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PubMed1998

The LuxR regulator protein controls synthesis of polyhydroxybutyrate in Vibrio harveyi.

The LuxR regulatory protein of Vibrio harveyi has been shown to control synthesis of polyhydroxybutyrate (PHB) as well as luminescence so as to occur at high cell density, suggesting that it is a general regulatory protein. Mutants defective in the production of LuxR (D1, D34, and MR1130) were found to be missing PHB, whose synthesis could be restored by complementation with luxR. Triparental mating with a V. harveyi genomic library revealed the presence of three genomic clones (G1, G2 and G3) that could also restore PHB synthesis and luminescence to cells which express low levels of luxR (D1 and D34) but not to luxR- cells (MR1130) suggesting that luxR expression was being stimulated. Analyses of luxR mRNA levels by mRNA dot blot hybridization and by primer extension confirmed that luxR mRNA levels were increased 4 to 7-fold in the D1 and D34 cells by the G1, G2 and G3 fragments and show that expression of a single genomic copy of luxR is sufficient to restore synthesis of PHB. The results demonstrate that V. harveyi LuxR controls the induction of a process not intimately involved in the bioluminescence system and clearly distinguishes its role in V. harveyi from that of LuxR from Vibrio (Photobacterium) fischeri, which has only been associated with regulation of light emission.

Hydroxybutyrates↗
PubMed1998

Identification and cloning of a developmentally regulated Cryptosporidium parvum gene by differential mRNA display PCR.

To identify Cryptosporidium parvum genes expressed during intracellular development, differential mRNA display was used to detect differences in gene expression between mock-infected and C. parvum-infected human epithelial cells. A reproducible band present only in C. parvum-infected cells, ddHC-23, was isolated and cloned. Southern blot analysis demonstrated that ddHC-23 represented a C. parvum gene. RT-PCR revealed that HC-23 mRNA levels decreased from 6 to 12h post-infection (pi), were maximally expressed at 24h pi, and returned to low levels at 48 and 72h pi. Northern blot analysis determined that the approx. 3.6kb transcript is expressed by sporozoites prior to invasion of epithelial cells. Screening of a C. parvum genomic library with ddHC-23 isolated a genomic subclone which contained a 2790bp ORF, uninterrupted by introns. Sequence analysis indicated that the encoded protein, which displayed no similarity to any sequences in the public databases, contained a high proportion of polar amino acids, with the most abundant being Asp (17.3%), Ser (15.8%) and Gly (8.1%). Numerous potential sites for posttranslational modification were present including: casein kinase II and protein kinase C phosphorylation sites, N-myristolation sites and N-glycosylation sites. These findings demonstrate the usefulness of differential mRNA display for identifying developmentally regulated C. parvum genes within the background of genes expressed by the host cell. 1998 Elsevier Science B.V.

Amino Acid Sequence↗
PubMed1994

Human cytochrome b561: a revised hypothesis for conformation in membranes which reconciles sequence and functional information.

Cytochrome b561 is a major transmembrane protein of catecholamine and neuropeptide secretory vesicles. In this report, we describe the cloning and properties of a full-length cDNA encoding human neuroendocrine cytochrome b561 from a human caudate cDNA library and a human peripheral blood genomic library. The human cDNA contains two major transcription start sites and only one translation start site that codes for an apocytochrome b561, which is 22 amino acid residues smaller than the previously deduced amino acid sequence from bovine cDNA. This smaller version of cytochrome b561 may contain only five transmembrane segments rather than the previously proposed six segments. The new model is in agreement with our previous results on transmembrane topology of the gene product. Northern-blot analysis shows an expanded tissue distribution of cytochrome mRNA expression where previous immunological assays were negative. These results support the hypothesis that cytochrome b561 is a marker for peptidergic and adrenergic tissues.

Amino Acid Sequence↗
PubMed2002

Rapid isolation of CA microsatellites from the tilapia genome.

We have developed (CA)n microsatellite markers for the cichlid fish, Oreochromis niloticus using a variation of the hybrid capture method. The resulting genomic library was highly enriched in repetitive DNA with 96% of clones containing CA repeats. The number of repeats ranged from four to 45 with an average of 19. Two-thirds of the sequenced clones had 12 or more repeats and sufficient flanking sequence to design primers. The resulting markers were tested in an F2 cross of O. niloticus x O. aureus. Nearly 90% of the markers amplified in this cross and 74% of these were informative. This work demonstrates the importance of minimizing the number of polymerase chain reaction (PCR) amplification cycles before and after the enrichment steps to reduce PCR recombination and the generation of chimaeric clones.

Animals↗
PubMed1982

Single-copy sequence hybridizes to polymorphic and homologous loci on human X and Y chromosomes.

Use of a 4.5-kilobase-pair (kb) segment of single-copy DNA from a human genomic library as a hybridization probe of genomic human DNAs revealed allelic Taq I restriction fragments 10.6, 11.8, and 14.6 kb long. Among 12 unrelated individuals, all 6 males exhibited the 14.6-kb fragment in addition to one of the other fragments. Three of the females displayed 10.6- and 11.8-kb fragments, and the other three displayed only one fragment length; none had the 14.6-kb fragment. Hybridization of this probe to Taq I-digested DNAs from human-rodent hybrid cell lines (which have partial complements of human chromosomes) demonstrated segregation of the 14.6-kb fragment with the human Y chromosome and segregation of the 10.6- and 11.8-kb fragments with the human X chromosome. Furthermore, hybridization of this probe to Taq I-digested DNAs from 48 members of a single kindred revealed Y-linked inheritance of the 14.6-kb fragment and X-linked inheritance of the 10.6- and 11.8-kb fragments. These experiments demonstrate homology between single-copy sequences on the human X and Y chromosomes.

Alleles↗
PubMed1992

Cloning, sequencing, and disruption of the gene encoding sterol C-14 reductase in Saccharomyces cerevisiae.

A sterol C-14 reductase (erg24-1) mutant of Saccharomyces cerevisiae was selected in a fen1, fen2, suppressor background on the basis of nystatin resistance and ignosterol (ergosta-8,14-dienol) production. The erg24-1 allele segregated genetically as a single, recessive gene. The wild-type ERG24 gene was cloned by complementation onto a 12-kb fragment from a yeast genomic library, and subsequently subcloned onto a 2.4-kb fragment. This was sequenced and found to contain an open reading frame of 1,314 bp, predicting a polypeptide of 438 amino acids (M(r) 50,612). A 1,088-bp internal region of the ERG24 gene was excised, replaced with a LEU2 gene, and integrated into the chromosome of the parental strain, FP13D (fen1, fen2) by gene replacement. The ERG24 null mutant produced ergosta-8,14-dienol as the major sterol, indicating that the delta 8-7 isomerase, delta 5-desaturase and the delta 22-desaturase were inactive on sterols with the C14 = 15 double bond.

Amino Acid Sequence↗
PubMedDate not supplied

Isolation and characterization of the Drosophila ornithine decarboxylase locus: evidence for the presence of two transcribed ODC genes in the Drosophila genome.

The polymerase chain reaction (PCR) was used to isolate two Drosophila ornithine decarboxylase (ODC) genes. Two mixtures of degenerate oligonucleotides corresponding to peptides that are fully conserved among ODCs from widely diverged species were used as opposing primers in the PCR with cDNA or genomic DNA as templates. Sequence analysis of the resulting DNA products confirmed their identity as ODC fragments. The genomic PCR product was then used as a probe for screening a Drosophila genomic library, resulting in the isolation of genomic clones representing two distinct ODC genes (dODC1 and dODC2). Sequence analysis of both genes demonstrated that although varying at their coding and noncoding regions, their overall structure is extremely similar containing 6 exons and 5 short introns. Southern blot and sequence analyses revealed that the two ODC genes are arranged in a tandem head-to-tail configuration. Both ODC genes were assigned by in situ hybridization analysis to position 44A on the right arm of the second chromosome. The isolation of cDNA clones corresponding to these two ODC genes demonstrated that both are transcribed in the adult fly. We hope that the isolation of genomic and cDNA clones of Drosophila ODC will permit the investigation of the expression of ODC during Drosophila development and the role of polyamines in this process.

Amino Acid Sequence↗
PubMed1986

Molecular cloning of a steroid-regulated 108K heat shock protein gene from hen oviduct.

The natural gene for a steroid inducible 108K heat shock protein has been isolated from a lambda genomic library prepared from hen oviduct tissue. Genomic DNA blots indicate that it exists as a single copy gene in the chick oviduct haploid genome. The 9.9 kilobase gene codes for a messenger RNA of 2733bp (21) and is split into 18 exons as established by sequence comparison of cDNA and genomic clones. The 3' end of the gene contains a repetitive element which shares homology with the CR1 family of repeats. The first exon contains both the untranslated leader and coding regions of the gene. The promoter region is rich in G + C residues (70%) and the dinucleotide CG. This 5' flanking segment contains bases similar both in sequence and location to the Goldberg-Hogness TATA homology and consensus sequence CCAAT. A consensus sequence located upstream of steroid hormone responsive chicken genes is found at -267 and on a reverse orientation at -593. The structure of this gene is of interest since the presence of introns in heat shock genes is rare in any species examined to date. Furthermore, this gene lacks the previously described heat shock promoter consensus sequence (C-GAA-TTC-G) present in other species.

Animals↗
PubMed1994

cDNA sequence and structure of a gene encoding trout testis high-mobility-group-1 protein.

Perchloric acid extraction of trout testis nuclei revealed the presence of two large high-mobility-group (HMG) proteins, HMG-T1 and HMG-T2. The sequence of a complete cDNA (1407 bp) for trout testis HMG-1 protein (referred as to HMG-T1) has been determined. The deduced HMG-T1 protein contains 203 amino acids with more than 86% similarity to mammalian HMG-1 proteins. A single-sized mRNA for HMG-T1 has been detected by Northern-blot analysis consistent with the size derived from the HMG-T1 cDNA. Amplification of human and trout genomic DNAs by polymerase chain reaction using primers specific for trout and human HMG-1 cDNAs revealed that unlike the human genome, which contains predominantly intronless HMG-1 sequences, intronless HMG-T1 sequences were not found in the fish genome. Southern-blot analysis suggested that the trout testis HMG-1 gene is encoded by at least two sequences with high similarity. A gene encoding HMG-T1 protein has been isolated from a trout testis genomic library and by PCR of trout genomic DNA (3879 bp). The trout testis HMG-1 gene is organized into five exons (four exons corresponding to the protein-coding region) and its exon/intron boundaries are identical to those of the human HMG-2 gene [Shirakawa, H. & Yoshida, M. (1992). J. Biol. Chem. 267, 6641-6645] suggesting the evolution of HMG-1 and HMG-2 genes from a common ancestor.

Amino Acid Sequence↗
PubMed1992

A conjugation procedure for Bdellovibrio bacteriovorus and its use to identify DNA sequences that enhance the plaque-forming ability of a spontaneous host-independent mutant.

Wild-type bdellovibrios are obligate intraperiplasmic parasites of other gram-negative bacteria. However, spontaneous mutants that can be cultured in the absence of host cells occur at a frequency of 10(-6) to 10(-7). Such host-independent (H-I) mutants generally display diminished intraperiplasmic-growth capabilities and form plaques that are smaller and more turbid than those formed by wild-type strains on lawns of host cells. An analysis of the gene(s) responsible for the H-I phenotype should provide significant insight into the nature of Bdellovibrio host dependence. Toward this end, a conjugation procedure to transfer both IncQ and IncP vectors from Escherichia coli to Bdellovibrio bacteriovorus was developed. It was found that IncQ-type plasmids were capable of autonomous replication in B. bacteriovorus, while IncP derivatives were not. However, IncP plasmids could be maintained in B. bacteriovorus via homologous recombination through cloned B. bacteriovorus DNA sequences. It was also found that genomic libraries of wild-type B. bacteriovorus 109J DNA constructed in the IncP cosmid pVK100 were stably maintained in E. coli; those constructed in the IncQ cosmid pBM33 were unstable. Finally, we used the conjugation procedure and the B. bacteriovorus libraries to identify a 5.6-kb BamHI fragment of wild-type B. bacteriovorus DNA that significantly enhanced the plaque-forming ability of an H-I mutant, B. bacteriovorus BB5.

Bdellovibrio↗
PubMed1993

Identification of an early-stage gene of Chlamydia psittaci 6BC.

Chlamydiae are parasitic bacteria characterized by a temporally regulated developmental cycle. In the early stage of the cycle, metabolically inert elementary bodies reorganize to dividing reticulate bodies, a process about which little is known. The purpose of this investigation was to identify and clone chlamydial genes that are expressed preferentially during the early stage of the developmental cycle of Chlamydia psittaci 6BC. Several potential early genes were cloned with highly radioactive, host-free-generated RNA probes to screen a genomic library. One clone appeared to encode a gene that was particularly well expressed at 1 h postinfection. In further characterization, we found that it encodes two complete open reading frames and one partial open reading frame of 370 codons. The partial open reading frame, designated gltX, is very similar to bacterial glutamyl-tRNA synthetases and was demonstrated to be transcribed in vivo at 24 h postinfection by primer extension analysis. A lysine-rich open reading frame (LRO) of 117 codons was found upstream and divergent from gltX. The LRO lacks homology to known proteins, and we were unable to demonstrate that it is transcribed in vivo. The third open reading frame, of 182 codons, was found to be convergent with and partially overlap the LRO. It was confirmed to be preferentially expressed within the first 1.5 h of infection by Northern (RNA) blot analysis and was designated the early upstream open reading frame (EUO). Like the LRO, the EUO is not homologous to known proteins. A major potential transcription start site of the EUO was identified by primer extension analysis. However, the sequence upstream of the site does not closely resemble the consensus recognition sequences of bacterial sigma factors even though it is AT rich. The EUO is the first chlamydial gene specific to the early stage to be cloned and sequenced.

Amino Acid Sequence↗
PubMed1998

Proviral structure, chromosomal location, and expression of HERV-K-T47D, a novel human endogenous retrovirus derived from T47D particles.

We previously described that type B retrovirus-like particles released from the human mammary carcinoma cell line T47D are pseudotypes and package retroviral RNA of different origins (W. Seifarth, H. Skladny, F. Krieg-Schneider, A. Reichert, R. Hehlmann, and C. Leib-Mösch, J. Virol. 69:6408-6416, 1995). One preferentially packaged retroviral sequence, ERV-MLN, has now been used to isolate the corresponding full-length provirus from a human genomic library. The 9,315-bp proviral genome comprises a complete retroviral structure except for a 3' long terminal repeat (LTR) truncation. A lysine tRNA primer-binding site and phylogenetic analyses assign this human endogenous retroviral element, now called HERV-K-T47D, to the HML-4 subgroup of the HERV-K superfamily. The gag, prt, pol, and env genes exhibit 40 to 60% amino acid identity to HERV-K10. HERV-K-T47D is located on human chromosome 10, with five closely related elements on chromosomes 8, 9, 15, 16, and 19 and several hundred HERV-K-T47D-related solitary LTRs dispersed over the human genome. HERV-K-T47D-related sequences are detected in the genomes of higher primates and Old World monkeys but not in those of New World monkeys. High HERV-K-T47D transcription levels were observed in human placenta tissue, whereas transcription in T47D cells was strictly steroid dependent.

Amino Acid Sequence↗
PubMed2004

Functional genomics analysis of Singapore grouper iridovirus: complete sequence determination and proteomic analysis.

Here we report the complete genome sequence of Singapore grouper iridovirus (SGIV). Sequencing of the random shotgun and restriction endonuclease genomic libraries showed that the entire SGIV genome consists of 140,131 nucleotide bp. One hundred sixty-two open reading frames (ORFs) from the sense and antisense DNA strands, coding for lengths varying from 41 to 1,268 amino acids, were identified. Computer-assisted analyses of the deduced amino acid sequences revealed that 77 of the ORFs exhibited homologies to known virus genes, 23 of which matched functional iridovirus proteins. Forty-two putative conserved domains or signatures were detected in the National Center for Biotechnology Information CD-Search database and PROSITE database. An assortment of enzyme activities involved in DNA replication, transcription, nucleotide metabolism, cell signaling, etc., were identified. Viruses were cultured on a cell line derived from the embryonated egg of the grouper Epinephelus tauvina, isolated, and purified by sucrose gradient ultracentrifugation. The protein extract from the purified virions was analyzed by polyacrylamide gel electrophoresis followed by in-gel digestion of protein bands. Matrix-assisted laser desorption ionization-time of flight mass spectrometry and database searching led to identification of 26 proteins. Twenty of these represented novel or previously unidentified genes, which were further confirmed by reverse transcription-PCR (RT-PCR) and DNA sequencing of their respective RT-PCR products.

Animals↗
PubMed1996

Thyroid hormone and retinoic acid induce the synthesis of insulin-like growth factor-binding protein-4 in mouse osteoblastic cells.

Thyroid hormone (T3) is a known regulator of the transcription rate of specific genes. By subtractive hybridization of T2-treated osteoblastic cells, differentially expressed messenger RNAs (mRNAs) were enriched in the form of double stranded complementary DNA (cDNA) fragments. Sequencing of a differentially expressed cDNA that detects a 2.6-kilobase mRNA in Northern blots revealed to homology in the EMBL-Genebank data bases. A mouse genomic library was screened, and the isolated genomic DNA was identified as part of the insulin-like growth factor-binding protein-4 (IGFBP-4) gene including the 3'-untranslated region to which the cloned cDNA fragment was mapped by sequencing. We observed an up-regulation of the 2.6-kilobase IGFBP-4 mRNA transcript in the presence of T3 or retinoic acid. The induction of the IGFBP-4 transcript persisted up to 48 h. This response was inhibited by cycloheximide as well as actinomycin D. Long term induction studies revealed that the T3 effect is present during the complete culture period, with a constant rise in IGFBP-4 mRNA levels until 14 days. Under these culture conditions, the DNA content of MC3T3-E1 cells were significantly reduced by T3 and retinoic acid, indicating the repressive effect of both hormones on cell growth. Western immunoblots showed that the transcriptional induction is consequently transduced to increased IGFBP-4 levels in the conditioned medium of T3-treated cells. Our data show that thyroid hormone and retinoic acid stimulate transcription of IGFBP-4 mRNA in osteoblasts, resulting in increased IGFBP-4 secretion into the medium. IGFBP-4, a known inhibitor of cellular proliferation, might contribute to the antiproliferative effect of T3 and retinoic acid on osteoblasts.

Animals↗
PubMed2006

Identification of microsatellite markers in the rye genome.

The rye genomic library, which consists of DNA fragments in the range of 0.5-1.1 kb, was screened for the presence of tri-and tetranucleotide and compound microsatellites. Of the 1,600,000 clones analysed, 102 clones were positive and 41 were suitable for SSR primer pair design. Twenty-six primer pairs amplified specific products, and six of them were capable of detecting polymorphism among 30 rye accessions of different genetic backgrounds. Using a set of Chinese Spring-Imperial wheat-rye addition lines, it was possible to locate 3 newly identified microsatellites on chromosomes 3R, 4R and 7R.

Chromosomes, Plant↗
PubMed1987

Unusual DNA sequences located within the promoter region and the first intron of the chicken pro-alpha 1(I) collagen gene.

Genomic clones corresponding to the amino-terminal propeptide and 5'-flanking sequences of the chicken pro-alpha 1(I) collagen gene were isolated as a first step in the identification of DNA sequences important for transcriptional regulation of the pro-alpha 1(I) collagen gene. Due to the failure to identify positive clones in either primary or amplified genomic libraries, a 5.1-kilobase pair StuI genomic fragment identified by Southern blotting was enriched by sucrose gradient fractionation of genomic DNA and cloned into lambda gt11. Comparison of the DNA sequence of the 5.1-kilobase pair StuI fragment to the DNA sequence of a cDNA clone encoding the amino-terminal propeptide, signal peptide, and the 5'-untranslated region identified the first four exons and most of the fifth. Exon size and intron position have been largely conserved between human and chicken alpha 1(I) genes. DNA sequence analysis of the region 5' to the transcription initiation site identified the canonical TATA and CAAT boxes. However, the 40-nucleotide pyrimidine stretch centered between -150 and -180 nucleotides, found in all previously isolated type I procollagen genes from chicken, mouse, and human, was absent in the chicken pro-alpha 1(I) collagen gene. This sequence corresponds to the in vivo DNase I hypersensitive site in the chicken pro-alpha 2(I) and mouse pro-alpha 1(I) collagen genes, as well as the in vitro S1 nuclease hypersensitive site in both chicken and mouse pro-alpha 2(I) collagen genes. Two unusual DNA sequences were identified within the chicken pro-alpha 1(I) collagen gene. Fifteen tandem repeats of the sequence GGGGAGA were identified within the first intron, 300 nucleotides 3' to the first exon. This sequence was identified due to its hypersensitivity to S1 nuclease in vitro in supercoiled plasmids. The second sequence located 5' to -180 contained at least 25 copies of a polymorphic, 23-base pair tandemly repeated sequence not identified in other type I procollagen genes. Both of these tandem repeat sequences were identified at other locations in the chicken genome by Southern blot hybridization.

Amino Acid Sequence↗
PubMed1995

Polysaccharide expression in Lactococci.

Lactococcus lactis produces polysaccharide under defined environmental conditions. Three approaches are being used to identify regulators of polysaccharide synthesis in the organism. i) Two new lactococcal vectors, each of which contains a promoterless reporter gene, have been developed. They are being used to select for chromosomal insertions that affect expression of polysaccharide ii) Genetic complementation with lactococcal genomic libraries identified two classes of lactococcal genomic activities, both of which affect capsule expression in Escherichia coli iii) Lon, a highly conserved protease, is a negative regulator of polysaccharide synthesis in E. coli. Antiserum specific to Lon protease reacted with a lactococcal protein similar in size to E. coli Lon. This reaction was strongest in lactococcal strains which could not be induced to express polysaccharide.

Cloning, Molecular↗
PubMed1997

Cloning and expression of a gene encoding Moraxella sp. CK-1 autolysin.

A gene of Moraxella sp. CK-1 encoding cell wall lytic activity has been cloned and expressed in E. coli. A genomic library of Moraxella sp. CK-1 was constructed in the multifunctional phagemid vector pT7T3 19u, with partial Sau3A digests of Moraxella sp. CK-1 genomic DNA inserted at the BamHI restriction site. Screening of about 5,000 transformants for cell lysis activity in LB agar plates containing Micrococcus Iuteus cells gave one positive clone harboring the 3.7 kb insert (pMXA282). Restriction mapping and deletion analysis of the recombinant plasmid carrying a 3.7 kb insert suggested that the autolysin gene was located within a 1.1 kb BamHI-PstI fragment. Analysis of extracts of E. coli clone harboring recombinant plasmids on renaturing SDS-polyacrylamide gels containing heat-killed Micrococcus luteus cells showed a clear zone around a polypeptide of about 32 kDa. Lytic activity against Micrococcus luteus cell walls by the cloned autolysin was maximum at pH 9.0. Even in conditions of over pH 10.0, this cloned autolysin showed a vigrous lytic activity. Southern blot analysis suggested the existence of other homologous regions in Moraxella sp. CK-1 genome.

Bacteriolysis↗