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PubMed1998

Properties and function of the P type ion pumps cloned from Helicobacter pylori.

Three distinct P type pumps were cloned from H. pylori 69A. Two of these pumps, ATPase 439 and ATPase 948 (CopA), were isolated by gene library screening using DNA oligonucleotide primers. Amino acid similarities found for the predicted proteins were about 50% to Cd2+/Cu2+ pumps. Gene disruption mutagenesis rendered the H. pylori knockout mutants more sensitive to Zn2+ and Cd2+ (ATPase 439) or Cu2+ (CopA). Some of the ATPase 439-deficient mutants were negative for urease activity while the majority of the mutants remained positive. Functional diversity of the pumps was also reflected by the ion affinities found for N-terminal peptides of CopA to Cu2+ and of ATPase 439 to Ni2+, Cu2+ and CO2+. The membrane domain of the two pumps were experimentally shown to consist of eight membrane spans. When ATPase 439 was expressed under control of a tac promoter in Escherichia coli, vanadate-sensitive phosphate accumulation was observed cytochemically along the membrane of the host cells. The third P type pump (ATPase 115) which also exhibited homology to transition metal ATPase was identified by sequencing a library of H. pylori membrane genes. The hydropathy plot of this pump was very similar to the former H. pylori ATPases whereas the N-terminal ion binding region was distinct. It was concluded that, in H. pylori, the presence of three transition metal ATPases with distinct ion specificity contributes to the adaptive mechanisms for gastric survival.

Adenosine Triphosphatases↗

PubMed1995

NiaA, the structural nitrate reductase gene of Phytophthora infestans: isolation, characterization and expression analysis in Aspergillus nidulans.

The nitrate reductase (NR) gene niaA of the oomycete Phytophthora infestans was selected from a gene library by heterologous hybridization. NiaA occurs as a single-copy gene ant its expression is regulated by the nitrogen source. The nucleotide sequence of niaA was determined and comparison of the deduced amino-acid sequence of 902 residues with NRs of higher fungi and plants revealed a significant homology, particularly within the three cofactor-binding domains for molybdenum, heme and FAD. The P. infestans niaA gene was used as a model gene to test whether oomycete genes are functional in the ascomycete Aspergillus nidulans, a fungus which is highly accessible for molecular genetic studies. The complete niaA gene was stably integrated into the genome of a nia- deletion mutant of A. nidulans. However, transformants containing one or more copies of the niaA gene were not able to complement the nia- mutant. This suggests that there is no functional expression of the introduced niaA gene in A. nidulans. In addition, the activity of two other oomycete gene promoters was analyzed in a transient expression assay. Plasmids containing chimaeric genes with the promoter of the P. infestans ubiquitin gene ubi3R, or the Bremia lactucae ham34 gene, fused to the coding sequence of the Escherichia coli beta-glucuronidase (GUS) reporter gene, were transferred to A. nidulans protoplasts. No significant GUS activity was detectable indicating that the ubi3R and ham34 promoters are not active in A. nidulans. Apparently, the regulatory sequences which are sufficient for gene activation in oomycetes are not functional in the ascomycete A. nidulans.

Amino Acid Sequence↗

PubMed1987

Primary structure of human pancreatic secretory trypsin inhibitor (PSTI) gene.

The human pancreatic secretory trypsin inhibitor (PSTI) gene was isolated from a human gene library. Restriction endonuclease mapping and DNA sequencing analysis revealed that this gene is approximately 7.5 kb long and is separated into four exons by three introns. The gene has multiple transcription start points and examination with a single-laser cell-sorter showed that it is located on chromosome 5.

Base Sequence↗

PubMed1989

Structure of mouse 2',3'-cyclic-nucleotide 3'-phosphodiesterase gene.

The mouse 2',3'-cyclic-nucleotide 3'-phosphodiesterase gene was isolated from a mouse gene library. Restriction endonuclease mapping and DNA sequencing analysis revealed that this gene is about 6 kb long and is separated into three exons by two introns. The transcription initiation site was identified. The mouse cDNA of 2374 bp was obtained and used for the screening and analysis of the gene.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗

PubMed1989

Cloning of the nitrate reductase gene (niaD) of Aspergillus nidulans and its use for transformation of Fusarium oxysporum.

An heterologous transformation system for the phytopathogenic fungus Fusarium oxysporum has been developed based on the use of the Aspergillus nidulans nitrate reductase gene (niaD). F. oxysporum nia- mutants were easily selected by chlorate resistance. The A. nidulans niaD gene was isolated from a gene library by complementation of an A. nidulans niaD mutant. The cloned gene is capable of transforming F. oxysporum nia- mutants at a frequency of up to ten transformants per microgram of DNA. Southern analysis of the DNA of the F. oxysporum transformants showed that transformation resulted in integration of one or more copies of the vector DNA into the genome.

Aspergillus nidulans↗

PubMed1993

Sequences of tRNA-encoding genes and associated open reading frames of Streptomyces lividans.

A gene library of Streptomyces lividans has been screened for tRNA-encoding genes with labeled Streptomyces tRNA as a probe. By sequence analysis of hybridizing fragments, two single genes have been identified which code for tRNA(Asp) and tRNA(Gly). Associated with the tRNA(Gly) gene, there are three open reading frames (ORFs) which might code for gene products possibly involved in active transport processes through the bacterial membrane. The transcriptional organization of tRNAGly and the following ORFs was examined by high-resolution S1 mapping. A third clone carried a cluster of genes which encode two tRNA(Gln) and three tRNA(Glu). This cluster corresponds to a similar cluster previously described for Streptomyces rimosus [Plohl and Gamulin, Mol. Gen. Genet. 222 (1990) 129-134].

Amino Acid Sequence↗

PubMed2004

VH3 gene expression in children with HIV infection.

OBJECTIVE: To study immunoglobulin heavy chain variable region (V(H)) gene expression in HIV-uninfected (HIV-) and HIV-infected (HIV+) children. METHODS: A retrospective, observational study was performed by PCR-ELISA to examine IgM and IgG V(H) gene family expression among peripheral CD19-positive B cells. The subjects were 10 HIV+ children with, and 11 HIV+ children without a history of invasive pneumococcal disease (IPD) and 12 HIV- children. The ages of the HIV+ and HIV- children, and the CD4 lymphocyte counts and viral loads (VL) of the HIV+ /IPD- and HIV+ /IPD- children were statistically similar. Rearranged V(H)3 gene libraries were constructed from 3 HIV+ /IPD- children to examine individual gene expression. RESULTS: The proportion of IgG V(H)3 expression among HIV+ /IPD- was lower than that of HIV- children, but the proportion of IgG V(H)3 expression among HIV+ /IPD+ children was not significantly different from that of HIV- children. IgG V(H)3 expression was positively, whereas IgM V(H)3 expression was inversely correlated with the VL among HIV+ /IPD+, but not HIV+ /IPD- children. IgM V(H) gene family expression did not differ between HIV+ and HIV- children. CONCLUSION: HIV+ children manifest qualitative and quantitative differences in V(H)3 expression, which may be influenced by IPD.

Bacteremia↗

PubMed2002

Development of a transformation system for the flavinogenic yeast Candida famata.

Riboflavin-overproducing mutants of the flavinogenic yeast Candida famata are used for industrial riboflavin production. This paper describes the development of an efficient transformation system for this species. Leucine-deficient mutants have been isolated from C. famata VKM Y-9 wild-type strain. Among them leu2 mutants were identified by transformation to leucine prototrophy with plasmids YEp13 and PRpL2 carrying the Saccharomyces cerevisiae LEU2 gene. DNA fragments (called CfARSs) conferring increased transformation frequencies and extrachromosomal replication were isolated from a C. famata gene library constructed on the integrative vector containing the S. cerevisiae LEU2 gene as a selective marker. The smallest cloned fragment (CfARS16) has been sequenced. This one had high adenine plus thymine (A+T) base pair content and a sequence homologous to the S. cerevisiae ARS Consensus Sequence. Methods for spheroplast transformation and electrotransformation of the yeast C. famata were optimized. They conferred high transformation frequencies (up to 10(5) transformants per microg DNA) with a C. famata leu2 mutant using replicative plasmids containing the S. cerevisiae LEU2 gene as a selective marker. Riboflavin-deficient mutants were isolated from the C. famata leu2 strain and their biochemical identification was carried out. Using the developed transformation system, several C. famata genomic fragments complementing mutations of structural genes for riboflavin biosynthesis (coding for GTP cyclohydrolase, reductase, dihydroxybutanone phosphate synthase and riboflavin synthase, respectively) have been cloned.

Base Sequence↗

PubMed2002

Analysis of NLS and rRNA binding motifs in the L25 ribosomal protein from Leishmania (viannia) braziliensis: investigation of its diagnostic capabilities.

A cDNA clone codifying ribosomal protein L25 was isolated from a Leishmania braziliensis cDNA gene library. The alignment of the amino acid sequence deduced from this gene with other proteins revealed that this protein is related to the L23/25 rihosomal protein family. This is so because this protein shows, in its C-terminal end, the rRNA binding domains characteristic of these proteins and at the N-terminal end the NLS sequence necessary for its entry into the nucleus. Southern blot analysis showed 2 copies of gene L25 per genome arranged in tandem position and pointing in the same direction. Northern blot analysis showed that this gene is transcribed in 2 mRNAs when parasite promastigotes are in the logarithmic phase. In order to analyse the antigenic properties of L. braziliensis RPL25, it was purified as a recombinant protein and ELISA-tested against cutaneous, mucocutaneous and Chagasic sera. The results indicate that the recombinant RPL25 from L. braziliensis presents a non-specific reaction that disqualifies it for the diagnosis of cutaneous leishmaniasis. In contrast, some of the synthetic peptides derived from its sequence may serve as promising tools for the diagnosis of this disease.

Amino Acid Sequence↗

PubMed1981

Terminal direct repeats in a retrovirus-like repeated mouse gene family.

The mouse genome contains multiple copies of a dispersed gene family known individually as VL30 genes which are thought to be associated with retroviruses. The copies consist of closely related 5.2-kilobase DNA sequences flanked by unrelated cellular DNA, and although no sequence homology has been found between the genes and the exogenous or endogenous retroviruses so far tested, the 30S RNA transcripts expressed by the genes are efficiently packaged into virions, recovered from infected cells and transmitted to other cells by pseudo-type infection. Stimulated by recent reports of the similarity between retroviruses and transposons (from which retroviruses may have evolved), and in particular by the recognition that both types of genetic elements are characterized by a large terminal repeat (LTR), we set out to determine whether the VL30 genes are also distinguished by this property. Using cloned DNAs from a mouse gene library and heteroduplex analysis, we have now found that the VL30 genes do indeed carry terminal direct repeats 400 base pairs long.

Animals↗

PubMed2005

Achromobacter xylosoxidans endophthalmitis diagnosed by polymerase chain reaction and gene sequencing.

PURPOSE: To report a patient with Achromobacter xylosoxidansendophthalmitis that was diagnosed using polymerase chain reaction and gene sequencing. METHODS: Case report. A patient with culture-negative endophthalmitis underwent an anterior chamber tap. Polymerase chain reaction was performed on the aqueous sample using the 16S ribosomal DNA primer set to detect and amplify bacterial DNA. The amplified DNA was sequenced and compared to archived sequences in a gene library using the BLAST search program. RESULTS: A 214-base pair gene sequence was amplified and matched with the gene sequence for A. xylosoxidans. Antimicrobial treatment was instituted with resolution of hypopyon, anterior chamber cells, and vitreous cells one month after treatment. CONCLUSIONS: Polymerase chain reaction of 16S rDNA combined with gene sequencing may be an alternative method of diagnosing culture-negative bacterial endophthalmitis.

Achromobacter denitrificans↗

PubMed1983

Molecular cloning of the human gastrin gene.

A genomic clone that contains the human gastrin gene was isolated from a human gene library. Restriction endonuclease mapping and DNA sequencing analysis revealed that this gene is about 0.7 kb long, and has an intron. The intron is located at a position that separates the coding region into the peptide region essential for biological activities of gastrin and the non-essential, N-terminal peptide region.

Amino Acid Sequence↗

PubMed1995

Identification and properties of the largest subunit of the DNA-dependent RNA polymerase of fish lymphocystis disease virus: dramatic difference in the domain organization in the family Iridoviridae.

Cytoplasmic DNA viruses encode a DNA-dependent RNA polymerase (DdRP) that is essential for transcription of viral genes. The amino acid sequences of the known largest subunits of DdRPs from different species contain highly conserved regions. Oligonucleotide primers, deduced from two conserved domains (RQP[T/S]LH and NADFDGDE) were used for detecting the corresponding gene of fish lymphocystis disease virus (FLCDV), a member of the family Iridoviridae, which replicates in the cytoplasm of infected cells of flatfish. The gene coding for the largest subunit of the DdRP was identified using a PCR-derived probe. The screening of the complete EcoRI gene library of the viral genome led to the identification of the gene locus of the largest subunit of the DdRP within the EcoRI DNA fragment B (12.4 kbp, 0.034 to 0.165 map units). The nucleotide sequence of a part (8334 bp) of the EcoRI DNA fragment B was determined and a large ORF on the lower strand (ATG = 5787; TAA = 2190) was detected which encodes a protein of 1199 amino acids. Comparison of the amino acid sequences of the largest subunits of the DdRP (RPO1) of FLCDV and Chilo iridescent virus (CIV) revealed a dramatic difference in their domain organization. Unlike the 1051 aa RPO1 of CIV, which lacks the C-terminal domain conserved in eukaryotic, eubacterial and other viral RNA polymerases, the 1199 aa RPO1 of FLCDV is fully collinear with its cellular and viral homologues. Despite this difference, comparative analysis of the amino acid sequences of viral and cellular RNA polymerases suggests a common origin for the largest RNA polymerase subunits of FLCDV and CIV.

Amino Acid Sequence↗

PubMed1995

glmS of Thermus thermophilus HB8: an essential gene for cell-wall synthesis identified immediately upstream of the S-layer gene.

A 30 kbp chromosomal region containing the S-layer gene (slpA) from Thermus thermophilus HB8 was cloned from a lambda phage gene library. DNA sequence analysis of the region upstream to the slpA gene revealed the presence of an open reading frame (ORF) which coded for a 604-amino-acid protein highly homologous to the glucosamine-6-P synthases (EC 2.6.1.16) of both prokaryotic and eukaryotic origin. The identification of this ORF as the glucosamine-6-P synthase gene from T. thermophilus (glmSth) has been carried out using three different strategies: (i) complementation of an Escherichia coli glmS mutant; (ii) in vivo insertional inactivation of the gene; and (iii) in vitro synthesis of glucosamine-6-P at 60 degrees C by a cytoplasmic extract of an overproducing E. coli strain. The glmSth gene is transcribed divergently from slpA in a 2.0 kb mRNA which probably also includes a tryptophan tRNA gene (trpTth) identified at its 3' extreme. As the products of both the glmSth and the slpA genes are main components of the cell envelope of T. thermophilus, their unusual clustering in the chromosome could be related to the existence of specific mechanisms for their coordinate expression.

Amino Acid Sequence↗

PubMed2006

Protein kinase C (PkcA) of Aspergillus nidulans is involved in penicillin production.

The biosynthesis of the beta-lactam antibiotic penicillin in the filamentous fungus Aspergillus nidulans is catalyzed by three enzymes that are encoded by the acvA, ipnA, and aatA genes. A variety of cis-acting DNA elements and regulatory factors form a complex regulatory network controlling these beta-lactam biosynthesis genes. Regulators involved include the CCAAT-binding complex AnCF and AnBH1. AnBH1 acts as a repressor of the penicillin biosynthesis gene aatA. Until now, however, little information has been available on the signal transduction cascades leading to the transcription factors. Here we show that inhibition of protein kinase C (Pkc) activity in A. nidulans led to cytoplasmic localization of an AnBH1-enhanced green fluorescent protein (EGFP) fusion protein. Computer analysis of the genome and screening of an A. nidulans gene library revealed that the fungus possesses two putative Pkc-encoding genes, which we designated pkcA and pkcB. Only PkcA showed all the characteristic features of fungal Pkc's. Production of pkcA antisense RNA in A. nidulans led to reduced growth and conidiation in Aspergillus minimal medium, while in fermentation medium it led to enhanced expression of an aatAp-lacZ gene fusion, reduced pencillin production, and predominantly cytoplasmic localization of AnBH1. These data agree with the finding that inhibition of Pkc activity prevented nuclear localization of AnBH1-EGFP. As a result, repression of aatA expression was relieved. The involvement of Pkc in penicillin biosynthesis is also interesting in light of the fact that in the yeast Saccharomyces cerevisiae, Pkc plays a major role in maintaining cell integrity.

Amino Acid Sequence↗

PubMed1984

Cloning of nitrate reductase genes from the cyanobacterium Anacystis nidulans.

Anacystis nidulans, a non-nitrogen-fixing cyanobacterium, can fulfill its nitrogen requirement by the assimilation of nitrate. The first step in the pathway, the reduction of nitrate to nitrite, is catalyzed by the molybdo-protein nitrate reductase. In this study, newly developed techniques for gene cloning in A. nidulans R2 were used for the isolation of two genes involved in nitrate reduction. One gene was cloned by complementation of the corresponding mutant; the other gene was picked up from a cosmid gene library by using a restriction fragment containing the transposon-inactivated gene as a probe. Both genes were unlinked single-copy chromosomal genes. Transformation studies provided evidence for the existence of a third locus involved in nitrate reduction.

Cloning, Molecular↗

PubMed1989

Nostoc commune UTEX 584 gene expressing indole phosphate hydrolase activity in Escherichia coli.

A gene encoding an enzyme capable of hydrolyzing indole phosphate was isolated from a recombinant gene library of Nostoc commune UTEX 584 DNA in lambda gt10. The gene (designated iph) is located on a 2.9-kilobase EcoRI restriction fragment and is present in a single copy in the genome of N. commune UTEX 584. The iph gene was expressed when the purified 2.9-kilobase DNA fragment, free of any vector sequences, was added to a cell-free coupled transcription-translation system. A polypeptide with an Mr of 74,000 was synthesized when the iph gene or different iph-vector DNA templates were expressed in vitro. When carried by different multicopy plasmids and phagemids (pMP005, pBH6, pB8) the cyanobacterial iph gene conferred an Iph+ phenotype upon various strains of Escherichia coli, including a phoA mutant. Hydrolysis of 5-bromo-4-chloro-3-indolyl phosphate was detected in recombinant E. coli strains grown in phosphate-rich medium, and the activity persisted in assay buffers that contained phosphate. In contrast, indole phosphate hydrolase activity only developed in cells of N. commune UTEX 584, when they were partially depleted of phosphorus, and the activity associated with these cells was suppressed partially by the addition of phosphate to assay buffers. Indole phosphate hydrolase activity was detected in periplasmic extracts from E. coli (Iph+) transformants.

Cyanobacteria↗

PubMed1986

Immunoglobulin K chain genes in mouse hybridoma PTF-02 and parent myeloma. Insertion of hybridoma K gene into the plasmid pSV2-gpt.

The mouse myeloma line P3-X63-Ag8.653 currently used as the parent line for hybridoma construction contains only one (non-productive) gene for immunoglobulin K chains. The allelic gene is lost. In the mouse hybridoma PTF-02 two K genes can be found. One is identical with the gene of the myeloma parent line, the other originates in lymphocytes and is transcribed and translated in the K chain of the antibody secreted by the hybridoma. From the gene library of hybridoma PTF-02 in phage Charon 28 both K genes were isolated. Restriction endonuclease mapping and Southern blot hybridization demonstrated that the fragment comprising the lymphocyte gene was of the size (7.5 kb) sufficient to carry all exons, transcription and translation units. This gene was then recloned in the plasmid pBR322 and shuttle plasmid pSV2-gpt, which opens up possibilities for transfection of lymphoid cells and for study of the regulation of individual gene expression.

Animals↗