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At least 1,135 records · Page 63Linked to original sources
PubMed2006

ARFA: a program for annotating bacterial release factor genes, including prediction of programmed ribosomal frameshifting.

UNLABELLED: Correct annotation of genes encoding release factors in bacterial genomes is often complicated by utilization of +1 programmed ribosomal frameshifting during synthesis of release factor 2, RF2. In the absence of robust computational approaches for predicting ribosomal frameshifting, the success of proper annotation depends on annotators' familiarity with this phenomenon. Here we describe a novel computer tool that allows automatic discrimination of genes encoding class-I bacterial release factors, RF1, RF2 and RFH. Most usefully, this program identifies and automatically annotates +1 frameshifting in RF2 encoding genes. Comparison of ARFA performance with existing annotations of bacterial genomes revealed that only 20% of RF2 genes utilizing ribosomal frameshifting during their expression are annotated correctly. AVAILABILITY: The PHP based web interface of ARFA and the source code are located at http://recode.genetics.utah.edu/arfa

Base Sequence↗
PubMed2004

Characterization of two membrane-associated protease genes obtained from screening out-membrane protein genes of Flavobacterium columnare G4.

In order to identify genes encoding the outer membrane proteins (OMPs) of the myxobacter Flavobacterium columnare G(4), the expression library of the bacterium was screened by using rabbit antisera developed against its OMPs. Positive colonies of Escherichia coli M15 containing fragments encoding the bacterial OMPs were selected for cloning the relevant genes by genomic walking methods. Two genes encoding a membrane-associated zinc metalloprotease and prolyl oligopeptidase are reported in this paper. The membrane-associated zinc metalloprotease gene (map) is 1800 bp in length, coding for 449 amino acids (aa). Despite the presence of a conserved motif HEXXH for all metalloproteases, the special HEXXH approximately 32 aa approximately E motif of the F. columnare G(4) Map and its low level of identity with other reported zinc-containing metalloproteases may imply that the membrane-associated zinc metalloprotease of F. columnare G(4) represents a new family of zincins. The gene encoding prolyl oligopeptidase (Pop), a serine proteinase, is 2352 bp in length, coding for 649 aa. Sequence homology analysis revealed that the Pop is also novel as it has < 50% identity with other reported prolyl oligopeptidase family proteins. The present study represents the first to employ anti-fish bacterial OMP sera to screen genes of membrane-associated proteases of fish pathogenic bacteria, and to provide necessary information for the examination of the role of the two genes in the infection and pathogenesis of F. columnare.

Amino Acid Motifs↗
PubMed1995

Lactose-specific enzyme II of the phosphoenolpyruvate-dependent phosphotransferase system of Staphylococcus aureus. Purification of the histidine-tagged transmembrane component IICBLac and its hydrophilic IIB domain by metal-affinity chromatography, and functional characterization.

The lactose-specific integral-membrane-protein enzyme II (IICBLac) of the bacterial phosphoenolpyruvate-dependent phosphotransferase system of Staphylococcus aureus catalyses the uptake and phosphorylation of lactose. It consists of an N-terminal membrane-spanning IIC domain and a C-terminal hydrophilic IIB domain. IICBLac was fused with a C-terminal tag of six histidine residues using recombinant DNA technology. The resulting protein, IICBLac-His, was produced in Escherichia coli and purified under nondenaturing conditions to homogenity. The purification procedure consists of a NaOH extraction step followed by solubilisation with Triton X-100, and metal-affinity chromatography using Ni(2+)-nitrilotriacetic acid resin. The purified recombinant His-tagged protein possessed substrate specificity identical to that of the wild-type protein. To investigate the hydrophilic IIB domain, the DNA sequence coding for IIB and the His tag were fused in-frame to a DNA sequence specific for an initiation signal. The overproduced recombinant IIBLac-His was obtained by metal-affinity chromatography in pure form. Bacterial phosphotransferase-system-dependent phosphorylation of IIB-His was demonstrated in a photometric assay and by urea/polyacrylamide gel electrophoresis. The phosphorylation activity of the mutant protein [C476S]-IICBLac, containing the mutagenized phosphorylation site, was restored in the presence of IIBLac-His in a phosphorylation assay.

Base Sequence↗
PubMed1993

Identification of Rickettsia prowazekii using the polymerase chain reaction.

Polymerase chain reaction (PCR) was used to identify Rickettsia prowazekii, the etiologic agent of epidemic typhus. For the PCR, Thermus thermophilus thermostable DNA polymerase was applied with buffer containing a relatively low Mg2+ concentration (1.5-2 mM with dNTP's at 250 microM each). A primer pair used to amplify a 448-base-pair (bp) fragment of R. prowazekii genome was synthesized on the basis of the DNA sequence of gene rpa14/16, coding for a precursor of the mature polypeptides of molecular weight (Mr) 14,000 and/or 16,000 (16kD) from R. prowazekii strain E. For determining the specificity of the primer pair, purified genomic DNAs of 16 rickettsial and 10 other bacterial strains were used.

Animals↗
PubMed1999

An Lrp-like protein of the hyperthermophilic archaeon Sulfolobus solfataricus which binds to its own promoter.

Regulation of gene expression in the domain Archaea, and specifically hyperthermophiles, has been poorly investigated so far. Biochemical experiments and genome sequencing have shown that, despite the prokaryotic cell and genome organization, basal transcriptional elements of members of the domain Archaea (i.e., TATA box-like sequences, RNA polymerase, and transcription factors TBP, TFIIB, and TFIIS) are of the eukaryotic type. However, open reading frames potentially coding for bacterium-type transcription regulation factors have been recognized in different archaeal strains. This finding raises the question of how bacterial and eukaryotic elements interact in regulating gene expression in Archaea. We have identified a gene coding for a bacterium-type transcription factor in the hyperthermophilic archaeon Sulfolobus solfataricus. The protein, named Lrs14, contains a potential helix-turn-helix motif and is related to the Lrp-AsnC family of regulators of gene expression in the class Bacteria. We show that Lrs14, expressed in Escherichia coli, is a highly thermostable DNA-binding protein. Bandshift and DNase I footprint analyses show that Lrs14 specifically binds to multiple sequences in its own promoter and that the region of binding overlaps the TATA box, suggesting that, like the E. coli Lrp, Lrs14 is autoregulated. We also show that the lrs14 transcript is accumulated in the late growth stages of S. solfataricus.

Amino Acid Sequence↗
PubMed2001

DNA polymerase eta undergoes alternative splicing, protects against UV sensitivity and apoptosis, and suppresses Mre11-dependent recombination.

Polymerase eta (pol eta) is a low-fidelity DNA polymerase that is the product of the gene, POLH, associated with the human XP variant disorder in which there is an extremely high level of solar-induced skin carcinogenesis. The complete human genomic sequence spans about 40 kb containing 10 coding exons and a cDNA of 2.14 kb; exon I is untranslated and is 6 kb upstream from the first coding exon. Using bacterial artificial chromosomes (BACs), the gene was mapped to human chromosome band 6p21 and mouse band 17D. The gene is expressed in most tissues, except for very low or undetectable levels in peripheral lymphocytes, fetal spleen, and adult muscle; exon II, however, is frequently spliced out in normal cells and in almost half the transcripts in the testis and fetal liver. Expression of POLH in a multicopy episomal vector proved nonviable, suggesting that overexpression is toxic. Expression from chromosomally integrated linear copies using either an EF1-alpha or CMV promoter was functional, resulting in cell lines with low or high levels of pol eta protein, respectively. Point mutations in the center of the gene and in a C-terminal cysteine and deletion of exon II resulted in inactivation, but addition of a terminal 3 amino acid C-terminal tag, or an N- or C-terminal green fluorescent protein, had no effect on function. A low level of expression of pol eta eliminated hMre11 recombination and partially restored UV survival, but did not prevent UV-induced apoptosis, which required higher levels of expression. Polymerase eta is therefore involved in S-phase checkpoint and signal transduction pathways that lead to arrest in S, apoptosis, and recombination. In normal cells, the predominant mechanism of replication of UV damage involves pol eta-dependent bypass, and Mre11-dependent recombination that acts is a secondary, backup mechanism when cells are severely depleted of pol eta.

Alternative Splicing↗
PubMed1995

High-level bacterial expression of human glutathione transferase P1-1 encoded by semisynthetic DNA.

A cDNA clone, lambda GTHP1del, encoding glutathione transferase (GST) P1-1, was isolated from a human K562 erythroleukemia cell line cDNA library. The coding sequence was lacking the codons for the N-terminal 34 amino acids. A DNA segment was designed in order to obtain the missing portion and a structure representing the entire protein. The synthetic DNA sequence was constructed to achieve efficient base pairing with Escherichia coli 16S ribosomal RNA, avoidance of internal secondary structure, and optimal codon usage for high-level protein expression in accord with the known preferences in E. coli. The truncated GST P1-1 cDNA sequence and the synthetic segment were ligated into a plasmid to give an inducible expression system. Among the resulting clones a limited number was selected by immunodetection for highest yield of GST P1-1. Maximal expression was obtained from a spontaneously mutated sequence with altered as well as deleted bases as compared to the original construct. This clone, pKXHP1, allowed heterologous expression in E. coli in yields of > 200 mg enzyme per liter culture medium. The physicochemical and catalytic properties of the recombinant protein were indistinguishable from those of the enzyme purified from human placenta.

Base Sequence↗
PubMed1994

Yeast artificial chromosome cloning.

Yeast artificial chromosome (YAC) cloning systems enable the cloning of DNA stretches of 50 to well over 2000 kb. This makes it possible to study large intact regions of DNA in detail, by restriction mapping the YAC to produce a physical map and by examining the YAC for coding sequences or genes. YACs are important for their ability to clone the complete sequences of large genes or gene complexes that exceed the size limit for cloning in conventional bacterial cloning vectors like plasmids (up to 10 kb), bacteriophage (15 kb), and cosmids (50 kb). A major advantage of cloning in yeast, a eukaryote, is that many sequences that are unstable, underrepresented, or absent when cloned into prokaryotic systems, remain stable and intact in YAC clones. It is possible to reintroduce YACs intact into mammalian cells where the introduced mammalian genes are expressed and used to study the functions of genes in the context of flanking sequences. The correct protein processing mechanisms are present in the mammalian cells to ensure that a viable protein product is produced.

Animals↗
PubMed1993

RPII15 codes for the M(r) 15,000 subunit 9 of Drosophila melanogaster RNA polymerase II.

The RPII15 gene product of Drosophila melanogaster, which has recently been identified by sequence comparison, possesses a high similarity to subunit 9 of yeast RNA polymerase II. Using the polymerase chain reaction the coding region of RPII15 was isolated from genomic DNA of adult flies. Sequence analysis shows four amino acid substitutions in comparison to the previously reported sequence. Antisera were generated against bacterially expressed RPII15 and were used for immunoblotting experiments with RNA polymerase II of Drosophila melanogaster. This analysis identified the M(r) 15,000 subunit 9 as gene product of RPII15.

Amino Acid Sequence↗
PubMed1982

Transcription of the bacterial beta-lactamase gene in Saccharomyces cerevisiae.

We have examined the transcription in yeast of Escherichia coli-yeast 2-micrometers DNA recombinant plasmids carrying the bacterial beta-lactamase (bla) gene. In Saccharomyces cerevisiae both strands of the gene are transcribed giving multiple RNA species of distinct lengths. At least one RNA transcript derived from the coding strand initiates at a yeast promoter on the 2-micrometers DNA segment. Another mRNA of 1.1 kb starts right in front of the gene on the bacterial DNA sequence. Deletion experiments have shown that expression of the bacterial bla gene is dependent on the presence of bacterial sequences right in front of the gene. Mutants lacking the bacterial promoter region do not give detectable gene products in yeast. The expression can be restored by substituting for the deleted sequence a DNA fragment which carries the E. coli lac promoter-operator region. We conclude that the bacterial promoter region of the bla gene as well as the lac promoter-operator fragment have promoter activity in yeast and that yeast-bla fusion transcripts cannot be used as a functional messenger for beta-lactamase in yeast.

Chromosome Mapping↗
PubMed1996

Structural feature of the initiator tRNA gene from Pyrodictium occultum and the thermal stability of its gene product, tRNA(imet).

Pyrodictium occultum is a hyperthermophilic archaeum that grows optimally at 105 degrees C. To study how tRNA molecules in P occulrum are thermally stabilized, we isolated the initiator tRNA gene from the organism using a synthetic DNA probe of 74 bp containing the known nucleotide sequences that are conserved in archaeal initiator tRNAs. A HindIII fragment of 700 bp containing the Pyrodictium initiator tRNA gene was cloned and sequenced by cycle sequencing. The nucleotide sequence revealed that the Pyrodictium initiator tRNA gene has no introns, and that the 3'CCA terminus is encoded. The tRNA gene also contained a unique TATA-like sequence, AAGCTTATAA, which is likely the promoter proposed for archaeal rRNA genes, 450 bp upstream of the 5' end of the tRNA coding region. In the region adjacent to the 3' end of the tRNA coding region, there was a sig G-C base pair inverted repeat followed by a C-rich sequence like the p-independent transcription termination signal of bacterial genes. The Pyrodictium initiator tRNA sequence predicted from the gene sequence contained all of the nucleotide residues A1, A37, U54, A57, U60, and U72, in addition to three G-C base pairs in the anticodon stem region, which are characteristic of archaeal initiator tRNAs. The melting temperature (Tm) of the unmodified initiator tRNA synthesized in vitro using the cloned tRNA gene as a template was 80 degrees C, which is only two degrees lower than that calculated from the G-C content in the stem regions of the tRNA. In contrast, the Tm of the natural initiator tRNA isolated from P occultum was over 100 degrees C. Analysis of digests of purified Pyrodictium initiator tRNA by means of HPLC-mass spectrometry and [32P] post-labeling, indicated that the tRNA contains a variety of modified nucleosides. These results suggest that the extraordinarily high melting temperature of P occultum tRNA(Met)i is due to posttranscriptional modification.

Archaea↗
PubMed1992

Expression cloning of a Na(+)-independent neutral amino acid transporter from rat kidney.

Uptake of long-chain and aromatic neutral amino acids into cells is known to be catalyzed by the Na(+)-independent system L transporter, which is ubiquitous in animal cells and tissues. We have used a Xenopus oocyte expression system to clone the cDNA of a system L transporter from a rat kidney cDNA library. The 2.3-kilobase cDNA codes for a protein of 683 amino acids. The transporter has four putative membrane-spanning domains and bears no sequence or structural homology to any known animal or bacterial transporter. When transcribed and expressed in Xenopus oocytes, the transporter exhibits many, but not all, of the characteristics of L-system transporters, suggesting that this represents one of several related L-system transporters.

Amino Acid Sequence↗