes, and includes 6 kb of 5'-upstream sequence. The gene contains four exons which code for the secreted form of the IL-1ra, however, our clone does not contain the alternative first exon used to generate an intracellular form of the protein as the protein as found in epithelial cells. Analysis of the sequence reveals a consensus TATA box, and three Alu repeats, two of which are in the upstream region and one in intron 3. The sequence also reveals an 86 bp motif tandomly repeated four times within intron 2, and may reflect the polymorphism known to exist in this region of the gene. By in-situ fluorescence hybridization we have shown that the IL-1ra gene is found on the long arm of chromosome 2 and maps to 2q13-14.1. Previous studies have revealed that IL-1 alpha, and IL-1 beta and both type I and type II forms of the IL-1 receptor all map close to this region of chromosome 2.

Amino Acid Sequence↗
PubMed1993

Molecular cloning and characterization of the hblA gene encoding the B component of hemolysin BL from Bacillus cereus.

Previous evidence suggests that hemolysin BL, which consists of a binding component, B, and two lytic components, L1 and L2, is the enterotoxin responsible for the diarrheal form of gastroenteritis caused by food-borne strains of Bacillus cereus. To prove that hemolysin BL and the enterotoxin are the same requires large amounts of these components free of other B. cereus proteins. For this purpose, we sought to clone the gene encoding the B component and to express it in Escherichia coli. A partial genomic library was constructed and a 29-base, 1,152-fold-degenerate oligonucleotide probe, designed from the N-terminal amino acid sequence of the B component, was used to identify recombinant clones containing the gene. Detection of gene products reactive with a monoclonal antibody specific for the B component and analysis of the nucleotide sequence confirmed that isolated clones, reactive with the oligonucleotide probe, did contain the gene encoding the B component. The protein, expressed in E. coli, apparently from the B. cereus promoter, produces a ring-shaped zone of hemolysis when combined with purified L components from B. cereus, a reaction typical of hemolysin BL. Northern (RNA) blot analysis of B. cereus RNA showed a large (5.1-kb) transcript which hybridized with a 500-bp probe internal to the B-component-coding sequence, suggesting that the hblA gene encoding the B component may be transcribed as part of a polycistronic message, possibly including the structural genes for the two lytic components. Higher levels of expression and disruption of the hblA gene are being pursued to resolve whether hemolysin BL is indeed the enterotoxin.

Amino Acid Sequence↗
PubMed1991

Overexpression of RPI1, a novel inhibitor of the yeast Ras-cyclic AMP pathway, down-regulates normal but not mutationally activated ras function.

A high-copy-number plasmid genomic library was screened for genes that when overexpressed down-regulate Ras protein activity in Saccharomyces cerevisiae. We report on the structure and characterization of one such gene, RPI1, which potentially encodes a novel 46-kDa negative regulator of the Ras-cyclic AMP pathway. Three lines of evidence suggest that the RPI1 gene product operates upstream to negatively regulate the activity of normal but not mutationally activated Ras proteins: (i) overexpressed RPI1 lowers cyclic AMP levels in wild-type yeast cells but not in yeast cells carrying the RAS2Val-19 mutation, (ii) overexpressed RPI1 suppresses the heat shock sensitivity phenotype induced by overexpression of normal RAS2 but does not suppress the same phenotype induced by RAS2Val-19, and (iii) disruption of RPI1 results in a heat shock sensitivity phenotype which can be suppressed by mutations that lower normal Ras activity. Thus, RPI1 appears to encode an inhibitor of Ras activity that shares a common feature with Ras GTPase-activating proteins in that it fails to down-regulate activated RAS2Val-19 function. We present evidence that the down-regulatory effect of RPI1 requires the presence of one of the two Ras GTPase activators, IRA1 and IRA2.

Amino Acid Sequence↗
PubMed1998

Nucleotide sequence of the ring3 gene in the class II region of the mouse MHC and its abundant expression in testicular germ cells.

The RING3 (NAT) gene is the first and only locus with no obvious function associated with the immune system in the class II region of the human major histocompatibility complex. This gene is a homologue of the Drosophila homeotic gene female sterile homeotic (fsh) and encodes a nuclear serine-threonine kinase. To study more about the physiological function of the RING3 gene, we isolated a mouse homologue from a genomic library, determined its gene structure, and investigated its expression profile. The mouse Ring3 gene spans approximately 8 kb and consists of 12 exons encoding a 798-amino-acid protein, sharing as high as 96% amino acid identity with the human RING3 protein. Northern hybridization revealed that the Ring3 gene abundantly produced 3.8- and 3.0-kb transcripts in the testis but was weakly expressed with 4.6- and 3.8-kb transcripts in somatic tissues. It appears that testis-specific 3.0-kb transcript gives rise to a smaller size Ring3 protein resulting from the usage of the second ATG codon for translational initiation compared to the almost ubiquitous 4.6-kb transcript. In RNAs isolated from fractionated testicular germ cells, the two testicular mRNAs were detected exclusively in the fractions containing a large population of round spermatids and pachytene spermatocytes. Furthermore, in situ hybridization on cross sections of seminiferous tubules in the testis showed that the expression of the Ring3 gene was initiated at the pachytene spermatocyte stage during meiosis and persisted throughout the round spermatid stage during spermiogenesis. These results suggest that the Ring3 gene plays an important role in spermatogenesis.

Amino Acid Sequence↗
PubMed1994

The human DNA polymerase beta gene structure. Evidence of alternative splicing in gene expression.

DNA polymerase beta (beta-pol) is a single-copy gene that is considered to be part of the DNA repair machinery in mammalian cells. Using two human genomic libraries we have cloned the complete human beta-pol gene and determined the organization of the beta-pol coding sequence within the gene. The human beta-pol gene spans 33 kb and contains 14 exons that range from 50 to 233 bp. The 13 introns vary from 96 bp to 6.5 kb. Information derived from this study was used in defining the location of a deletion/insertion type restriction fragment length polymorphism (RFLP) 5' to exon I of the human beta-pol gene. This RFLP was utilized in linkage analysis of DNAs from CEPH families and the results confirm the previous assignment of the human beta-pol gene to chromosome 8 (p12-p11). Analysis of mRNA from six human cell lines using the polymerase chain reaction showed the expression of two beta-pol transcripts. Sequence analysis revealed that the size difference in these transcripts was due to deletion of the 58 bp sequence encoded by exon II, suggesting that the smaller transcript results from an alternative splicing of the exon II sequence during processing of the beta-pol precursor RNA.

Animals↗
PubMed2003

Cloning and analysis of a DNA fragment stimulating avermectin production in various Streptomyces avermitilis strains.

To isolate a gene for stimulating avermectin production, a genomic library of Streptomyces avermitilis ATCC 31267 was constructed in Streptomyces lividans TK21 as the host strain. An 8.0-kb DNA fragment that significantly stimulated actinorhodin and undecylprodigiosin production was isolated. When wild-type S. avermitilis was transformed with the cloned fragment, avermectin production increased approximately 3.5-fold. The introduction of this fragment into high-producer (ATCC 31780) and semi-industrial (L-9) strains also resulted in an increase of avermectin production by more than 2.0- and 1.4-fold, respectively. Subclones were studied to locate the minimal region involved in stimulation of pigmented-antibiotic and avermectin production. An analysis of the nucleotide sequence of the entire DNA fragment identified eight complete and one incomplete open reading frame. All but one of the deduced proteins exhibited strong homology (68 to 84% identity) to the hypothetical proteins of Streptomyces coelicolor A3(2). The orfX gene product showed no significant similarity to any other protein in the databases, and an analysis of its sequence suggested that it was a putative membrane protein. Although the nature of the stimulatory effect is still unclear, the disruption of orfX revealed that this gene was intrinsically involved in the stimulation of avermectin production in S. avermitilis.

Bacterial Proteins↗
PubMed1998

Identification and sequencing of the groE operon and flanking genes of Lawsonia intracellularis: use in phylogeny.

Proliferative enteropathy (PE) is a complex of diseases of commercial importance to the pig industry. The obligate intracellular bacterium Lawsonia intracellularis is consistently associated with PE and pure cultures of this bacterium have been used to reproduce PE in pigs. In this study L. intracellularis bacteria were purified directly from PE-affected tissue. DNA extracted from purified bacteria was used to construct a partial genomic library which was screened using sera from L. intracellularis-immunized rabbits. Two seroreactive recombinant clones were identified, one of which expressed proteins of 10 and 60 kDa. The sequence of the insert from this clone, pISI-2, revealed ORFs with sequence similarity to the groES/EL operon of Escherichia coli, the 505 ribosomal proteins L21 and L27 of E. coli, a GTP-binding protein of Bacillus subtilis and a possible protoporphyrinogen oxidase, HemK, of E. coli. Primers designed from unique sequences from the pISI-2 insert amplified DNA from infected, but not non-infected, porcine ilea; the amplicon sequence obtained from tissue-cultured L. intracellularis was identical to the corresponding sequence in pISI-2, confirming the origin of the clone. The sequence of L. intracellularis GroEL and other GroEL sequences in the databases were used to construct a partial phylogenetic tree. Analysis of the GroEL sequence relationship suggested that L. intracellularis is not significantly related to other organisms whose GroEL sequences are held in the databases and supports previous data from 16S sequence analyses suggesting that L. intracellularis is a member of a novel group of enteric pathogens.

Amino Acid Sequence↗
PubMed1995

Cloning of a Candida albicans peptide transport gene.

A Candida albicans peptide transport gene, CaPTR2, was cloned from a C. albicans genomic library by functional complementation of a peptide transport deficient mutant (strain ptr2-2) of Saccharomyces cerevisiae. CaPTR2 restored peptide transport to transformants as determined by uptake of radiolabelled dileucine, growth on dipeptides as sources of required amino acids, and restoration of growth inhibition by toxic peptides. Plasmid curing experiments demonstrated that the peptide transport phenotype was plasmid borne. CaPTR2 was localized to chromosome R of C. albicans by contour-clamped homologous electric field gel chromosome blots. Deletion subclones and frameshift mutagenesis were used to narrow the peptide transport complementing region to a 5.1 kb DNA fragment. DNA sequencing of the complementing region identified an ORF of 1869 bp containing an 84 nucleotide intron. The deduced amino acid sequence predicts a protein of 70 kDa consisting of 623 amino acids with 12 hydrophobic segments. A high level of identity was found between the predicted protein and peptide transport proteins of S. cerevisiae and Arabidopsis thaliana. This study represents the first steps in the genetic characterization of peptide transport in C. albicans and initiates a molecular approach for the study of drug delivery against this pathogen.

Amino Acid Sequence↗
PubMed1995

A 17 kDa outer-membrane protein (Omp4) from Serratia marcescens confers partial resistance to bacteriocin 28b when expressed in Escherichia coli.

A cosmid-based genomic library of Serratia marcescens N28b was introduced into Escherichia coli and clones were screened for a bacteriocin 28b insensitive phenotype. One clone was found that showed partial resistance to bacteriocin 28b. By using Tn5tac1 insertions it was shown that this phenotype was due to the expression in E. coli of an outer-membrane protein of 17 kDa (Omp4). The DNA region defined by insertion mutagenesis was sequenced and found to contain an ORF of 515 bp. The deduced amino acid sequence has 172 residues with a theoretical molecular mass of 18.4 kDa. The protein contains an N-terminal signal sequence of 24 amino acid residues and, when compared to other enterobacterial outer-membrane proteins, most closely resembles a family of small outer-membrane proteins of Enterobacteriaceae whose known functions appear to be related with virulence. Immunoblotting experiments showed that Omp4 is present in 15 biotypes of S. marcescens. The bacteriocin 28b resistance phenotype conferred on E. coli by Omp4 appears to be pleiotropic since overexpression of the Omp4-encoding gene leads to a decrease in the amount of OmpA, OmpF and/or OmpC; OmpA and OmpF are the receptors for bacteriocin 28b in E. coli.

Amino Acid Sequence↗
PubMed1996

Identification and characterization of the eps (Exopolysaccharide) gene cluster from Streptococcus thermophilus Sfi6.

We report the identification and characterization of the eps gene cluster of Streptococcus thermophilus Sfi6 required for exopolysaccharide (EPS) synthesis. This report is the first genetic work concerning EPS production in a food microorganism. The EPS secreted by this strain consists of the following tetrasaccharide repeating unit:-->3)-beta-D-Galp-(1-->3)-[alpha-D-Galp-(1-->6)]-beta-D- D-Galp-(1-->3)-alpha-D-Galp-D-GalpNAc-(1-->. The genetic locus The genetic locus was identified by Tn916 mutagenesis in combination with a plate assay to identify Eps mutants. Sequence analysis of the gene region, which was obtained from subclones of a genomic library of Sfi6, revealed a 15.25-kb region encoding 15 open reading frames. EPS expression in the non-EPS-producing heterologous host, Lactococcus lactis MG1363, showed that within the 15.25-kb region, a region with a size of 14.52 kb encoding the 13 genes epsA to epsM was capable of directing EPS synthesis and secretion in this host. Homology searches of the predicted proteins in the Swiss-Prot database revealed high homology (40 to 68% identity) for epsA, B, C, D, and E and the genes involved in capsule synthesis in Streptococcus pneumoniae and Streptococcus agalactiae. Moderate to low homology (37 to 18% identity) was detected for epsB, D, F, and H and the genes involved in capsule synthesis in Staphylococcus aureus for epsC, D, and E and the genes involved in exopolysaccharide I (EPSI) synthesis in Rhizobium meliloti for epsC to epsJ and the genes involved in lipopolysaccharide synthesis in members of the Enterobacteriaceae, and finally for eps K and lipB of Neisseria meningitidis. Genes (epsJ, epsL, and epsM) for which the predicted proteins showed little or no homology with proteins in the Swiss-Prot database were shown to be involved in EPS synthesis by single-crossover gene disruption experiments.

Bacterial Proteins↗
PubMed1998

Identification of a gene sequence encoding a putative pyruvate oxidoreductase in Serpulina pilosicoli.

Serpulina pilosicoli is a recently described species of intestinal spirochaete which can be identified using a species-specific monoclonal antibody BJL/AC1 reactive with a 29-kDa protein located in the cell envelope. A genomic library of the type strain of S. pilosicoli P43/6/78T was created in lambda zap express and screened using BJL/AC1. Single positive clones were isolated and excised into the phagemid vector pBK-CMV. Phagemid DNA was purified and a single clone was selected for sequencing. The size of spirochaetal DNA insert was determined by digestion with restriction endonucleases EcoRI and PstI as being approximately 2.6 kb. The nucleotide sequence of the gene encoding the protein with which the antibody reacted was determined by cycle sequencing. The insert contained an open reading frame of 285 nucleotides. Translation of the nucleotide sequence into amino acid (aa) residues showed a sequence of 275 aa. Comparison of this sequence with databases revealed homology to pyruvate oxidoreductases from various organisms found in the gastroinestinal tract. These included the pyruvate ferredoxin oxidoreductase (POR) alpha submit of Helicobacter pylori (38.8% identity in 250 aa), pyruvate-flavodoxin oxidoreductase of Escherichia coli (28.7% identify in 258 aa) and Giardia intestinalis (25.1% identity in 251 aa). A significant level of homology was also observed with hyperthermophilic bacteria such as the POR of Thermatoga maritima (38.6% in 254 aa) and the 2-ketovalerate-ferredoxin oxidoreductase of Pyrococcus furiosus (34% in 262 aa).

Amino Acid Sequence↗