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PubMed1997

Sequencing analysis of prion genes from red deer and camel.

An abnormal isoform of the prion protein (PrP) appears to be the agent responsible for transmissible spongiform encephalopathies (TSE). The normal isoform of PrP is host-encoded and expressed in the central nervous system. The recent bovine spongiform encephalopathy (BSE) epidemic in the UK and the incidence of prion-related diseases in other animals could indicate that ruminants are highly susceptible to infection via ingestion of prion-contaminated food. Sequence analysis of PrP gene open reading frames from red deer and camel was carried out to investigate sequence variability of these genes among ruminants.

Amino Acid Sequence↗

PubMed1998

Cloning and sequence analysis of four t(9;11) therapy-related leukemia breakpoints.

The t(9;11)(p22;q23) is the most common chromosomal translocation in topoisomerase II inhibitor therapy-related acute myeloid leukemia (tAML). This translocation fuses the MLL and AF9 proto-oncogenes producing a novel chimeric protein. In order to gain insight into the mechanism generating the t(9;11) and to clarify the role topoisomerase II inhibition may play in that mechanism we have cloned and sequenced the breakpoints from four tAML patients with the t(9;11). This sequence analysis identifies topoisomerase II consensus binding sequences near or at the chromosome 11 and chromosome 9 breakpoints in all four patients. One patient also had the consensus binding sequence for the TRANSLIN DNA-binding protein at the 9p22 and 11q23 breakpoints. Our results further support a direct role for topoisomerase II in the genesis of these tAML translocations.

Acute Disease↗

PubMed1995

Sequence analysis of putative pVIII, E3 and fibre regions of porcine adenovirus type 3.

Sequence analysis of a region of the genome of porcine adenovirus type 3 from map unit 79.5 to map unit 92 was performed. Homology studies revealed genes coding for the hexon-associated protein pVIII on the left and for the fibre protein on the right of the sequenced region. By analogy with the genomic organization of other adenoviruses, the 1179 bp sequence between the pVIII and fibre open reading frames, extending from map unit 81.3 to map unit 84.7, was identified as the equivalent of the E3 region of human adenoviruses. The deduced amino acid sequence of one of the three open reading frames of the putative E3 region showed homology with the 13.3K E3 protein of canine adenovirus type 2. The primary structure of the putative fibre protein was similar to that described for human adenovirus types 2 and 5, with a 14 pseudorepeat motif in the shaft region of the fibre. A 742 bp tandem repeat starting in the middle of the fibre gene and extending beyond the termination codon of this gene was observed.

Adenoviridae↗

PubMed1994

Molecular cloning and sequence analysis of an Azospirillum brasilense indole-3-pyruvate decarboxylase gene.

Azospirillum brasilense isolated from the rhizosphere of different plants has the ability to excrete indole-3-acetic acid (IAA) into the culture media. Cosmid p0.2, isolated from an A. brasilense Sp245 genome library in pLAFR1, complements the Tn5-induced mutant SpM7918 of A. brasilense Sp6 which excretes reduced amounts of IAA. Restriction mapping and gene expression studies identified a BglII-EcoRI 4.3 kb fragment of p0.2 sufficient for the restoration of high levels of IAA production in mutant SpM7918. Tn5 mutagenesis localized the gene responsible on a 1.8 kb SmaI fragment. Nucleotide sequence analysis revealed that this fragment contains one complete open reading frame. The predicted protein sequence shows extensive homology with the indole-3-pyruvate decarboxylase of Enterobacter cloacae and the pyruvate decarboxylases of Saccharomyces cerevisiae and Zymomonas mobilis. The A. brasilense mutant Sp245a, constructed by homogenotization of a Tn5 insertion derivative of the 1.8 kb SmaI fragment, also displayed reduced IAA production. Introduction of the cloned wild-type gene into Rhizobium meliloti 1021 resulted in increased IAA production. Cell-free extracts prepared from R. meliloti and A. brasilense transconjugants harboring this gene could convert indole-3-pyruvic acid to indole-3-acetaldehyde and tryptophol. These results clearly demonstrate that IAA production in A. brasilense is mediated by indole-3-pyruvate decarboxylase.

Amino Acid Sequence↗

PubMed1999

DNA sequence analysis of spontaneous tonB deletion mutations in a polA1 strain of Escherichia coli K12.

By DNA sequence analysis, we have determined a spectrum of 61 spontaneous mutations occurring in the endogenous tonB gene in the polA1 strain of Escherichia coli. The overall mutation frequency was approximately 2.4-fold higher in the polA1 strain and this was attributable to enhanced rates of deletion and frameshift mutations. Among 39 deletions, a hot spot (17 mutations) was detected: a 13-bp deletion presumably directed by a 3-bp repeated sequence at its end points. The remaining 22 were distributed among 19 different mutations either flanked (16/19) or not flanked (3/19) by repeated sequences. Single-base frameshifts accounted for 8 mutations of either repeated (3/8) or nonrepeated (5/8) bases among which 6 were minus one frameshift. In contrast to previous reports, we did not frequently observe a 5'-GTGG-3' sequence in the vicinity of the deletions and frameshifts. The results presented here indicated an anti-deletion and anti-frameshift role for DNA polymerase I.

Bacterial Proteins↗

PubMed2000

Informatics issues in large-scale sequence analysis: elucidating the protein kinases of C. elegans.

With the availability of the nearly complete genomic sequence of C. elegans, the first multicellular organism to be sequenced, molecular biology has definitely entered the postgenomic era. Annotation of the genomic sequence, which refers to identifying the genes and other biologically relevant sections of the genome, is an important and nontrivial next step. A first-pass annotation will be necessarily incomplete but will drive further biological experiments, which in turn will help to annotate the genome better. Given the scale of the genome sequence analysis, it is clear that the annotation should be automated as much as possible without sacrificing the quality of analysis. In this work, we outline our approach to identifying the protein kinases of C. elegans from the genomic sequence. We describe new tools we have developed for analysis, management and visualization of genomic data. By developing modular and scalable solutions, this study has provided a framework for future analysis of the Drosophila and human genomes.

Animals↗

PubMed1993

Isolation, characterization and sequence analysis of the scrK gene encoding fructokinase of Streptococcus mutans.

A gene encoding an ATP-dependent fructokinase from Streptococcus mutans GS-5 was identified within a 2 kb DNA fragment immediately downstream from the scrA gene. The gene cloned in Escherichia coli also expressed mannokinase activity. Insertional inactivation of this gene in S. mutans markedly decreased both fructokinase and mannokinase activities. Nucleotide sequence analysis of the 2 kb fragment revealed an ORF starting 199 bp downstream from the scrA gene, preceded by potential ribosome-binding (Shine-Dalgarno) and promoter-like sequences. This ORF specified a putative protein of 293 amino acids with a calculated M(r) of 31,681. The deduced amino acid sequence of the fructokinase gene, scrK, from S. mutans exhibited no significant similarity to fructokinase genes from Klebsiella pneumoniae, E. coli plasmid pUR400 or Vibrio alginolyticus, but was similar to a comparable gene from Zymomonas mobilis.

Amino Acid Sequence↗

PubMed1984

Primary structure of rabbit 18S ribosomal RNA determined by direct RNA sequence analysis.

The primary structure of rabbit 18S ribosomal RNA was determined by nucleotide sequence analysis of the RNA directly. The rabbit rRNA was specifically cleaved with T1 ribonuclease, as well as with E. coli RNase H using a Pst 1 DNA linker to generate a specific set of overlapping fragments spanning the entire length of the molecule. Both intact and fragmented 18S rRNA were end-labeled with [32P], base-specifically cleaved enzymatically and chemically and nucleotide sequences determined from long polyacrylamide sequencing gels run in formamide. This approach permitted the detection of both cistron heterogeneities and modified bases. Specific nucleotide sequences within E. coli 16S rRNA previously implicated in polyribosome function, tRNA binding, and subunit association are also conserved within the rabbit 18S rRNA. This conservation suggests the likelihood that these regions have similar functions within the eukaryotic 40S subunit.

Animals↗

PubMed2005

Cloning and sequence analysis of the M gene of porcine epidemic diarrhea virus LJB/03.

Porcine epidemic diarrhea virus (PEDV) LJB/03 was isolated from the fece of piglets infected with PEDV on a pig farm, Heilongjiang province, China. The M gene of LJB/03 was amplified from the RNA extracted directly from the fece samples by RT-PCR and cloned into pMD18-T vector. The M gene cDNA was sequenced and encompasses an open reading frame of 681 nucleotides, encoding a 226-amino acid protein. The LJB/03 M gene has a base composition of 152 adenines (22%), 153 cytosines (23%), 161 guanines (24%), and 214 thymines (31%). Sequence comparison with other PEDV strains selected from GenBank revealed that the LJB/03 M gene has a high sequence homology to those of other PEDV isolates, 97.80% with JMe2, 96.92% with KPEDV-9 (Korean field isolate), 97.36% with KPEDV-9 (Korean), 97.80% with Br1/87, and 97.94% with CV777. The encoded protein shared 97.79% amino acid identities compared with CV777, 97.35% with Br1/87, 97.79% with JMe2, 96.90% with KPEDV-9 (Korean field isolate), 96.46% with KPEDV-9 (Korean). Sequence analysis of the M gene, including genetic distance measurement, phylogenetic tree analysis, and residue substitution analysis, showed that all other PED viruses analyzed fell into three groups, and the LJB/03 itself branched in an independent group. These data revealed that the M gene nucleotide sequence of LJB/03 has some mutations in comparison with the other PED viruses.

Animals↗

PubMed1993

Cloning and sequence analysis of cDNAs encoding human hippocampus N-methyl-D-aspartate receptor subunits: evidence for alternative RNA splicing.

Several cDNA clones encoding human N-methyl-D-aspartate receptor (hNR1) subunit polypeptides were isolated from a human hippocampus library. Degenerate oligodeoxyribonucleotide (oligo) primers based on the published rat NR1 (rNR1) amino acid (aa) sequence [K. Moriyoshi et al. Nature 354 (1991) 31-37] amplified a 0.7-kb fragment from a human hippocampus cDNA library, via the polymerase chain reaction (PCR). This fragment was used as a probe for subsequent hybridization screening. DNA sequence analysis of 28 plaque-purified clones indicated three distinct classes, designated hNR1-1, hNR1-2 and hNR1-3, presumably generated by alternative RNA splicing. One of these clones, hNR1-1(5A), was isolated as a full-length cDNA. The hNR1-2 and hNR1-3 cDNAs represented 66.8 and 98.9%, respectively, of the total aa coding information predicted for the polypeptides. The hNR1 cDNAs demonstrated an 84-90.8% nucleotide (nt) identity with the corresponding rodent cDNAs. The nt sequences of hNR1-1, hNR1-2 and hNR1-3 would encode 885-, 901- and 938-aa proteins, respectively, that have 99.1-99.8% identity with the corresponding rodent NR1 (roNR1) subunits. The changes between the predicted aa sequences of hNR1 and the corresponding roNR1 subunits are confined to the extracellular N-terminal regions. We have also identified two possible allelic variations of the hNR1-3 cDNA that result in aa substitutions in the extracellular N- and C-terminal regions. One of these naturally occurring aa variations is situated within a potential glutamate-binding site.

Alternative Splicing↗

PubMed1997

Sequence analysis of the small cryptic Xanthomonas campestris pv. vesicatoria plasmid pXV64 encoding a Rep protein similar to gene II protein of phage 12-2.

The complete nucleotide sequence (1,851 bp) was determined for the Xanthomonas campestris pv. vesicatoria plasmid pXV64. Sequence analysis revealed an intergenic region (IG) of 355 bp and two oppositely running open reading frames, ORF1 and ORF2, encoding polypeptides of 39 and 16 kDa, respectively. While the function of ORF2 is not known, ORF1 is suggested to be the gene encoding Rep protein based on (i) similarity in amino acid sequence to that of the gene II protein (gIIP) of filamentous phage 12-2, (ii) presence of a sequence in the ori-containing region which is similar to the sequence around the Rep nicking site in some rolling circle-replicating replicons, and (iii) ability to support replication in trans of the region containing pXV64 ori (392 bp) which is located within the region including IG and a short stretch in the N-terminus of ORF2.

Amino Acid Sequence↗

PubMed1994

Mouse plasma glutathione peroxidase. cDNA sequence analysis and renal proximal tubular expression and secretion.

A mouse kidney cDNA isolated by differential screening was found to be highly homologous to rat, human, and bovine plasma glutathione peroxidase (GPx) sequences. Analysis of the full-length coding region sequence demonstrated an in-frame selenocysteine-encoding opal codon and putative signal sequence, suggesting that the sequence represents the mouse homolog of plasma GPx. The level of expression of plasma GPx in various mouse tissues and during development was investigated by Northern blot analysis. Plasma GPx mRNA was observed to be very abundant in kidney compared with placenta, epididymis, intestine, lung, heart, testis, ovary, salivary gland, spleen, thymus, stomach, brain, and fetal kidney and could not be detected in pancreas or in liver except from pregnant mice. In addition, plasma GPx mRNA levels were shown to increase during postnatal development of the kidney. In situ hybridization localized plasma GPx mRNA to proximal tubules, while primary cell culture demonstrated that plasma GPx is synthesized and secreted by proximal tubular epithelial cells. The relative abundance of plasma GPx mRNA in mouse kidney suggests that proximal tubules may be the primary source of the enzyme detectable in plasma and further suggests that plasma GPx has an important function in protecting the kidney from oxidative damage.

Amino Acid Sequence↗

PubMed1999

Cloning and nucleotide sequence analysis of gyrB of Bacillus cereus, B. thuringiensis, B. mycoides, and B. anthracis and their application to the detection of B. cereus in rice.

As 16S rRNA sequence analysis has proven inadequate for the differentiation of Bacillus cereus from closely related species, we employed the gyrase B gene (gyrB) as a molecular diagnostic marker. The gyrB genes of B. cereus JCM 2152(T), Bacillus thuringiensis IAM 12077(T), Bacillus mycoides ATCC 6462(T), and Bacillus anthracis Pasteur #2H were cloned and sequenced. Oligonucleotide PCR primer sets were designed from within gyrB sequences of the respective bacteria for the specific amplification and differentiation of B. cereus, B. thuringiensis, and B. anthracis. The results from the amplification of gyrB sequences correlated well with results obtained with the 16S rDNA-based hybridization study but not with the results of their phenotypic characterization. Some of the reference strains of both B. cereus (three serovars) and B. thuringiensis (two serovars) were not positive in PCR amplification assays with gyrB primers. However, complete sequencing of 1.2-kb gyrB fragments of these reference strains showed that these serovars had, in fact, lower homology than their originally designated species. We developed and tested a procedure for the specific detection of the target organism in boiled rice that entailed 15 h of preenrichment followed by PCR amplification of the B. cereus-specific fragment. This method enabled us to detect an initial inoculum of 0.24 CFU of B. cereus cells per g of boiled rice food homogenate without extracting DNA. However, a simple two-step filtration step is required to remove PCR inhibitory substances.

Amino Acid Sequence↗

PubMed2004

Outbreak of infection with hepatitis A virus (HAV) associated with a foodhandler and confirmed by sequence analysis reveals a new HAV genotype IB variant.

An outbreak of infection with hepatitis A virus associated with a foodhandler and involving 26 subjects occurred in Southern Italy. Sequence analysis of the VP3-VP1 and VP1-P2A junctions confirmed that the outbreak was due to a point source and allowed the identification of a new genotype IB variant. This report confirms the usefulness of sequence-based molecular fingerprinting during outbreaks.

Amino Acid Sequence↗

PubMed1997

Sequence analysis of a 45-kb segment in the 19 degrees-23 degrees region of the Bacillus subtilis chromosome containing glpT and mpr loci.

The nucleotide sequence of a 45,137-bp segment covering the 19 degrees and 23 degrees region in the 360 degrees map of the Bacillus subtilis genome was determined. This region contained 45 open reading frames (ORFs) including 7 which corresponded to the products of genes with known functions that had been previously sequenced. The known genes were: glpT and glpQ for glycerol utilization pathway; purT for a part of the purine synthesis pathway; mpr for an extracellular metalloprotease; pss and psd for the parts of the phospholipid synthesis pathway; and gltP for a glutamine transporter. Deduced amino acid sequences of the 22 newly identified ORFs showed significant homologies to known gene products in the database such as a Methicillin-resistant Staphylococcus aureus (MRSA) gene which is related to drug resistance, a two-component response regulator, a series of amino acid permeases, transcriptional regulators, beta-lactamase, the phosphotransferase system (PTS) enzyme II for sugar uptake, and the eukaryotic ECA39 gene which is associated with cancer and apoptosis, etc. The remaining 16 ORFs did not show any significant sequence similarities to known gene products in the database.

Animals↗

PubMed1996

Purification and sequence analysis of 4-methyl-5-nitrocatechol oxygenase from Burkholderia sp. strain DNT.

4-Methyl-5-nitrocatechol (MNC) is an intermediate in the degradation of 2,4-dinitrotoluene by Burkholderia sp. strain DNT. In the presence of NADPH and oxygen, MNC monooxygenase catalyzes the removal of the nitro group from MNC to form 2-hydroxy-5-methylquinone. The gene (dntB) encoding MNC monooxygenase has been previously cloned and characterized. In order to examine the properties of MNC monooxygenase and to compare it with other enzymes, we sequenced the gene encoding the MNC monooxygenase and purified the enzyme from strain DNT. dntB was localized within a 2.2-kb ApaI DNA fragment. Sequence analysis of this fragment revealed an open reading frame of 1,644 bp with an N-terminal amino acid sequence identical to that of purified MNC monooxygenase from strain DNT. Comparison of the derived amino acid sequences with those of other genes showed that DntB contains the highly conserved ADP and flavin adenine dinucleotide (FAD) binding motifs characteristic of flavoprotein hydroxylases. MNC monooxygenase was purified to homogeneity from strain DNT by anion exchange and gel filtration chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single protein with a molecular weight of 60,200, which is consistent with the size determined from the gene sequence. The native molecular weight determined by gel filtration was 65,000, which indicates that the native enzyme is a monomer. It used either NADH or NADPH as electron donors, and NADPH was the preferred cofactor. The purified enzyme contained 1 mol of FAD per mol of protein, which is also consistent with the detection of an FAD binding motif in the amino acid sequence of DntB. MNC monooxygenase has a narrow substrate specificity. MNC and 4-nitrocatechol are good substrates whereas 3-methyl-4-nitrophenol, 3-methyl-4-nitrocatechol, 4-nitrophenol, 3-nitrophenol, and 4-chlorocatechol were not. These studies suggest that MNC monooxygenase is a flavoprotein that shares some properties with previously studied nitrophenol oxygenases.

Adenosine Diphosphate↗

PubMed1999

Separation of strains of the yeasts Xanthophyllomyces dendrorhousand Phaffia rhodozyma based on rDNA IGS and ITS sequence analysis.

The type strains of the anamorph Phaffia rhodozyma (CBS 5905) and the teleomorph Xanthophyllomyces dendrorhous (VKM Y-2786) were analyzed by nucleotide sequence analysis and compared to the sequences found in three additional strains (ATCC 24228, ATCC 24230 and CBS 6938). The results of ribosomal DNA Internal transcribed spacer (ITS) and Intergenic spacer (IGS) region analyses indicate that P. rhodozyma, which was isolated from a beech tree, is a distinct species from the other four strains. The latter that were collected from birch trees are considered to be strains of X. dendrorhous. These individual strains of X. dendrorhous, which have geographically distinct isolation sources, can be distinguished by nucleotide substitutions and deletion/insertion gaps in sub-repeat regions of the Intergenic spacer. The conclusions demonstrate that differences in the IGS region provide molecular markers for denoting strains that may differ in their biochemical and physiological capabilities. The hypothesis is presented that strain differences in the IGS region may be useful to demonstrate geographic and host specificity.

Base Sequence↗

PubMed1993

Cloning and sequence analysis of an Escherichia coli gene conferring bicyclomycin resistance.

We have cloned and sequenced DNA from Escherichia coli that, when present in a high-copy-number plasmid, confers resistance to the diketopiperazine antibiotic, bicyclomycin (Bc). The DNA includes a 378-amino-acid open reading frame (ORF), disruption of which results in the loss of Bc resistance. This ORF contains the BcR gene. Studies using the minicell expression system reveal that a polypeptide of 31 kDa is produced from this cloned region. The ORF maps at 47.1 min on the E. coli genome map. Sequence comparison between the translated ORF and a protein database reveal between 26.5 and 23.4% aa sequence homology to bacterial transmembrane (TM) proteins including those mediating chloramphenicol (Cm) and tetracycline (Tc) resistance and an arabinose-proton symport protein. Sequence analysis using the Diagon program showed the BcR gene product (BcR) had homology with the N-terminal regions of the CmR and TcR-encoded proteins and weak N-terminal homology with the arabinose-proton symport protein. Hydropathy profiles of the BcR protein and CmR products show a striking similarity, both having twelve predicted TM domains.

Amino Acid Sequence↗