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At least 595 records · Page 33Linked to original sources
PubMed1992

Alternative promotion of aromatase P-450 expression in the human placenta.

We have previously reported the isolation and characterization of the human gene encoding aromatase cytochrome P-450 (P-450AROM). The gene had been demonstrated to span at least 52 kb and contain ten exons, the first of which, exon I.1, is untranslated. Here we report the isolation and characterization of a P-450AROM cDNA from a human placental primer-extended cDNA library which contains a unique 5' sequence. This cDNA has been isolated and sequences used to screen a human placental genomic library for the presence of a unique first exon. The exon (exon I.2) lies 9 kb 5' of the second, ATG-containing exon (exon II) and is spliced onto exon II at the same site as that reported for exon I.1. DNA sequence analysis indicates that exon I.2 has a putative TATA (TAAA) sequence 33 base pairs (bp) upstream from a putative transcription start site and putative CAAT (CATT) binding sequence beginning at 54 bp upstream from this start site. Polymerase chain reaction (PCR) amplification experiments indicate that mRNA containing exon I.2-specific sequences can be demonstrated in tissues of fetal, but not adult, origin. These data have been confirmed by Northern analysis in the placenta. Characterization of this genomic clone containing exons I.2 and II now establishes the P-450AROM gene to be at least 72 kb in length and raises new questions regarding tissue specific and developmental control of aromatase expression in the human.

Aromatase↗
PubMed2006

Isolation and characterization of SSR sequences from the genome and TAC clones of common wheat using the PCR technique.

We have developed the 2-step PCR method, a kind of suppression PCR procedure, to isolate simple sequence repeats (SSRs) from common wheat (Triticum aestivum L.) in a more convenient manner. This system requires neither genomic library screening nor the SSR-enrichment procedure. As a result, we designed 131 primer pairs based on isolated SSRs from not only genomic DNA, but also transformation-competent artificial chromosome (TAC) clones. It has been demonstrated that 34 of the 131 SSR markers developed were polymorphic among 8 wheat lines. Four of 34 polymorphic SSR markers were derived from TAC clones, indicating that this method could be applied to the targeted development of unique SSR markers in large genomic DNA libraries such as those composed of bacterial artificial chromosomes (BACs). A considerable number of isolated SSR clones had similarities with part of several long terminal repeats of retrotransposons (LTR-RTs) identified in various Triticeae genome sequences. Most of those SSRs showed smear amplification profiles, suggesting that a considerable number of dysfunctional SSRs originating from repetitive DNA components, especially LTR-RTs, might exist in the common wheat genome.

Base Sequence↗
PubMed1994

Cloning, characterization, and genetics of the juvenile hormone esterase gene from Heliothis virescens.

The gene for juvenile hormone esterase (JHE) was cloned from Heliothis virescens (Lepidoptera: Noctuidae). A genomic library was constructed from embryonic DNA and screened with a homologous N-terminal probe from the JHE cDNA. Five genomic clones were isolated and analyzed by dot blot hybridization using regions of the JHE cDNA as probes. Clone C hybridized to both 5' and 3' probes from the JHE cDNA, suggesting that clone C contains both ends of JHE gene. This was verified by sequencing the ends of the JHE gene from clone C using primers from both the 5' and 3' ends of the JHE cDNA. Additional sequencing and restriction mapping were used to characterize the gene. The gene is c. 8 kb long and contains four introns with consensus intron-exon junctions. One of the introns is relatively large (4 kb) and is situated near the extreme 5' end of the gene. Genetic analysis of RFLP variation in interspecific and intraspecific crosses shows that the JHE locus is single-copy with no closely related paralogs and is autosomally encoded in Heliothis. Therefore the developmental pattern of expression of this gene and the previously documented sequence variation in cDNA clones is not explainable by reference to a JHE gene family with distinct structural loci for the different forms.

Animals↗
PubMed1991

Most peripheral B cells in mice are ligand selected.

Using amplified cDNA and genomic libraries, we have analyzed the VH gene repertoire of pre-B cells and various B cell subsets of conventional mice at the level of VH genes belonging to the J558 VH gene family. The sequence data were evaluated on the basis of a newly established list of 67 J558 VH genes that comprise approximately two-thirds of the J558 VH genes of the murine IgHb haplotype. The results of the analysis demonstrate that VH gene utilization in pre-B cells, although biased to some extent by B cell autonomous VH gene selection, scatters over the whole range of J558 VH genes present in the germline. In contrast, in mature, peripheral B cells comprising long-lived mu + delta high B cells as well as Ly-1 B cells, small overlapping sets of germline VH genes are dominantly expressed. The data indicate that the recruitment of newly generated B cells into the long-lived peripheral B cell pool is mediated through positive selection by internal and/or external antigens. Because of the absence of immunoglobulin class switching and somatic hypermutation, this process is different from the selection of memory B cells in T cell-dependent immune responses.

Animals↗
PubMed2002

Identification of a NifL-like protein in a diazotroph of the beta-subgroup of the Proteobacteria, Azoarcus sp. strain BH72.

NifA, the transcriptional activator of nitrogenase (nif) genes, has up to now been described to be regulated in its activity via the sensor NifL only for members of the gamma-subgroup of the PROTEOBACTERIA: This paper reports a functionally similar NifL-like protein outside this group in Azoarcus sp. strain BH72, a diazotrophic grass endophyte belonging to the beta-subgroup of the PROTEOBACTERIA: Its structural genes for nitrogenase (nifHDK) are regulated in response to combined nitrogen and O(2) and expressed endophytically inside rice roots. In order to characterize nitrogen-regulatory genes, an Azoarcus sp. BH72 genomic library was used to select cosmids that complemented a nifA mutation in Azotobacter vinelandii. Sequence analysis of the 3.4 kb genomic region complementing nifA showed two ORFs with sequence identities of 44% to NifL and 61% to NifA of Azotobacter vinelandii. According to Northern blot and reverse transcriptase PCR analysis, the nifLA transcript was more abundant at low combined nitrogen and O(2) levels, results which were corroborated by GUS (beta-glucuronidase) assays using a transcriptional nifL::gusA fusion. N(2) fixation was abolished in a NifLA(-) and a NifA(-) mutant, wild-type fixation being restored by nifLA in trans. The NifLA(-) mutant also failed to activate nifH::gus expression, indicating that NifA is the obligate transcriptional activator for nifHDK. A nifL mutant was diazotrophic and did not show repression of nifH::gusA by ammonium or O(2), suggesting that NifL of Azoarcus sp. strain BH72 has a similar role in inactivating NifA in response to O(2) and combined nitrogen as NifL in bacteria of the gamma-PROTEOBACTERIA:

Azoarcus↗
PubMed2000

A genomic sample sequence of the entomopathogenic bacterium Photorhabdus luminescens W14: potential implications for virulence.

Photorhabdus luminescens is a pathogenic bacterium that lives in the guts of insect-pathogenic nematodes. After invasion of an insect host by a nematode, bacteria are released from the nematode gut and help kill the insect, in which both the bacteria and the nematodes subsequently replicate. However, the bacterial virulence factors associated with this "symbiosis of pathogens" remain largely obscure. In order to identify genes encoding potential virulence factors, we performed approximately 2,000 random sequencing reads from a P. luminescens W14 genomic library. We then compared the sequences obtained to sequences in existing gene databases and to the Escherichia coli K-12 genome sequence. Here we describe the different classes of potential virulence factors found. These factors include genes that putatively encode Tc insecticidal toxin complexes, Rtx-like toxins, proteases and lipases, colicin and pyocins, and various antibiotics. They also include a diverse array of secretion (e.g., type III), iron uptake, and lipopolysaccharide production systems. We speculate on the potential functions of each of these gene classes in insect infection and also examine the extent to which the invertebrate pathogen P. luminescens shares potential antivertebrate virulence factors. The implications for understanding both the biology of this insect pathogen and links between the evolution of vertebrate virulence factors and the evolution of invertebrate virulence factors are discussed.

Animals↗
PubMed1994

Molecular cloning of the human homeobox gene goosecoid (GSC) and mapping of the gene to human chromosome 14q32.1.

Goosecoid is a homeobox gene first isolated from a Xenopus dorsal lip cDNA library. Homologous genes have been isolated from mouse, zebrafish, and chick. In all species examined, the gene is expressed and plays an important role during the process of gastrulation in early embryonic development. We report here the cloning of the human goosecoid gene (GSC) from a genomic library and the sequence of its encoded protein. The genomic organization and protein sequence of the human gene are highly conserved with respect to those of its Xenopus and mouse counterparts: all three genes consist of three exons, with conserved exon-intron boundaries; the sequence of the homeodomain is 100% conserved in most vertebrates. Using somatic cell hybrid and chromosomal in situ hybridization, the gene was mapped to chromosome 14q32.1.

Amino Acid Sequence↗
PubMed1999

Iterative selection from a Salmonella typhimurium cosmid library can lead to the isolation of an atypically small plasmid.

We have constructed a DNA library from a virulent Salmonella typhimurium strain, in an avirulent strain. The process of selecting the components of interest from the library involved iterative growth in liquid culture. This resulted, after four cycles, in the culture becoming homogeneous for a single plasmid, which was much smaller than the average size for the library. We have identified the larger precursor of this plasmid which has two regions of sufficient homology to allow recombination resulting in the formation of the small plasmid. S. typhimurium carrying the small plasmid have a smaller genetic burden than other members of the library and survive better in spent culture medium, facilitating selection on repeated subculture. Such rapid adventitious selection has important implications for isolation of clones of interest from genomic libraries.

Cosmids↗
PubMed2005

[Cloning and structural analysis of mouse genomic nucleophosmin gene].

Nucleophosmin (NPM) is an abundant nucleolar phosphoprotein. NPM gene involved chromosomal translocations were found in the patients with anaplastic large cell lymphomas (ALCL), myelodysplastic syndrome (MDS), acute myeloid leukemia (AML) and acute promyelocytic leukemia (APL). To generate NPM gene knockout mice and study its biological function in vivo, we screened the lambda phage genomic library derived from 129S1 mice with mouse NPM cDNA probe. A positive phage clone which contained the full-length NPM genomic DNA was obtained and the insert of 15.3 kb genomic DNA in this clone was sequenced with shotgun method. BLAST analysis showed that the sequence of insert are 99.8% identity to that of NPM gene of C57BL/6 mouse strain. Based on the sequence, bioinformatics analysis on genomic structure of NPM and the transcription factor binding sites in the NPM 5' flanking region were performed.

5' Flanking Region↗
PubMed1994

The structure of the urokinase-type plasminogen activator receptor gene.

The cellular receptor for urokinase-type plasminogen activator (uPAR) is a glycosylphosphatidylinositol (GPI)-anchored membrane protein that plays a central role in pericellular plasminogen activation. It contains 313 amino acid residues, including 28 cysteine residues in a pattern of three homologous repeats. The cysteine residue pattern suggests that uPAR belongs to a superfamily of proteins including CD59, murine Ly-6, and a variety of elapid snake venom toxins. A novel 1.7-kb uPAR cDNA was isolated that is missing exon 5 and that contains 380 bp not previously reported at the 5' end. This cDNA was used to probe a human genomic library from which three clones were isolated and analyzed. The uPAR gene consists of 7 exons spread over 23 kb of genomic DNA. Exons 2, 4, and 6 code for homologous domains within the mature protein, as do exons 3, 5, and 7; CD59-like homologous pairs are encoded by exons 2-3, 4-5, and 6-7, respectively. The structure of the gene for uPAR further confirms the relationship of this molecule to the superfamily containing CD59, Ly-6, and the elapid snake venom toxins.

Amino Acid Sequence↗
PubMed1995

Genomic structure of the mouse delta opioid receptor gene.

Using mouse delta opioid receptor (DOR) cDNA sequence to probe genomic libraries in bacteriophage lambda and P1 vectors, clones traversing the entire DOR coding sequence and 5' and 3' flanking regions were isolate. Genomic sequence encoding mature DOR message, including 5' and 3' untranslated sequence, is divided by two introns of 26 kb and 3 kb, resulting in the gene occupying 32 kb of chromosomal DNA. Multiple putative transcription initiation sites were located, by RNase protection assay, in TATA-less G+C rich sequence between 390 and 140 nucleotides upstream from the ATG translation start codon. A polyadenylation site was located 1.24 kb downstream from the TGA translation stop codon. Examination of 1.3 kb of 5'flanking sequence revealed potential binding sites for several known transcription factors including: Sp1, Ap-2, NF-kappa B, NF-IL6, and NGFI-B.

Animals↗
PubMed1994

Structure of the human cyclo-oxygenase-2 gene.

Cyclo-oxygenase (Cox), a rate-limiting enzyme in the synthesis of prostanoids, is encoded by two genes, Cox-1 and Cox-2, which are differentially expressed and regulated. Human Cox-1 and -2 polypeptides share 61% primary sequence identity. While the expression of Cox-1 is maximal in quiescent cells. Cox-2 expression is induced by growth factors and cytokines. We have screened a human genomic library with a probe from the 5'-untranslated region (UTR) of the human Cox-2 (hCox-2) cDNA and isolated two overlapping genomic clones. We have determined the DNA sequence of 0.8 kb upstream of the transcription start site, 6 kb of protein coding region, which includes 10 exons and 9 introns, as well as 2.5 kb of the 3'-UTR. The structures of the hCox-1 and hCox-2 and the murine TIS10 (Cox-2) genes are highly conserved, with a few exceptions. The 3'-UTRs of the Cox-1 and -2 genes are distinct; for example, the largest exon in the Cox-2 gene encodes the entire 3'-UTR, containing 22 copies of the 'AUUUA' RNA instability element. Sequence analysis of the 5'-flanking region has shown several potential transcription regulatory sequences, including a TATA box, a C/EBP motif, two AP-2 sites, three SP1 sites, two NF-kappa B sites, a CRE motif and an Ets-1 site. These efforts serve as a basis for future studies on transcriptional and post-transcriptional mechanisms of Cox-2 gene regulation.

Amino Acid Sequence↗
PubMed1994

Molecular cloning, structure, and chromosomal localization of the human inducible nitric oxide synthase gene.

Nitric oxide, a multifunctional effector molecule synthesized by nitric oxide synthase (NOS) from L-arginine, conveys signals for vasorelaxation, neurotransmission, and cytotoxicity. Three different NOS isoforms have been identified which fall into two distinct types, constitutive and inducible. The inducible NOS (iNOS) isoform is expressed in a variety of cell types and tissues in response to inflammatory agents and cytokines. The human iNOS (NOS2) gene was isolated on overlapping cosmid clones from a human genomic library using both the murine macrophage and the human hepatocyte iNOS cDNAs as probes. All isolated cosmids were part of a single genomic locus and no other genomic loci were identified or isolated. Analysis of this locus indicated that the human iNOS gene is approximately 37 kilobases in length and consists of 26 exons and 25 introns. Primer extension analysis of lipopolysaccharide and cytokine-stimulated human hepatocyte RNA mapped the transcriptional initiation site 30 base pairs downstream of a TATA sequence, and a 400-base pair 5'-flanking region was found to be structurally similar to the recently described murine iNOS promoter. Polymerase chain reaction analysis of a human/rodent genomic DNA somatic cell hybrid panel and fluorescent in situ hybridization indicated that the human iNOS gene is located on chromosome 17 at position 17cen-q11.2.

Amino Acid Oxidoreductases↗
PubMed1993

Gene rearrangement and truncated mRNA in cell lines with 11q23 translocation.

We have previously demonstrated that the breakpoints of t(11;19)(q23;p13) leukemias are within 360 kb of the CD3 gene. One of the phage clones, 6n, which was isolated from the yeast artificial chromosome clone yB22B2 containing CD3, was found to be within 60 kb of t(11;19) breakpoints. In this study, gene walking was conducted and two phage clones (lambda Hp8-3 and lambda Hp23-13) were isolated from a human placenta genomic library. Southern blot analysis with a genomic probe from lambda Hp8-3 detected gene rearrangements in t(4;11) and t(11;19) cell lines with BamHI digestion. Subsequently, using reiterated sequence-free probes from both ends of 6n that detected transcriptional units in various hematopoietic cells, we isolated cDNA clones. These cDNA clones were classified into two groups (designated MLL-a and MLL-b), which do not hybridize to each other. Northern blot analysis with MLL-a cDNA detected 15-, 14- and 12-kb mRNAs, while MLL-b detected the additional 9.7- and 5-kb mRNAs in peripheral blood lymphocytes. MLL-b cDNA detected a truncated form of 12.5-kb mRNA in t(4;11) cell lines and a truncated form of 10-kb or 9.2-kb mRNA in t(11;19) cell lines. MLL-a did not demonstrate a truncated form of mRNA, but the stronger 14-kb signal was noted in t(4;11) cells, while this signal was very weak in t(11;19) cells. By Southern blot analysis, MLL-b cDNA detected gene rearrangement in cell lines with t(4;11) and t(11;19), whereas MLL-a did not. Furthermore, chimeric cDNA clones were isolated from cDNA libraries of t(4;11) and t(11;19) cell lines with a MLL-b cDNA probe. These results indicate that the MLL-b cDNA is derived from the common target gene involved in 11q23 translocation with 4q21 or 19p13.

Adult↗
PubMed1995

Generation of immunoglobulin light chain gene diversity in Raja erinacea is not associated with somatic rearrangement, an exception to a central paradigm of B cell immunity.

In all vertebrate species examined to date, rearrangement and somatic modification of gene segmental elements that encode portions of the antigen-combining sites of immunoglobulins are integral components of the generation of antibody diversity. In the phylogenetically primitive cartilaginous fishes, gene segments encoding immunoglobulin heavy and light chain loci are arranged in multiple clusters, in which segmental elements are separated by only 300-400 bp. In some cases, segmental elements are joined in the germline of nonlymphoid cells (joined genes). Both genomic library screening and direct amplification of genomic DNA have been used to characterize at least 89 different type I light chain gene clusters in the skate, Raja. Analyses of predicted nucleotide sequences and predicted peptide structures are consistent with the distribution of genes into different sequence groups. Predicted amino acid sequence differences are preferentially distributed in complementarity-determining versus framework regions, and replacement-type substitutions exceed neutral substitutions. When specific germline sequences are related to the sequences of individual cDNAs, it is apparent that the joined genes are expressed and are potentially somatically mutated. No evidence was found for the presence of any type I light chain gene in Raja that is not germline joined. The type I light chain gene clusters in Raja appear to represent a novel gene system in which combinatorial and junctional diversity are absent.

Amino Acid Sequence↗
PubMed1994

Assignment of 30 microsatellite loci to the linkage map of Arabidopsis.

Thirty microsatellite loci were assigned to the Arabidopsis linkage map. Several microsatellite sequences in Arabidopsis DNA were found by searching the EMBL and GenBank databases, and a number of these were subsequently found to detect polymorphisms between different Arabidopsis strains by the polymerase chain reaction (PCR). After the presence of microsatellites in Arabidopsis and their utility for genetic mapping had been demonstrated, systematic screening for (CA)n and (GA)n sequences was carried out on marker-selected plasmid libraries and a small-insert genomic library. Positive clones were sequenced, PCR primers flanking the repeats were synthesized, and PCR was carried out on different strains to look for useful polymorphisms. Surprisingly, of 18 (CA)n repeats (n > 13), only one was polymorphic. In contrast, 25 of 30 (GA)n repeats, 2 of 3 (AT)n repeats, and 2 of 4 (A)n repeats were polymorphic. The majority of the (CA)n repeats were complex, with adjacent short di-, tri-, or tetranucleotide repeats, whereas most of the (GA)n, (TA)n, and (A)n repeats were simple. The (CA)n repeats were also refractory to PCR analysis, requiring extensive optimization of PCR conditions, whereas the other repeat classes were mostly amplified with a single set of standard conditions. When polymorphisms were detected, the microsatellites were mapped using a set of recombinant inbred lines originating from a cross between the strains Columbia and Landsberg erecta.

Alleles↗
PubMed1991

Gene and precursor structures of human C-type natriuretic peptide.

We have isolated the gene for human C-type natriuretic peptide (CNP) from a human genomic library using a cloned porcine CNP genomic DNA fragment as probe. Human CNP gene consists of at least two coding blocks and an intron, and encodes a 126-residue CNP precursor (human prepro-CNP). From a comparison of the amino acid sequences of porcine and rat prepro-CNPs, human prepro-CNP is found to be processed to generate 22-and 53-residue peptides (human CNP-22 and human CNP-53, respectively) as major endogenous CNPs in human. Interestingly, human CNP-53 has two amino acid substitutions as compared to the porcine and rat CNP-53s, whereas human CNP-22 is identical to the porcine and rat CNP-22s. Intravenous injection of human CNP-53 into anesthetized rats induces diuretic-natriuretic and hypotensive activities with same potencies as porcine CNP-53 does, although these activities were considerably lower (about 1/100) than those of human alpha-ANP.

Amino Acid Sequence↗
PubMed1997

A DNA fingerprinting system for the ectoparasite Sarcoptes scabiei.

We describe multiple hypervariable microsatellites that will provide a highly informative genetic marker system for the sarcoptid mite Sarcoptes scabiei. Eighteen positive clones containing the highly repetitive sequence (GA)n were isolated from a partial genomic library of S. scabiei. Ten of these clones were characterised by sequencing and primers were designed from the unique sequences flanking eight microsatellite loci. Genomic DNA was subsequently extracted from individual mites and the repeat blocks were amplified by way of [gamma 33P] ATP end-labelled polymerase chain reaction. Fragment length polymorphisms were revealed in three of the loci when resolved on polyacrylamide sequencing gels. The high levels of allelic variability demonstrated between individual mites enable these three loci to form a DNA fingerprinting system that will be suitable for epidemiological and taxonomic studies both within and between host species.

Alleles↗