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PubMed1997

Placenta growth factor: identification and characterization of a novel isoform generated by RNA alternative splicing.

We report the isolation and characterization of a third isoform of placenta growth factor (PlGF), generated by alternative splicing of its RNA transcript. This novel form of PlGF, PlGF-3, was cloned by the polymerase chain reaction technique using a human cDNA library prepared from the terminal placental tissue. PlGF-3 contains an in-frame insertion of 72-amino acids near the C-terminal portion of PlGF-1. Southern blot analysis revealed that a single gene encoded PlGF-2 and PlGF-3. Nucleic acid sequence analysis found the insertion of 216 nucleotides of PlGF-3 between exon 4 and exon 5 of the PlGF gene. Northern blot and tissue distribution studies discovered two mRNA species for PlGF-3, which were both uniquely expressed in the placenta. Transient expression of PlGF-3 cDNA in mammalian cells showed PlGF-3 was detected in the conditioned medium as both dimers and monomers. Unlike PlGF-2, PlGF-3 lacked heparin-binding affinity. Thus, alternative splicing of PlGF RNA produces at least three polypeptides with different secretion pattern, heparin-binding affinity and dimerization properties.

Alternative Splicing↗
PubMed1990

Identification and characterisation of a Leishmania donovani antigen belonging to the 70-kDa heat-shock protein family.

A Leishmania donovani promastigote cDNA library was screened with serum obtained from a patient infected with visceral leishmaniasis. Sequence analysis of a clone obtained from this library revealed that the 600-bp insert corresponded to the carboxy-terminal region of an antigen related to the 70-kDa heat-shock protein family. The full-length sequence of the corresponding gene (1959 nucleotides) was determined after isolation of genomic clones. Genes encoding the antigen are present on a single chromosome as a series of approximately twelve 3.7-kb direct tandem repeats. The antigen can be identified as a 70-kDa heat-shock cognate protein by virtue of its molecular mass, sequence and constitutive expression during heat shock. It is expressed at all stages of the parasite life-cycle. Antibodies against the lambda gt11 fusion protein were detected in more than 50% of serum samples obtained from patients with visceral leishmaniasis, but were not detected in sera from patients with cutaneous leishmaniasis or Chagas' disease.

Amino Acid Sequence↗
PubMed1991

Tumorigenesis in transgenic mice: identification and characterization of synergizing oncogenes.

Transgenic mice carrying oncogenes present a useful model with which to assess the tissue-specific action of oncogenes. These mice are usually predisposed to a specific type of neoplastic growth. The tumors that arise are usually monoclonal in origin and become only apparent after a variable latency period, suggesting that additional events are required for tumor formation. Identification of these additional events is highly relevant: it might give access to the genes that can synergize with a preselected oncogene in tumorigenesis and could facilitate the identification of the biochemical pathways in which these genes act. Retroviruses can be instrumental in identifying cooperating oncogenes. Proto-oncogene activation or tumor suppressor gene inactivation by insertional mutagenesis is an important mechanism by which the non-acute transforming retroviruses can induce tumors in several species. Owing to the sequence tag provided by the provirus, the relevant proto-oncogene can be directly identified by cloning of the DNA flanking the proviral insertion site. We have exploited this potential of retroviruses by infecting E mu-pim-1 and E mu-myc transgenic mice, which are predisposed to lymphomagenesis, with Moloney murine leukemia virus (MuLV). A strong acceleration of tumor induction ensued upon infection of these mice with MuLV. More importantly, it allowed us to identify a number of additional common insertion sites marking both previously known as well as new (putative) oncogenes. In a significant portion of the tumors more than one oncogene was found to be activated, indicating that within this system the synergistic effect of at least three genes can be established.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

MotY, a component of the sodium-type flagellar motor.

Energy to power the rotation of bacterial flagella can be derived from the proton or sodium transmembrane potential. Until now, genes encoding a bacterial sodium-type flagellar motor have not been defined. A gene, motY, encoding one component of the sodium-type flagellar motor of Vibrio parahaemolyticus was cloned by complementation of a Mot- mutant strain. Sequencing revealed an open reading frame of 879 nucleotides in which a transposon conferring a motility defect mapped. Overexpression of motY in Escherichia coli allowed identification of a product 33 kDa in apparent size on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This size correlated well with the predicted molecular mass of 33,385 Da. Unlike mot genes identified in other bacteria, localized transposon mutagenesis suggested that the locus was not an extended region containing multiple genes required for swimming motility. Sequencing upstream and downstream of motY confirmed that the gene maps alone and placed it within a locus homologous to the E. coli rnt locus. Although data bank searches failed to reveal significant similarity to known motility components, the carboxyl terminus of MotY showed extensive homology to a number of outer membrane proteins known to interact with peptidoglycan, including OmpA and peptidoglycan-associated lipoproteins. To a limited extent, this domain could also be identified in the Bacillus subtilis MotB protein. This finding suggests that MotY plays the role of a stator in the sodium flagellar motor, stabilizing the force-generating unit through direct interaction with the cell wall.

Amino Acid Sequence↗
PubMed2003

Identification and characterization of the carAB genes responsible for encoding carbamoylphosphate synthetase in Halomonas eurihalina.

Halomonas eurihalina is a moderately halophilic bacterium which produces exopolysaccharides potentially of great use in many fields of industry and ecology. Strain F2-7 of H. eurihalina synthesizes an anionic exopolysaccharide known as polymer V2-7, which not only has emulsifying activity but also becomes viscous under acidic conditions, and therefore we consider it worthwhile making a detailed study of the genetics of this strain. By insertional mutagenesis using the mini-Tn 5 Km2 transposon we isolated and characterized a mutant strain, S36 K, which requires both arginine and uracil for growth and does not excrete EPS. S36 K carries a mutation within the carB gene that encodes the synthesis of the large subunit of the carbamoylphosphate synthetase enzyme, which in turn catalyzes the synthesis of carbamoylphosphate, an important precursor of arginine and pyrimidines. We describe here the cloning and characterization of the carAB genes, which encode carbamoylphosphate synthetase in Halomonas eurihalina, and discuss this enzyme's possible role in the pathways for the synthesis of exopolysaccharides in strain F2-7.

Amino Acid Sequence↗
PubMed1995

Sequence-based identification of T-DNA insertion mutations in Arabidopsis: actin mutants act2-1 and act4-1.

A method is presented to facilitate the isolation of mutations in plant genes, which requires knowledge of the target gene or protein sequence, and is independent of mutant phenotype. The polymerase chain reaction was used to amplify the junctions between a T-DNA insert and the gene of interest from pools of mutant plant lines. The approach was used to identify mutations in Arabidopsis thaliana actin genes. The Arabidopsis genome encodes 10 actins in six ancient subclasses each with distinct expression patterns. Primers in the T-DNA border and highly degenerate actin primers, designed from conserved amino acid motifs, were used to prime the amplification. The PCR products were transferred to filters and probed for actin at low stringency. Thus, mutations in all 10 actin genes were screened for simultaneously. Mutations in the vegetative constitutive actin gene, ACT2, and the pollen-specific actin gene, ACT4, were identified in a population of 5300 lines containing approximately 1.5 T-DNA insertions per line. The screen was sensitive enough that actin insertion alleles were easily distinguished among pools of 100 plant lines. PCR techniques were used which accelerated the purification of mutant lines, and segregation, physical mapping, and sequencing of the act2-1 and act4-1 mutations. This strategy should be generally useful in screening mutant libraries made with a variety of plant insertion elements for mutations in any known sequence.

Actins↗
PubMed1995

Identification of novel insertion elements, restriction fragment length polymorphism patterns, and discontinuous 23S rRNA in Lyme disease spirochetes: phylogenetic analyses of rRNA genes and their intergenic spacers in Borrelia japonica sp. nov. and genomic group 21038 (Borrelia andersonii sp. nov.) isolates.

Borrelia spp. associated with Lyme disease possess an rRNA gene organization consisting of a single 16S rRNA gene followed by a spacer of several kilobases and a tandem repeat of a 23S (rrl)-5S (rrf) rRNA gene cluster. The restriction fragment length polymorphism (RFLP) patterns for these genes have been widely used to classify Lyme disease spirochete isolates. We analyzed the rRNA gene organization and sequences for two Ixodes ovatus isolates from Japan (IKA2 and HO14) and two group 21038 isolates associated with Ixodes dentatus ticks or rabbits from North America (isolates 21038 and 19857). This analysis revealed unique polymorphisms not previously described in other Lyme disease spirochete isolates. The molecular basis of these polymorphisms was determined by Southern blotting and PCR analyses. Only one continuous copy of the rrl-rrf gene cluster was identified in isolates IKA2, 19857, and 21038. The second rrl-rrf gene cluster is entirely absent from the IKA2 genome. In isolates 19857 and 21038, an intervening sequence is present, resulting in a fragment rrlB gene. The insertion site of this intervening sequence element differed in each isolate. While isolates 19857 and 21038 were found to carry a fragmented rrlB gene, they lacked rrfB. To determine if these rRNA polymorphisms were indicative of an underlying phylogenetic divergence, sequence analysis of the 16S rRNA (rrs) genes was conducted. The phylogenies inferred from rrs sequence analysis suggest that the polymorphisms resulted from recent mutational events. In addition, the phylogenetic analyses also support the proposed species status of Borrelia japonica sp. nov. and indicate that isolates of genomic group 21038 belong to a previously undescribed species for which we propose the nomenclature Borrelia andersonii sp. nov.

Base Sequence↗
PubMed2005

Occurrence of insertion sequences within the genomes and Tn1546-like elements of glycopeptide-resistant enterococci isolated in Brazil, and identification of a novel element, ISEfa5.

Insertion sequences (IS) occur widely within the Tn1546-like elements responsible for VanA glycopeptide resistance in enterococci from several countries. As such insertions can be used as epidemiological markers and for studying horizontal transfer of gene clusters, we investigated the distribution of IS6770, IS1542, IS1216V, IS1476, and IS1251 elements in 26 VanA Enterococcus faecium and 21 VanA Enterococcus faecalis from Brazil. PCR, using genomic DNA as a template, indicated that most of the isolates contained IS6770 (97%), IS1216V (87%) and IS1476 (72%) elements. IS1251 was also detected, but at a higher frequency in E. faecium (80%) than in E. faecalis (14%). None of the isolates harboured IS1542. Only two of 47 isolates had IS elements within their Tn1546-like elements; one possessed IS1251 between vanS and vanH, as reported in the United States; another possessed a novel IS element, designated ISEfa5, located between vanX and van Y. This novel element was found in the genomic DNA of 25 (96%) E. faecium and II (52%) E. faecalis. In stability studies, no IS-mediated changes were detected in the Tn1546-like elements of 25 vancomycin-resistant enterococci (VRE) monitored over 11 months. These results suggest that the occurrence of IS in Brazilian isolates is similar to that reported in American isolates, but that these elements occur rarely within the vanA gene clusters. As patterns of IS carriage did not correlate with the PFGE type of the VRE, the prevalence of IS elements in genomic DNA of VRE is not a useful epidemiological marker. However, the presence of IS-modified Tn1546-like elements, which appear to be rare in Brazil, could be a useful molecular marker in local epidemiological studies to monitor the evolution and horizontal transmission of VanA elements.

Anti-Bacterial Agents↗
PubMed1997

Large scale identification of genes involved in cell surface biosynthesis and architecture in Saccharomyces cerevisiae.

The sequenced yeast genome offers a unique resource for the analysis of eukaryotic cell function and enables genome-wide screens for genes involved in cellular processes. We have identified genes involved in cell surface assembly by screening transposon-mutagenized cells for altered sensitivity to calcofluor white, followed by supplementary screens to further characterize mutant phenotypes. The mutated genes were directly retrieved from genomic DNA and then matched uniquely to a gene in the yeast genome database. Eighty-two genes with apparent perturbation of the cell surface were identified, with mutations in 65 of them displaying at least one further cell surface phenotype in addition to their modified sensitivity to calcofluor. Fifty of these genes were previously known, 17 encoded proteins whose function could be anticipated through sequence homology or previously recognized phenotypes and 15 genes had no previously known phenotype.

Cell Membrane↗
PubMed1990

Transposon Tn917lacZ mutagenesis of Bacillus subtilis: identification of two new loci required for motility and chemotaxis.

We have used Tn917lacZ to mutagenize the Bacillus subtilis chromosome and have isolated mutants that are defective in chemotaxis and motility. Mapping of the transposon inserts identified two new loci. Mutations in one of these loci generated mutants that had paralyzed flagella. Accordingly, we designate this a mot locus. The other locus is closely linked to the first and encodes proteins specifying chemotaxis functions. This locus is designated the cheX locus. Both the mot and cheX loci map close to ptsI. An additional transposon insert that maps in the hag locus was obtained. The pattern of beta-galactosidase expression from some of the transposons suggested that the mot locus is regulated by sigD, a minor sigma factor of B. subtilis. The cheX locus appeared to be under the control of vegetative sigA. Four transposon inserts were mapped to a previously characterized che locus near spcB. These mutants did not produce flagellin and were defective in the methylation of the methyl-accepting chemotaxis proteins. This locus probably encodes proteins required for flagellum biosynthesis and other proteins that are required for the methylation response.

Bacillus subtilis↗
PubMed1984

Identification of sites required for repression of a silent mating type locus in yeast.

There are three loci in the yeast Saccharomyces, each containing one of two possible genetic elements that can determine cell type. At one of these loci, MAT, this information is expressed to establish the mating type of the cell. At the other two loci, HML and HMR, this same information is phenotypically and transcriptionally silent, even though a large amount of identical sequence flanks MAT, HML and HMR coding regions. Transcriptional repression of HML and HMR requires the trans active gene products of four loci, designated variously as MAR or SIR, that are unlinked to each other or to MAT, HML or HMR. We have examined the phenotypic expression of a cloned, plasmid-borne copy of HML and of various deletion and insertion derivatives of this plasmid following their reintroduction into Mar+/Sir+ yeast strains. From these data, we have identified two sites flanking the locus, both of which are required for MAR/SIR repression of the locus. In addition, we demonstrate that each of these sites promotes autonomous replication in yeast. Abraham et al. (1984) have presented evidence demonstrating that a similar regulatory structure exists at the other silent locus, HMR. From an analysis of the sequences of these four regulatory sites, we have identified several specific sequences that may be involved in mediating repression of these loci and in promoting replication in yeast. These results are discussed in the context of potential models for the mechanism of regulation of the silent mating type loci.

Base Sequence↗
PubMed2006

Identification of transposons, retroelements, and a gene family predominantly expressed in floral tissues in chromosome 3DS of the hexaploid wheat progenitor Aegilops tauschii.

A multigene family expressed during early floral development was identified on the short arm of wheat chromosome 3D in the region of the Ph2 locus, a locus controlling homoeologous chromosome pairing in allohexaploid wheat. Physical, genetic and molecular characterisation of the Wheat Meiosis 1 (WM1) gene family identified seven members that localised within a region of 173-kb. WM1 gene family members were sequenced and they encode mainly type Ia plasma membrane-anchored leucine rich repeat-like receptor proteins. In situ expression profiling suggests the gene family is predominantly expressed in floral tissue. In addition to the WM1 gene family, a number of other genes, gene fragments and pseudogenes were identified. It has been predicted that there is approximately one gene every 19-kb and that this region of the wheat genome contains 23 repetitive elements including BARE-1 and Wis2-1 like sequences. Nearly 50% of the repetitive elements identified were similar to known transposons from the CACTA superfamily. Ty1-copia, Ty3-gypsy and Athila LTR retroelements were also prevalent within the region. The WM1 gene cluster is present on 3DS and on barley 3HS but missing from the A and B genomes of hexaploid wheat. This suggests either recent generation of the cluster or specific deletion of the cluster during wheat polyploidisation. The evolutionary significance of the cluster, its possible roles in disease response or floral and early meiotic development and its location at or near the Ph2 locus are discussed.

DNA Transposable Elements↗
PubMed2003

Comparison of polymerase chain reaction and bacteriological culture for the diagnosis of sheep brucellosis using aborted fetus samples.

PCR assay has been shown to be a promising option for the diagnosis of brucellosis. However, few studies have been performed with field samples in order to evaluate the assay as a diagnostic tool. In this study, routine use of a species-specific PCR assay previously developed for the identification of Brucella cultures was assessed for the detection of Brucella DNA directly from the stomach contents of aborted sheep fetuses. The assay is based on the insertion sequence IS711 in the Brucella chromosome. In the study, during 3 successive lambing seasons (1998-1999, 1999-2000 and 2000-2001) 126 aborted fetus samples each from different flocks and locations were examined. Brucella strains were isolated from 39 (31%) of the samples and all of the strains were identified as Brucella melitensis by biochemical characteristics, agglutination with monospecific A and M sera and PCR. Thirty-seven of 39 B. melitensis isolates were biotyped as biotype 3, and 2 isolates as biotype 1. From 38 of 39 culture positive fetal stomach contents B. melitensis-specific DNA was detected by PCR. PCR was found negative in all of the culture negative samples. Compared with culture, sensitivity and specificity of PCR were determined as 97.4 and 100%, respectively. The results indicate that this PCR procedure has a potential for use in routine diagnosis of sheep brucellosis.

Aborted Fetus↗
PubMed2004

Insertion of green fluorescent protein into nonstructural protein 5A allows direct visualization of functional hepatitis C virus replication complexes.

Hepatitis C virus (HCV) replicates its genome in a membrane-associated replication complex, composed of viral proteins, replicating RNA and altered cellular membranes. We describe here HCV replicons that allow the direct visualization of functional HCV replication complexes. Viable replicons selected from a library of Tn7-mediated random insertions in the coding sequence of nonstructural protein 5A (NS5A) allowed the identification of two sites near the NS5A C terminus that tolerated insertion of heterologous sequences. Replicons encoding green fluorescent protein (GFP) at these locations were only moderately impaired for HCV RNA replication. Expression of the NS5A-GFP fusion protein could be demonstrated by immunoblot, indicating that the GFP was retained during RNA replication and did not interfere with HCV polyprotein processing. More importantly, expression levels were robust enough to allow direct visualization of the fusion protein by fluorescence microscopy. NS5A-GFP appeared as brightly fluorescing dot-like structures in the cytoplasm. By confocal laser scanning microscopy, NS5A-GFP colocalized with other HCV nonstructural proteins and nascent viral RNA, indicating that the dot-like structures, identified as membranous webs by electron microscopy, represent functional HCV replication complexes. These findings reveal an unexpected flexibility of the C-terminal domain of NS5A and provide tools for studying the formation and turnover of HCV replication complexes in living cells.

Amino Acid Sequence↗
PubMed1991

Identification of posttranscriptionally active inhibitory sequences in human immunodeficiency virus type 1 RNA: novel level of gene regulation.

cis-acting inhibitory region (IR) sequences were identified within the gag/pol gene of the human immunodeficiency virus type 1 (HIV-1) by using a novel feedback-stimulated, rev-independent tat reporter gene to screen HIV-1 sequences in transient expression assays. Two regions, a 1,295-nucleotide segment in the gag gene (IR-1) and a 1,932-nucleotide segment of the pol gene (IR-2), each inhibited reporter gene expression 10- to 20-fold. IR-1 and IR-2 both contained subsequences which inhibited reporter gene expression. Introduction of IR sequences into a heterologous reporter plasmid, pCMV-CAT, resulted in decreased chloramphenicol acetyltransferase expression, suggesting that the inhibitory effect was not restricted to a reporter gene under the control of the HIV-1 promoter. The presence of HIV IR sequences in cis did not alter relative levels of reporter gene RNA; however, fractionation studies revealed IR-containing RNA accumulated in the nucleus. These findings demonstrate that IR sequences within the gag/pol region affect gene expression by altering the cellular distribution of viral RNA.

Base Sequence↗
PubMed2004

Comparative evaluation of PCR assays for the robust molecular detection of Mycobacterium avium subsp. paratuberculosis.

Mycobacterium avium subsp. paratuberculosis (MAP) can cause a very serious, often-fatal disease, namely paratuberculosis, in several animal species, especially ruminants. Recently, it has also been implicated in the pathogenesis of Infectious Bowel Disease of man. The aim of this study was to develop a molecular method for the routine detection and identification of MAP, from tissue samples of animal origin. The proposed assay would have to combine optimum performance and cost, with high reproducibility. To this goal, three laboratories in Greece and the Czech Republic undertook different parts of a study that involved evaluation of DNA extraction procedures, and PCR assays, for MAP detection. For DNA extraction we used one in-house, and one commercial method, and for the PCR we assessed a number of different assays, starting with the evaluation of primer specificity with an extended GenBank database search. Based on these results, we chose to assess a one-tube nested, 2 two-tube nested, and a single PCR assay, targeted to different genomic regions of the IS900 element. These four methods were applied on positive and negative control samples, consisted of pure bacterial cultures and formalin-fixed paraffin-embedded (FFPE) tissue samples collected from cattle with paratuberculosis and chickens with M. avium subsp. avium infection. Based on the criteria of reliability and cost, the procedure that performed better was the one-tube nested PCR assay combined with the in-house DNA extraction method. The agreement of the results obtained by the three collaborating laboratories indicates the reliability of the proposed assay even under different laboratory conditions.

Animals↗
PubMed1999

Multiple lysophosphatidic acid acyltransferases in Neisseria meningitidis.

Lysophosphatidic acid (LPA) and phosphatidic acid (PA) are critical phospholipid intermediates in the biosynthesis of cell membranes. In Escherichia coli, LPA acyltransferase (1-acyl-sn-glycerol-3-phosphate acyltransferase; EC 2.3.1.51) catalyses the transfer of an acyl chain from either acyl-coenzyme A or acyl-acyl carrier protein onto LPA to produce PA. While E. coli possesses one essential LPA acyltransferase (PlsC), Neisseria meningitidis possesses at least two LPA acyltransferases. This study describes the identification and characterization of nlaB (neisserial LPA acyltransferase B), the second LPA acyltransferase identified in N. meningitidis. The gene was located downstream of the Tn916 insertion in N. meningitidis mutant 469 and differed in nucleotide and predicted amino acid sequence from the previously characterized neisserial LPA acyltransferase homologue nlaA. NlaB has specific LPA acyltransferase activity, as demonstrated by complementation of an E. coli plsC(Ts) mutant in trans, by decreased levels of LPA acyltransferase activity in nlaB mutants and by lack of complementation of E. coli plsB26,X50, a mutant defective in the first acyltransferase step in phospholipid biosynthesis. Meningococcal nlaA mutants accumulated LPA and demonstrated alterations in membrane phospholipid composition, yet retained LPA acyltransferase activity. In contrast, meningococcal nlaB mutants exhibited decreased LPA acyltransferase activity, but did not accumulate LPA or display any other observable membrane changes. We propose that N. meningitidis possesses at least two LPA acyltransferases to provide for the production of a greater diversity of membrane phospholipids.

1-Acylglycerol-3-Phosphate O-Acyltransferase↗
PubMed1987

Identification of a gene that regulates expression of M protein, the major virulence determinant of group A streptococci.

By using Tn916 insertional mutagenesis, we have identified mry (M protein RNA yield), a gene required for high-level expression of the M protein, an essential virulence determinant of the group A streptococcus. The mry::Tn916 mutation causes a reduction by a factor of approximately equal to 50 in the amount of M protein produced, in the original strain and in a nonmutagenized host to which the mutation was transferred by transduction. The insertion is located approximately 1.8 kilobases upstream of the structural gene for M protein (emm) and its promoter. The genomic region including mry::Tn916 and emm was cloned on a cosmid vector and introduced into Escherichia coli. When the transposon excised precisely from the chimeric cosmid in E. coli, the resulting streptococcal DNA showed the same restriction pattern as the homologous chromosomal region in the parental nonmutagenized streptococcus. The sequence of the promoter for emm was not altered in the mry mutant. The reduction in protein correlates with a decrease in the amount of M protein-specific mRNA, indicating that mry regulates transcription of emm.

Antigens, Bacterial↗