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PubMed1994

Direct and rapid detection of Erysipelothrix rhusiopathiae DNA in animals by PCR.

Erysipelothrix rhusiopathiae is a gram-positive rod capable of causing erysipelas in swine. To establish a method for specifically detecting E. rhusiopathiae for practical applications, such as for the inspection of slaughterhouses, the feasibility of using primers derived from the DNA sequence coding for 16S rRNA in a PCR-specific detection system was investigated. Oligonucleotide primers were designed to amplify a 407-bp DNA fragment by PCR. The amplification was specific to the Erysipelothrix DNA but not to that of other bacterial genera tested. This PCR-based method efficiently and specifically detected the Erysipelothrix DNA sequence in joint and spleen samples from mice within 6 h, and application of the 407-bp DNA segment from samples containing very low numbers of bacteria (< 20 bacteria per spleen from mice) was possible. Although this PCR amplification is specific for the Erysipelothrix genus, which contains at least two species, E. rhusiopathiae and E. tonsillarum, it can be concluded that all Erysipelothrix strains detected by this PCR system in diseased pigs are E. rhusiopathiae because only E. rhusiopathiae is virulent for pigs. These results show that this PCR amplification system using the DNA sequence coding for 16S rRNA is very rapid and reliable and avoids cumbersome and lengthy cultivation steps, demonstrating that this system could be used for practical applications.

Animals↗
PubMed1992

Primary structure of a chitinase-encoding gene (chi1) from the filamentous fungus Aphanocladium album: similarity to bacterial chitinases.

Chitinase 1 (Chi1) is the major extracellular chitinase from the hyperparasitic fungus, Aphanocladium album. We determined the complete sequence of the chromosomal and cDNA copies of the structural gene (chi1) coding for Chi1. The coding region is interrupted by three short introns (55, 53 and 49 bp long). Chi1 is 423 aa long and begins with a stretch of 34 aa not found in the mature protein. The Chi1 sequence presents overall similarities with bacterial chitinases from Serratia marcescens and Bacillus circulans. Compared with other chitinases, A. album Chi1 has only two short similarity regions (12 and 8 aa long), which are also found in bacterial, yeast and some plant chitinases.

Amino Acid Sequence↗
PubMed2002

Characterization of the porcine melanocortin 2 receptor gene (MC2R ).

A porcine bacterial artificial chromosome (BAC) clone, containing the melanocortin 2 receptor gene (MC2R) was isolated. The complete coding sequence of the MC2R gene, contained in 1 exon, was determined. Polymerase chain reaction-single stranded conformational polymorphism (PCR-SSCP) was performed on a 241-bp coding fragment. An AluI polymorphism, detecting a silent mutation, was found and typed on unrelated animals of five different pig breeds. The Meishan, Piétrain and Large White breeds differ significantly in allele frequencies from the Landrace and Czech Meat Pig breeds. The melanocortin 5 receptor gene (MC5R) was detected by PCR in the same BAC clone, as could be expected from the human and porcine mapping data. PCR-SSCP was performed on a 200-bp coding of MC5R, but no polymorphisms were detected. The BAC clone was mapped to Sscr6q27 by fluorescent in situ hybridization (FISH). A (CA)n microsatellite (SGU0002), isolated from the BAC, was localized on chromosome 6 by RH mapping near marker SW1473 and by linkage mapping on the MARC reference family at the same position as the marker SW2173 (97 cM). Allele frequencies, heterozygosity and polymorphism information contents (PIC) values were calculated for the five different pig breeds examined. The transcription of both genes in porcine liver, heart, kidney, fat, brain, pancreas, stomach, bladder, ovaries, lung, spleen, skin, adrenal gland and muscle tissues was examined by reverse transcriptase-polymerase chain reaction. Transcription was detected in skin and adrenal gland tissues for MC2R, while a positive signal was detected for MC5R in kidney, fat, pancreas, skin, adrenal gland and spleen tissues.

Amino Acid Sequence↗
PubMed1997

Bacterial scavengase p20 is structurally and functionally related to peroxiredoxins.

Scavengase p20 was recently identified as a novel family of bacterial antioxidant enzymes possessing thioredoxin-linked thiol peroxidase activity. In this study, the Escherichia coli gene coding for scavengase p20 was isolated from three different strains and the nucleotide sequence was determined. Multiple alignment of amino acid sequence revealed that a previously unidentified Cys-61 is most conserved among all bacterial p20 scavengases and corresponds to the active site in the well-characterized peroxiredoxins. Phylogenetic analysis further supported that scavengase p20 is a novel subfamily of peroxiredoxins. Site-directed mutagenesis studies demonstrated that Cys-61 is indispensable for the antioxidant activities of scavengase p20. Taken together, our findings strongly suggest that the p20 scavengases are structurally and functionally related to peroxiredoxins.

Amino Acid Sequence↗
PubMed1986

Nucleotide sequence of the luxB gene of Vibrio harveyi and the complete amino acid sequence of the beta subunit of bacterial luciferase.

The nucleotide sequence of the 1.30-kilobase EcoRI/BglII fragment from Vibrio harveyi carrying the majority of the luciferase beta subunit coding region (luxB gene) has been determined. The EcoRI/BglII fragment was derived from a 4.0-kilobase HindIII fragment carrying both luxA and luxB which was detected in a genomic clone bank based on the expression of bioluminescence from colonies of Escherichia coli carrying V. harveyi HindIII fragments in plasmid pBR322 (Baldwin, T. O., Berends, T., Bunch, T. A., Holzman, T. F., Rausch, S. K., Shamansky, L., Treat, M. L., and Ziegler, M. M. (1984) Biochemistry 23, 3663-3667). The entire alpha subunit coding sequence (luxA gene) and the amino-terminal 13 codons of the beta subunit sequence (luxB gene) were contained on a 1.85-kilobase EcoRI fragment, the sequence of which has been reported (Cohn, D. H., Mileham, A. J., Simon, M. I., Nealson, K. H., Rausch, S. K., Bonam, D., and Baldwin, T. O. (1985) J. Biol. Chem. 260, 6139-6146). The beta subunit coding sequence was found to terminate 972 bases past the start of the luxB coding sequence. The beta subunit had a calculated molecular weight of 36,349 and comprised a total of 324 amino acid residues; the alpha beta dimer had a molecular weight (alpha + beta) of 76,457. There were 27 base pairs separating the stop codon of the beta subunit structural gene and a 340-base open reading frame extending to (and beyond) the distal BglII site. Approximately two-thirds of the beta subunit was sequenced by protein chemical techniques. The amino acid sequence predicted from the DNA sequence, with few exceptions, confirmed the chemically determined sequence, and the measured amino acid composition was in excellent agreement with the composition implied from the DNA sequence.

Amino Acid Sequence↗
PubMed2001

Genome alignment, evolution of prokaryotic genome organization, and prediction of gene function using genomic context.

Gene order in prokaryotes is conserved to a much lesser extent than protein sequences. Only several operons, primarily those that code for physically interacting proteins, are conserved in all or most of the bacterial and archaeal genomes. Nevertheless, even the limited conservation of operon organization that is observed can provide valuable evolutionary and functional clues through multiple genome comparisons. A program for constructing gapped local alignments of conserved gene strings in two genomes was developed. The statistical significance of the local alignments was assessed using Monte Carlo simulations. Sets of local alignments were generated for all pairs of completely sequenced bacterial and archaeal genomes, and for each genome a template-anchored multiple alignment was constructed. In most pairwise genome comparisons, <10% of the genes in each genome belonged to conserved gene strings. When closely related pairs of species (i.e., two mycoplasmas) are excluded, the total coverage of genomes by conserved gene strings ranged from <5% for the cyanobacterium Synechocystis sp to 24% for the minimal genome of Mycoplasma genitalium, and 23% in Thermotoga maritima. The coverage of the archaeal genomes was only slightly lower than that of bacterial genomes. The majority of the conserved gene strings are known operons, with the ribosomal superoperon being the top-scoring string in most genome comparisons. However, in some of the bacterial-archaeal pairs, the superoperon is rearranged to the extent that other operons, primarily those subject to horizontal transfer, show the greatest level of conservation, such as the archaeal-type H+-ATPase operon or ABC-type transport cassettes. The level of gene order conservation among prokaryotic genomes was compared to the cooccurrence of genomes in clusters of orthologous genes (COGs) and to the conservation of protein sequences themselves. Only limited correlation was observed between these evolutionary variables. Gene order conservation shows a much lower variance than the cooccurrence of genomes in COGs, which indicates that intragenome homogenization via recombination occurs in evolution much faster than intergenome homogenization via horizontal gene transfer and lineage-specific gene loss. The potential of using template-anchored multiple-genome alignments for predicting functions of uncharacterized genes was quantitatively assessed. Functions were predicted or significantly clarified for approximately 90 COGs (approximately 4% of the total of 2414 analyzed COGs). The most significant predictions were obtained for the poorly characterized archaeal genomes; these include a previously uncharacterized restriction-modification system, a nuclease-helicase combination implicated in DNA repair, and the probable archaeal counterpart of the eukaryotic exosome. Multiple genome alignments are a resource for studies on operon rearrangement and disruption, which is central to our understanding of the evolution of prokaryotic genomes. Because of the rapid evolution of the gene order, the potential of genome alignment for prediction of gene functions is limited, but nevertheless, such predictions information significantly complements the results obtained through protein sequence and structure analysis.

Computational Biology↗
PubMed2004

Gene content and density in banana ( Musa acuminata) as revealed by genomic sequencing of BAC clones.

The complete sequence of Musa acuminata bacterial artificial chromosome (BAC) clones is presented and, consequently, the first analysis of the banana genome organization. One clone (MuH9) is 82,723 bp long with an overall G+C content of 38.2%. Twelve putative protein-coding sequences were identified, representing a gene density of one per 6.9 kb, which is slightly less than that previously reported for Arabidopsis but similar to rice. One coding sequence was identified as a partial M. acuminata malate synthase, while the remaining sequences showed a similarity to predicted or hypothetical proteins identified in genome sequence data. A second BAC clone (MuG9) is 73,268 bp long with an overall G+C content of 38.5%. Only seven putative coding regions were discovered, representing a gene density of only one gene per 10.5 kb, which is strikingly lower than that of the first BAC. One coding sequence showed significant homology to the soybean ribonucleotide reductase (large subunit). A transition point between coding regions and repeated sequences was found at approximately 45 kb, separating the coding upstream BAC end from its downstream end that mainly contained transposon-like sequences and regions similar to known repetitive sequences of M. acuminata. This gene organization resembles Gramineae genome sequences, where genes are clustered in gene-rich regions separated by gene-poor DNA containing abundant transposons.

Base Composition↗
PubMed2003

The human solute carrier SLC41A1 belongs to a novel eukaryotic subfamily with homology to prokaryotic MgtE Mg2+ transporters.

We report here the first identification and structural characterization of a eukaryotic protein with homology to the bacterial MgtE family of potential Mg(2+) transporters. This human protein, denoted solute carrier family 41 member 1 (SLC41A1), consists of 513 amino acids with an estimated molecular weight of 56kDa. Computer analysis of the protein structure reveals that the protein consists of 10 putative transmembrane domains and includes two distinct domains highly homologous to the integral membrane part of the bacterial MgtE protein family. The gene encoding SLC41A1 is found on chromosome 1 (1q31-32) and the protein coding sequence is found on 10 exons. A 5-kb long transcript is identified in various human tissues with highest expression levels in heart and testis. We have also identified 10 SLC41A1 homologs in Homo sapiens, Mus musculus, Drosophila melanogaster, Anopheles gambiae, and Caenorhabditis elegans, and propose that these hypothetical proteins belong to a novel eukaryotic gene family.

Amino Acid Sequence↗
PubMed1988

A complex androgen-responsive enhancer resides 2 kilobases upstream of the mouse Slp gene.

Neighboring genes encoding the mouse sex-limited protein (Slp) and fourth component of complement (C4) show extensive homology. In contrast to C4, however, Slp is regulated by androgen. One region of the Slp gene capable of hormonal response following transfection was located about 2 kilobases upstream of the transcription start site, where the C4 and Slp sequences diverge. This region, delimited here to a 0.75-kilobase fragment, showed cryptic promoter activity as well as androgen responsiveness in either orientation in front of the bacterial chloramphenicol acetyltransferase coding region. When this fragment was placed upstream of a viral long terminal repeat, increased chloramphenicol acetyltransferase expression derived from the viral promoter. Proteins from nuclear extracts specifically bound to four sequences within the region, near sites that are DNase I hypersensitive in vivo and reflect the hormonal and developmental regulation of Slp. Like several other cellular enhancers, this androgen-responsive element seems to be modular in nature and complex in its function.

Acetyltransferases↗
PubMed2001

Truncated and dispersed rpl2 and rps19 pseudogenes are co-transcribed with neighbouring downstream genes in wheat mitochondria.

The wheat mitochondrial genome contains only partial coding sequences for the L2 and S19 ribosomal proteins, unlike in rice or liverwort mitochondria, where these genes are functional and have a bacterial-type linkage. A single-copy stretch corresponding to the extreme 3' terminus of the wheat rpl2 gene is co-transcribed with the trans-splicing nad1 exon 4; and, at another unique location, the rps19 segment lacking the 5' coding region is co-transcribed with the downstream nad4L gene. In both cases, the 5' termini of these transcripts map to promoter consensus motifs acquired through genomic reorganization, enabling continued expression of essential downstream genes. In both wheat and rice, the rpl2 and rps19 genomic regions differ in their RNA profiles between germinating embryos and seedlings. The absence of intact rpl2 and rps19 genes in wheat mitochondria is consistent with their inactivation through DNA rearrangement/deletion after the successful transfer of functional copies to the nucleus.

Amino Acid Sequence↗
PubMed1981

The structure of the gene for the large subunit of ribulose 1,5-bisphosphate carboxylase from spinach chloroplast DNA.

A cloned fragment of spinach chloroplast DNA carrying the gene for the large subunit of ribulose bisphosphate (RuBP) carboxylase has been analysed by electron microscopy of R-loops, by hybridization to Northern blots of chloroplast RNA, by S1 nuclease mapping and by DNA sequencing. The transcribed region of the gene is 1690 +/- 3 nucleotides long and co-linear with its mRNA. It comprises a 178-179 bp 5' untranslated sequence, a 1425 bp coding region and an 85-88 bp 3' untranslated region. The deduced sequence of the 475 amino acids of the spinach large subunit protein shows 10% divergence from that of the maize large subunit protein (1). The nucleotide sequence divergence between spinach and maize over the same coding region is 16% but in the transcribed flanking regions it is 35%. Features of the spinach chloroplast gene which resemble those of bacterial genes include a 5-base Shine-Dalgarno sequence complementary to a sequence near the 3' end of chloroplast and bacterial 16S rRNA, a promoter region partially homologous to a consensus sequence of bacterial promoters, and a transcription termination region capable of forming a typical stem and loop structure.

Amino Acid Sequence↗
PubMed1971

Hybridization properties of DNA sequences directing the synthesis of messenger RNA and heterogeneous nuclear RNA.

The relationship of the DNA sequences from which polyribosomal messenger RNA (mRNA) and heterogeneous nuclear RNA (NRNA) of mouse L cells are transcribed was investigated by means of hybridization kinetics and thermal denaturation of the hybrids. Hybridization was performed in formamide solutions at DNA excess. Under these conditions most of the hybridizing mRNA and NRNA react at values of D(o)t (DNA concentration multiplied by time) expected for RNA transcribed from the nonrepeated or rarely repeated fraction of the genome. However, a fraction of both mRNA and NRNA hybridize at values of D(o)t about 10,000 times lower, and therefore must be transcribed from highly redundant DNA sequences. The fraction of NRNA hybridizing to highly repeated sequences is about 1.7 times greater than the corresponding fraction of mRNA. The hybrids formed by the rapidly reacting fractions of both NRNA and mRNA melt over a narrow temperature range with a midpoint about 11 degrees C below that of native L cell DNA. This indicates that these hybrids consist of partially complementary sequences with approximately 11% mismatching of bases. Hybrids formed by the slowly reacting fraction of NRNA melt within 4 degrees -6 degrees C of native DNA, indicating very little, if any, mismatching of bases. Hybrids of the slowly reacting components of mRNA, formed under conditions of sufficiently low RNA input, have a high thermal stability, similar to that observed for hybrids of the slowly reacting NRNA component. However, when higher inputs of mRNA are used, hybrids are formed which have a strikingly lower thermal stability. This observation can be explained by assuming that there is sufficient similarity among the relatively rare DNA sequences coding for mRNA so that under hybridization conditions, in which these DNA sequences are not truly in excess, reversible hybrids exhibiting a considerable amount of mispairing are formed. The fact that a comparable phenomenon has not been observed for NRNA may mean that there is less similarity among the relatively rare DNA sequences coding for NRNA than there is among the rare sequences coding for mRNA.

Animals↗
PubMed1992

Molecular cloning and promoter analysis of the rat liver farnesyl diphosphate synthase gene.

The isolation and characterization of the rat genomic clone encoding the cholesterogenic enzyme farnesyl diphosphate (FPP) synthase is reported. The gene is localized on a 15-kilobase (kb) genomic fragment, spans approximately 12 kb and contains eight exons. Sequences containing from 3.9 kb to 132 base pairs (bp) of the putative promoter were joined to the coding region of the bacterial reporter gene chloramphenicol acetyltransferase (CAT). The CAT activities or CAT mRNA levels of the hybrid genes were determined following either transient transfections into human hepatoma HepG2 cells or stable transfections into Chinese hamster ovary cells. The transient transfections identified a 319-bp fragment that was required for a 4-fold induction in the absence of sterols. Sequence analysis of this region showed it contained five potential copies of the sterol regulatory element (SRE-1) (Smith, J.R., Osborne, T.F., Brown, M.S., Goldstein, J.L., and Gil, G. (1988) J. Biol. Chem. 263, 18480-18487) previously identified in the promoters of the 3-hydroxy-3-methyl-coenzyme A (HMG-CoA) reductase, HMG-CoA synthase, and low density lipoprotein receptor genes. Further mutational and deletion analysis of the FPP synthase promoter-CAT constructs followed by stable transfection and primer extension of the CAT mRNA levels indicated that these potential SRE-1 regulatory elements were not involved in the sterol-mediated transcriptional regulation of the gene. Our analyses have identified a 115-bp region that is required for the transcriptional induction of FPP synthase in the absence of sterols. These results suggest that the FPP synthase gene may be regulated at the transcriptional level by a different mechanism than other sterol regulated genes.

Alkyl and Aryl Transferases↗
PubMed2002

Distinguishing the ORFs from the ELFs: short bacterial genes and the annotation of genomes.

A substantial fraction of hypothetical open reading frames (ORFs) in completely sequenced bacterial genomes are short, suggesting that many are not genes but random stretches of DNA. Although it is not feasible to authenticate the coding capacity of all such regions experimentally, comparisons of ORFs in related genomes can expose those that encode functional proteins.

Bacteria↗
PubMed2003

A group II intron has invaded the genus Azotobacter and is inserted within the termination codon of the essential groEL gene.

A group II intron that was previously identified within Azotobacter vinelandii by polymerase chain reac-tion with consensus primers has been completely sequenced, together with its flanking exons. In contrast to other bacterial members of group II, which are associated with mobile or other presumably non-essential DNA, the A. vinelandii intron is inserted within the termination codon of the groEL coding sequence, which it changes from UAA to UAG. Both the host gene and the intron appear to be functional as (i) the ribozyme component of the intron self-splices in vitro and (ii) both intron-carrying and intronless versions of the single-copy groEL gene from A. vinelandii complement groEL mutations in Escherichia coli. Moreover, analysis of nucleotide substitutions within and around a closely related intron sequence that is present at the same site in Azotobacter chroococcum provides indirect evidence of intron transposition posterior to the divergence of the two Azotobacter taxa. Somewhat surprisingly, however, analyses of RNA extracted from cells that had or had not undergone a heat shock show that the bulk of groEL transcripts end within the first 140 nucleotides of the intron. These findings are discussed in the light of our current knowledge of the biochemistry of group II introns.

Azotobacter vinelandii↗
PubMed2002

A GyrB-GyrA fusion protein expressed in yeast cells is able to remove DNA supercoils but cannot substitute eukaryotic topoisomerase II.

BACKGROUND: Type II topoisomerases are a highly conserved class of enzymes which transport one double-stranded DNA segment through a transient break in another. Whereas the eukaryotic enzymes are homodimers of a single polypeptide, their bacterial homologues are homodimers of two independently coded protein subunits. Unlike eukaryotic topoisomerase II and bacterial topoisomerase IV, DNA gyrase is a bacterial type II topoisomerase which specializes in intramolecular DNA transport. RESULTS: We have fused the Escherichia coli coding sequences for the proteins GyrB and GyrA, which comprise DNA gyrase. This fusion was expressed in yeast cells and yielded the expected full-length protein product. When it was expressed in Deltatop1- top2-4 yeast cells, the fusion protein compensated their slow growth and reverted their elevated chromosomal excision of ribosomal genes. Furthermore, it removed DNA positive supercoils. The fusion protein, however, was unable to complement the temperature-dependent lethality of top2-4 cells. CONCLUSION: Fusion of the E. coli GyrB and GyrA proteins leads to a catalytically active topoisomerase which compensates several phenotypic traits attributed to unconstrained DNA supercoiling in topoisomerase-deficient cells. However, since the fusion protein cannot substitute for topoisomerase II, it may be efficient in intramolecular but not intermolecular DNA passage, resembling the catalytic properties of DNA gyrase.

DNA Gyrase↗
PubMed1993

A gene involved in the biogenesis of c-type cytochromes is co-transcribed with a ribosomal protein gene in wheat mitochondria [corrected].

Sequence analysis of a transcribed region of the wheat mitochondrial (mt) genome revealed two open reading frames (orfs) coding for proteins of 589 and 174 amino acids. Both genes are co-transcribed in a 2.6-kb RNA. The largest orf codes for a hydrophobic protein which bears similarity to a bacterial protein involved in the biogenesis of c-type cytochromes. Its corresponding RNA sequence is fully edited at 34 positions. The second orf encodes a protein homologous to the amino-terminal third of E. coli ribosomal protein S1, corresponding to the ribosome-binding domain of this protein. Its RNA sequence is edited at four positions, one of the edits creating a stop codon. The presence of both proteins in wheat mitochondria was demonstrated using specific antibodies raised against fusion proteins obtained in E. coli from the corresponding cDNAs.

Amino Acid Sequence↗