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At least 829 records · Page 46Linked to original sources
PubMed2001

Outer sheath associated proteins of the oral spirochete Treponema maltophilum.

We recently cloned the major outer membrane protein of Treponema maltophilum [Heuner, K., Choi, B.K., Schade, R., Moter, A., Otto, A., Göbel, U.B., J. Bacteriol. 181, 1025-1029]. Here we report the localization of the major sheath protein (Msp)A protein in T. maltophilum by immunogold electron microscopy and its expression. Northern blot analysis revealed that mspA is expressed constitutively as a monocistronic unit. The transcription initiation site of the mspA gene was identified by primer extension analysis. A further screening of a genomic library of T. maltophilum with an anti-outer membrane fraction antibody was done. We were able to clone DNA regions of T. maltophilum encoding putative sugar transport operons and putative outer membrane proteins of this oral treponeme which has a high prevalence in periodontal lesions.

Bacterial Outer Membrane Proteins↗
PubMed1992

Identification of a novel Brugia pahangi beta-tubulin gene (beta 2) and a 22-nucleotide spliced leader sequence on beta 1-tubulin mRNA.

We have examined the expression of beta-tubulin genes in the parasitic nematode, Brugia pahangi. A genomic library was constructed and screened by hybridization with a Haemonchus contortus beta-tubulin cDNA fragment which recognizes several B. pahangi beta-tubulin sequences, including sequences which correspond to the previously characterized beta 1-tubulin gene. The B. pahangi beta 2-tubulin gene was isolated by selecting clones which hybridize to the H. contortus beta-tubulin gene but which do not hybridize to the beta 1-tubulin gene. A partial sequence of the beta 2-tubulin gene confirms that it codes for a distinct beta-tubulin. Southern hybridization analyses show that the beta 2-tubulin sequence exists as a single copy gene within the B. pahangi genome. Expression of the beta 2-tubulin gene is developmentally regulated and the message is found predominantly in adult male worms, whereas the beta 1-tubulin gene is expressed in microfilariae and approximately equal levels of the transcript are found in male and female adult worms. During mRNA maturation the beta 1-tubulin mRNA of microfilariae and adult worms acquires a trans-spliced leader identical to the SL1 of Caenorhabditis elegans.

Amino Acid Sequence↗
PubMed1993

Molecular cloning and expression of Rickettsia prowazekii genes for three outer membrane proteins in Escherichia coli.

Rickettsia prowazekii (virulent Breinl strain) random genomic DNA fragments were cloned in the lambda gt11 expression vector by using non-palindromic adaptors. Several immunoreactive clones were selected after screening 20,000 individual recombinant plaques with human convalescent serum. Some recombinants expressed complete 60 kDa polypeptide, and others expressed beta-galactosidase fusion polypeptides containing different epitopes of 134 kDa protein of the R. prowazekii outer membrane (OM). Amplified genomic library was screened with monospecific antibodies directed against abundant 31 kDa and 29.5 kDa OM proteins (OMPs). Several recombinant clones expressing full or part of 29.5 kDa polypeptide, and none expressing 31 kDa polypeptide were revealed. The serum of a patient convalescing from epidemic typhus did not react in Western blot with recombinant 29.5 kDa protein.

Antibodies, Bacterial↗
PubMed1997

The isolation of novel heat shock genes in Lactococcus lactis using RNA subtractive hybridization.

Lactococcus lactis is subjected to heat shock (hs) during cheese manufacturing. A number of conserved hs genes have been cloned and studied in this organism, although no regulatory gene, e.g. alternative sigma factor, has been identified. RNA subtractive hybridization was used to identify genes expressed very early when L. lactis MG1363 was shifted from 30 to 43 degrees C. 32P-labeled cDNA synthesized from RNA isolated from hs cells at 43 degrees C was mixed with an excess vegetative RNA and the mixture was directly used as a probe after a short hybridization step. Northern analysis revealed a moderate induction for the probes used, and low expression was also detected in non-hs cells, demonstrating the applicability of this technique for the detection of differentially expressed genes. The probes were used to identify genomic library clones containing the corresponding genes. Among the five clones studied, a cell division operon including a putative ftsZ homolog (pJAK2) was identified. Additionally, a putative hsp86 homolog (pJAK3), three different transposase encoding genes (pJAK1 and pJAK3), a gene coding for a deoR-like transcriptional repressor (pJAK4) and a putative regulatory gene that showed homology to an alkaline shock protein (pJAK5) were characterized.

DNA, Complementary↗
PubMed2001

cDNA-RNA subtractive hybridization reveals increased expression of mycocerosic acid synthase in intracellular Mycobacterium bovis BCG.

Identifying genes that are differentially expressed by Mycobacterium bovis BCG after phagocytosis by macrophages will facilitate the understanding of the molecular mechanisms of host cell-intracellular pathogen interactions. To identify such genes a cDNA-total RNA subtractive hybridization strategy has been used that circumvents the problems both of limited availability of bacterial RNA from models of infection and the high rRNA backgrounds in total bacterial RNA. The subtraction products were used to screen a high-density gridded Mycobacterium tuberculosis genomic library. Sequence data were obtained from 19 differential clones, five of which contained overlapping sequences for the gene encoding mycocerosic acid synthase (mas). Mas is an enzyme involved in the synthesis of multi-methylated long-chain fatty acids that are part of phthiocerol dimycocerosate, a major component of the complex mycobacterial cell wall. Northern blotting and primer extension data confirmed up-regulation of mas in intracellular mycobacteria and also revealed a putative extended -10 promoter structure and a long untranslated upstream region 5' of the mas transcripts, containing predicted double-stranded structures. Furthermore, clones containing overlapping sequences for furB, groEL-2, rplE and fadD28 were identified and the up-regulation of these genes was confirmed by Northern blot analysis. The cDNA-RNA subtractive hybridization enrichment and high density gridded library screening, combined with selective extraction of bacterial mRNA represents a valuable approach to the identification of genes expressed during intra-macrophage residence for bacteria such as M. bovis BCG and the pathogenic mycobacterium, M. tuberculosis.

Acyltransferases↗
PubMed1998

Extracellular release of antigenic proteins by Helicobacter pylori.

Screening a Helicobacter pylori genomic library with antisera raised against H. pylori broth culture supernatant resulted in the identification of six antigens: urease, HspB, Lpp20, DnaK, MsrA, and a cysteine-rich 28-kDa protein (designated HcpA). H. pylori antigens may be released into the extracellular space by multiple mechanisms, including specific secretion pathways, autolysis, and membrane vesicle formation.

Amino Acid Sequence↗
PubMed1995

Xylem-specific gene expression in loblolly pine.

Two genes preferentially expressed in differentiating xylem of loblolly pine (Pinus taeda L.) were cloned from cDNA and genomic libraries and designated PtX3H6 and PtX14A9. Transcripts of PtX3H6 and PtX14A9 are very abundant in differentiating xylem, less abundant in needles, and very low or non-detectable in embryos and megagametophytes. PtX3H6 contains a putative signal peptide, a threonine-rich region, a proline-rich region, and a hydrophobic tail. Repeats of Pro-Pro-Pro-Val-X-X are similar to repeats found in proline-rich cell wall proteins. The amino acid compositions of PtX3H6 and PtX14A9 are similar to those of arabinogalactan proteins (AGPs). PtX14A9 contains an 8 amino acid sequence similar to amino terminal sequences of ryegrass, carrot and rose AGPs. Upstream sequences have been determined from genomic clones encoding PtX3H6 and PtX14A9. A 7 bp sequence found in the 5' flanking regions of both genes has previously been shown to be involved in the vascular-specific expression of GRP 1.8, a glycine-rich protein found in bean. The sequence is also present upstream of another glycine-rich protein from bean, GRP 1.0, and may be partially responsible for the xylem-specific expression of pTx3H6 and PtX14A9.

Amino Acid Sequence↗
PubMed1998

A novel human luteinizing hormone receptor gene.

A novel human luteinizing hormone receptor (LHR) gene was isolated from a human placental genomic library. This gene (Gene II) differs from that previously isolated from a lymphocyte library (Gene I) by several base changes in the 5' flanking region and the deletion of 6 nucleotides in the coding region (+55 to +60). The sequence of the exon 1 coding region of gene II conforms to the sequence of the human ovarian LHR cDNA. Furthermore, primer extension and reporter gene analyses indicate that both the Gene II promoter and the human ovarian LHR mRNA transcriptional start sites are located within the -176 bp TATA-less 5' flanking domain. Additional upstream transcriptional start sites (> -176 bp) were identified in human testicular mRNA and the human choriocarcinoma JAR cell. Restriction enzyme analysis verifies that both LHR genes are present in the human genome, and gene dosing reveals four copies of the human LHR in contrast to a single copy in the rat genome. Chromosomal mapping localizes all copies of the human LHR to the chromosome 2p16-21 loci. These studies suggest that tissue-specific LHR promoter utilization and LHR gene expression may be correlated with gene diversity.

Amino Acid Sequence↗
PubMed1991

Genome analysis of imperfect fungi: electrophoretic karyotyping and characterization of the nuclear gene coding for glyceraldehyde-3-phosphate dehydrogenase (gpd) of Curvularia lunata.

The gene coding for glyceraldehyde-3-phosphate dehydrogenase (gpd) has been isolated from a genomic library of the filamentous fungus Curvularia lunata. The coding region of this gene consists of 1014 nucleotides and is interrupted by four introns. The gpd gene product shows a high degree of sequence identity with the corresponding proteins of various species belonging to both taxonomically related (e.g., Aspergillus nidulans), as well as more divergent, taxa. Using contour-clamped homogeneous electric field (CHEF) gel electrophoresis eight distinct chromosomal bands have been resolved, with two bands migrating as doublets and one as a triplet. Thus, the total number of chromosomes of C. lunata appears to be 12. The size of the chromosomes ranges from about 1.4 Mb to 4.0 Mb allowing an estimation of the genome to be approximately 29.7 Mb. By hybridization of fractionated chromosomes the gpd gene and the rDNA locus have been localized on individual chromosomes.

Amino Acid Sequence↗
PubMed1991

[Androgen-dependent protein secreted by mouse caput epididymis shows high homologies with different glutathione peroxidases].

Principal cells of the mouse caput epididymidis synthesize and secrete a 24 kDa protein able to bind to the head of the spermatozoa. Sequencing of several clones selected from cDNA and genomic libraries, combined with the microsequencing of the NH2 terminus of the protein allowed to reconstitute the entire primary structure of the mature 24 kDa protein. It revealed 81% homology with a human plasma glutathione peroxidase and 61% homology with a mouse erythrocyte glutathione peroxidase. This enzyme, once secreted in the epididymal fluid, might protect sperm membrane lipids, particularly those of the acrosomal part, against peroxidation.

Androgens↗
PubMed1994

Isolation of a potentially functional HPRT processed pseudogene from the hill kangaroo Macropus robustus.

A highly conserved hypoxanthine phosphoribosyltransferase processed pseudogene (KPH) has been isolated from a female kangaroo (Macropus robustus) lambda EMBL3 genomic library. The pseudogene contains only transcribed material with all of the introns precisely removed and has possible direct repeats at either end of the message. It has a 654-nucleotide open reading frame (ORF) from the Met start codon to the stop codon that contains no additions, deletions or premature stops relative to expressed HPRT genes and, therefore, the possibility exists that it is expressed in vivo. Possible CAAT and GC boxes are present in the region 5' to the ORF and a polyadenylation signal is present in the region 3' to the ORF. If not expressed, the age of the pseudogene is estimated to be 10.7 million years. We propose that integration into the genome occurred specifically in a homocopolymeric region within a highly repeated region unique to the kangaroo genome.

Amino Acid Sequence↗
PubMed1997

Development of a specific DNA probe and PCR for the detection of Mycoplasma bovis.

Mycoplasma bovis is responsible for several production diseases in cattle, including mastitis, arthritis, pneumonia, abortion and infertility. Current methodologies for detecting and identifying M. bovis are time consuming and difficult. Tests which rely on antigen or antibody detection have poor sensitivity and specificity. In this paper associated protocols for the development of a hybridization probe and PCR are described. A genomic library (SauIIIA digested) was prepared from M. bovis DNA (Colindale Reference Strain: NC10131:02) and cloned into pUC19. Colony hybridization, using a probe preparation made from purified M. bovis DNA, was used to identify colonies of interest. M. bovis DNA fragments were retrieved from recombinant plasmids by digestion with EcoRI and HindIII. This DNA was used to prepare randomly primed probes for dot blot hybridization analysis with immobilized DNA from M. bovis (two strains), M. dispar, M. agalactiae, M. bovigenitalium (two strains), M. ovipneumoniae, a Group 7 strain, M. arginini and bacteria belonging to different genera. Four probes were found to hybridize only with M. bovis and M. ovipneumoniae DNA, whereas one probe reacted with genomic DNA from only one of the two M. bovis strains. The level of sensitivity of the dot blot hybridization assay was 200 CFU (colony forming units)/mL. To enhance the sensitivity further, an M. bovis-specific PCR assay was developed. The primers were designed using sequences obtained from the probe DNA which discriminated M. bovis from all other Mycoplasma DNA tested. The minimum amount of target DNA that could be detected by the PCR assay was that isolated from 10-20 CFU/mL. The PCR assay was therefore 10 times more sensitive than dot blot hybridization.

Animals↗
PubMed1992

Amplification of human polymorphic sites in the X-chromosomal region q21.33 to q24: DXS17, DXS87, DXS287, and alpha-galactosidase A.

Methods for the PCR amplification of five polymorphic sites in the region Xq21.33 to Xq24 were developed and used to predict heterozygosity for Fabry disease in informative families. Clones containing polymorphic sites associated with DNA segments DXS17, DXS87, and DXS287, and the alpha-galactosidase A gene were isolated from genomic libraries. Surrounding nucleotide sequences and optimal conditions for amplification of each polymorphic site were determined. These amplifiable polymorphisms provided predictions of heterozygosity for Fabry disease and should be useful for diagnostic linkage analyses in Alport syndrome, X-linked cleft palate and ankyloglossia, Pelizaeus-Merzbacher disease, and X-linked agammaglobulinemia as well as sequence-tagged sites for gene mapping.

Base Sequence↗
PubMed1998

Characterization of the 5' flanking region of the Xenopus laevis transforming growth factor-beta 5 (TGF-beta 5) gene.

Transforming growth factors-beta are potent regulators of cellular proliferation, differentiation and morphogenesis. 2.41 kb of the 5' flanking region of the transforming growth factor-beta 5 (TGF-beta 5) gene has been isolated from a Xenopus laevis genomic library and sequenced. The transcription start site of this gene was determined by 5' RACE method. Promoter activity was demonstrated by transient transfection experiments using luciferase reporter gene constructs in XTC cells. A number of putative recognition sites for transcription factors were found in the 5' flanking region of the TGF-beta 5 gene.

Amino Acid Sequence↗
PubMed1994

A pyruvate decarboxylase gene from Aspergillus parasiticus.

A gene encoding a putative pyruvate decarboxylase (EC 4.1.1.1) was isolated from a genomic library of the filamentous fungus Aspergillus parasiticus strain SU-1. The deduced amino acid sequence showed 37% homology to PDC1 from Saccharomyces cerevisiae. Although A. parasiticus has an obligate growth requirement for oxygen, it produced ethanol in shake flask cultures indicating a response to anoxic conditions mediated by pyruvate decarboxylase.

Amino Acid Sequence↗
PubMed1993

Genomic DNA structure of a gene encoding cytosolic ascorbate peroxidase from Arabidopsis thaliana.

A genomic DNA clone encoding cytosolic ascorbate peroxidase was isolated from a genomic library of Arabidopsis thaliana, using a cDNA for the enzyme as a probe. Nucleotide sequence and primer extension analyses of this gene (APX1) revealed nine exons split by eight introns, one of which is inserted in the 5'-untranslated region. The exon/intron organization of the APX1 gene differs from that of the guaiacol peroxidase genes.

Amino Acid Sequence↗
PubMed1991

Rabbit serum amyloid a gene: cloning, characterization and sequence analysis.

A genomic DNA clone that encodes serum amyloid A protein has been isolated from a rabbit liver lambda EMBL3 genomic library. Southern blot analysis indicates that the cloned gene is a member of multiple SAA gene family in rabbit. The nucleotide sequence of this DNA shows that the cloned gene corresponds to a SAA cDNA recently isolated from an acute phase rabbit liver cDNA library (1). This rabbit SAA gene contains four exons which are located within a 4500-base pair region spanning a putative TATA box and an AATAAA polyadenylation signal. DNA sequence upstream of the major transcription start site also contains other structural elements such as the sequences for NF-kappa B, IL-6 receptor and Sp1 binding. Significance of these regulatory elements in SAA gene expression is discussed.

Animals↗
PubMed1995

Isolation of novel human endogenous retrovirus-like elements with foamy virus-related pol sequence.

A new class of reverse transcriptase coding sequences was detected in reverse-transcribed RNAs from human placenta by polymerase chain amplification with primers in highly conserved regions of the pol gene of mammalian retroviruses and retrotransposons. Using one of these novel sequences as a probe to screen a human genomic library, we isolated retrovirus-like elements bordered by long terminal repeats and having a potential leucine tRNA primer-binding site. Determination of the complete nucleotide sequence (6,591 bp) of one of these elements, termed HERV-L (for human endogenous retrovirus with leucine tRNA primer), revealed domains of amino acid similarities to retroviral reverse transcriptase and integrase proteins. In addition, a region with homologies to dUTPase proteins was found unexpectedly downstream from the integrase domain. Amino acid sequence and phylogenetic analysis indicate that the HERV-L pol gene is related to that of foamy retroviruses. HERV-L-related sequences are detected in several mammalian species and have expanded in primate and mouse genomes up to 100 to 200 copies.

Amino Acid Sequence↗