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PubMed1975

Formylatable methionine transfer RNA from Mycoplasma: purification and comparison of partial nucleotide sequences with those of other prokaryotic initiator tRNAs.

The major species of the formylatable methionine tRNA from Mycoplasma mycoides var capri has been purified. The 5'- and 3'-terminal sequences of the purified tRNA are pC-G- and C-A-A-C-C-AOH, respectively. Thus, this tRNA also contains the unique structural feature found in two other prokaryotic initiator tRNAs in that the first nucleotide at the 5'-end cannot form a Watson-Crick type of base-pair to the fifth nucleotide from the 3'-end. The Mycoplasma tRNA does not contain ribothymidine; however, a specific uridine residue in the sequence G-U-psi-C-G- can be enzymatically methylated by E. coli extracts to yield G-T-psi-C-G. Since ribothymidine is absent in crude tRNA from this strain of Mycoplasma, the absence of T is probably due to the lack of a U yields T modifying enzyme.

Base Sequence↗

PubMed1993

Antigenic variation in Mycoplasma hyorhinis: increased repertoire of variable lipoproteins expanding surface diversity and structural complexity.

VlpE is characterized as a new member in a family of variable surface lipoproteins (Vlps) of Mycoplasma hyorhinis. VlpE shows phenotypic variation in expression and size within isogenic lineages of some strains but is absent from lineages of other strains that express only three previously known Vlps. Expression of four Vlps in some cells further indicates the presence and usage of an expanded reservoir of Vlp coding sequences, which greatly increases the capacity for surface diversification.

Antibodies, Monoclonal↗

PubMed1994

Low efficiency of the 5' nontranslated region of hepatitis A virus RNA in directing cap-independent translation in permissive monkey kidney cells.

To characterize in vivo the translational control elements present in the 5' nontranslated region (5'NTR) of hepatitis A virus (HAV) RNA, we created an HAV-permissive monkey kidney cell line (BT7-H) that stably expresses T7 RNA polymerase and carries out cytoplasmic transcription of uncapped RNA from transfected DNA containing the T7 promoter. The presence of an internal ribosomal entry site (IRES) within the 5'NTR of HAV was confirmed by using BT7-H cells transcribing bicistronic RNAs in which the 5'NTR was placed within the intercistronic space, controlling translation of a downstream reporter protein (bacterial chloramphenicol acetyltransferase). However, translation directed by the 5'NTR in these bicistronic transcripts and in monocistronic T7 transcripts in which the HAV 5'NTR was placed upstream of the chloramphenicol acetyltransferase coding sequence was very inefficient compared with the translation of monocistronic transcripts containing either the IRES of encephalomyocarditis (EMC) virus or a short nonpicornavirus 5' nontranslated leader sequence. A large deletion within the HAV IRES (delta 355-532) eliminated IRES activity in bicistronic transcripts. In contrast, larger deletions within the IRES in monocistronic transcripts (delta 1-354, delta 1-532, delta 1-633, and delta 158-633) resulted in 4- to 14-fold increases in translation. In the latter case, this was most probably due to a shift from IRES-directed translation to translation initiation by 5'-end-dependent scanning. Translation of RNAs containing either the EMC virus IRES or the nonpicornavirus leader was significantly enhanced by cotransfection of the reporter constructs with pEP2A, which directs transcription of RNA containing the EMC virus IRES fused to the poliovirus 2Apro coding region. This 2Apro enhancement of cap-independent translation suggests a greater availability of limiting cellular translation factors following 2Apro-mediated cleavage of the p220 subunit of the eukaryotic initiation factor eIF-4F and subsequent shutdown of 5' cap-dependent translation. In contrast, pEP2A cotransfection resulted in severe inhibition of translation directed by the HAV IRES in either monocistronic or bicistronic transcripts. This inhibition was due to competition from the EMC virus IRES present in pEP-2A transcripts, as well as the expression of proteolytically active 2Apro. 2Apro-mediated suppression of HAV translation was not seen with transcripts containing large deletions in the HAV IRES (delta 158-633, delta 1-532, or delta 1-633). These data suggest that the HAV IRES may have a unique requirement for intact p220 or that it may be dependent on active expression of another cellular translation factor which is normally present in severely limiting quantities.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1999

[Sub-cloning and preliminary sequence analysis of the gene encoding of a cefoperazone hydrolyzing beta-lactamase isolated from Escherichia coli].

Escherichia coli HX88108, which is resistant to cefoperazone(CPZ), was isolated from a severely infected patient. We studied genetical basis of beta-lactamase produced in E. coli HX88108 by pFL25, one of the recombinant plasmid of pFC. Largescale pFL25 plasmid was extracted, purified, and cleaved with restriction endonuclease EcoR I, Sal I, Pvu I, then subcloned into vector pUC19 as 1.9 kb, 0.9 kb, 0.65 kb fragments respectively. Recomminant plasmids were selected by alpha-complementation and determined by restriction endonuclease analysis. DNA sequencing was performed by the dideoxy polymerase chain termination method. Partial nucleotide sequence(1-78 nucleotide position) of the gene was found to be highly homologous (97%) with the gene coding for TEM-52 extended spectrum beta-lactamase of K. pneumonise, suggesting that the beta-lactamase coded by the cefoperazone resistant gene might be derived from TEM-type beta-lactamase.

Anti-Bacterial Agents↗

PubMed1985

Expression of human terminal deoxynucleotidyl transferase in Escherichia coli.

A cloned DNA fragment related to pT17 containing a partial cDNA sequence of human terminal deoxynucleotidyl transferase was used as a probe to screen for the full length cDNA sequence of the enzyme in a lambda gt11 library constructed from human lymphoblastoid KM-3 cDNA. A recombinant containing a 2068-base pair insert was isolated and recloned into the EcoRI site of the sequencing plasmic pUC-8 as two subclones, pT711 and pT106. DNA sequencing and hybridization studies showed that pT711 contains the pT17 sequence and an additional 172 upstream nucleotides. pT711 represents the coding sequence for the carboxyl half of the terminal transferase protein. pT106, containing a 965-base pair insert, hybridizes to the same mRNA as pT711 on Northern blots and contains an open reading frame that is in phase with the reading frame of the insert in pT711. Amino acid sequencing of the 58-kDa peptide of the calf thymus terminal transferase failed, indicating that the N terminus is blocked. N-Terminal sequencing of a 56-kDa form of the protein produced 24 amino acids corresponding to the translated human cDNA coding sequence starting at residue 398 of the insert in pT106 with 83% homology between bovine and human sequence. The initiation codon is assigned to an ATG sequence at nucleotide 329 of the insert in pT106. Comparison of the translated human terminal transferase sequence with peptides from the calf thymus enzyme showed that the homology between the human and bovine enzyme is better than 90% among 263 amino acids determined. The coding sequences in pT106 and pT711 were recloned into an expression plasmid pUC-19 downstream from the lac promoter and in phase with the coding sequence of the lac Z gene. Lysates of bacteria carrying the reconstructed coding sequence of human terminal transferase contain a fused protein of 60 kDa that reacts with rabbit antibody to terminal transferase on immunoblots and exhibits enzyme activity. Isolation of this fused protein from bacterial lysates with mouse monoclonal antibody to human terminal transferase produces the expected protein of 60 kDa.

Amino Acid Sequence↗

PubMed1994

[Recognition and identification of unknown infectious agents].

The recognition and the identification of previously unrecognized infectious agents require a multidisciplinary approach to specify the nosologic entity of the disease and the epidemiological data, especially the modes of transmission and the risk factors, as well as to discover the microorganism in the laboratory. In the past 20 years, significant breakthroughs have been achieved in cellular cultures (growth factors), in immunology (monoclonal antibodies), and moreover in molecular biology, which have been widely used in the field of infectious diseases. Whereas the classical methods used to grow microorganisms remain of major interest in many cases, innovating strategies have been recently designed to identify previously unknown pathogens. The genomic amplification by polymerase chain reaction (PCR) of highly conserved bacterial genes (as those coding for ribosomal RNA), from tissue biopsies for example, allow to recognize unknown bacteria. The evolutionary distance between a newly recognized pathogen and known microorganisms can be calculated through sequencing of these genes, as described for Rochalimaea henselae or Tropheryma whipplelii. The constitution of cDNA banks from infected tissues is also a novel approach allowing to clone and sequence viral genes, such as those from hepatitis C or from hepatitis E. In the near future, noteworthy improvements will be achieved to rapidly detect microorganisms with highly sensitive and specific tests using monoclonal antibodies, molecular probes (including branched DNA) and with PCR (including Q beta replicase and ligase chain reaction), and to determine the genetic diversity of microbial pathogens by new methods as pulse field gel electrophoresis or arbitrarily primed PCR. This will result in a better knowledge of the pathophysiology of infectious diseases, in a better recognition of a typical, previously unrecognized clinical expression of pathogenicity, and also in a more precise assessment of the actual impact of a given pathogen in human populations by highly sophisticated diagnosis tests.

Animals↗

PubMed1997

Characterization, occurrence, and molecular cloning of a 39-kilodalton Brucella abortus cytoplasmic protein immunodominant in cattle.

Monoclonal antibodies and polyclonal antisera recognizing a 39-kDa protein (P39) of brucellin, a cytoplasmic extract from Brucella melitensis rough strain B115, were produced. The P39 was purified by anion-exchange chromatography. Eleven of fourteen Brucella-infected cows whose infections had been detected by the delayed-type hypersensitivity (DTH) test with brucellergen also developed a DTH reaction when purified P39 was used as the trigger. The T-cell proliferative responses to P39 of peripheral blood lymphocytes from Brucella-infected cows were also positive. None of the animals infected with other bacterial species that are presumed to induce immunological cross-reactions with Brucella spp. reacted to P39, either in DTH tests or in lymphocyte proliferation assays. A lambda gt11 genomic library of Brucella abortus was screened with a monoclonal antibody specific for P39, and the gene coding for this protein was subsequently isolated. The nucleotide sequence of the P39 gene was determined, and the deduced amino acid sequence is in accordance with the sequence of an internal peptide isolated from P39.

Allergens↗

PubMed2006

Cloning and organisation analysis of a hepcidin-like gene and cDNA from Japan sea bass, Lateolabrax japonicus.

A hepcidin gene was amplified from the liver of Lateolabrax japonicus challenged with a mixed bacterial suspension. Using RT-PCR and RACE, a full length cDNA sequence of the hepcidin like antimicrobial peptide was determined. The complete hepcidin cDNA Hepc2 is 581 bases and contains an ORF of 258 bases with a coding capacity of 86 amino acids. The deduced amino acid sequence, which shares eight cysteines at the identical conserved positions, and gene organisation are conserved between Japan sea bass and other fish species. The predicted molecular weight of the peptide is 9.4 kDa. The 3'-untranslated region is composed of 225 bp with a polyadenylation signal AATAAA sequence appearing at 189 nt and the poly(A) tail at 212 nt downstream of stop codon TGA. The predicted signal peptide cleavage site of its deduced peptide is between codons 24 and 25. Japan sea bass hepcidin-like genomic DNA hepc2 sequence including upstream and downstream regions was composed of two introns and three exons. The cloned 173-bp upstream sequence of Japan sea bass hepcidin-like gene contains putative regulatory elements and several binding motifs for transcription factors. High homologies with hepcidin cDNAs and peptides of white bass (Morone chrysops), human and other fish were shown. Hepc2 of Lateolabrax japonicus is a new member of the hepcidin gene family.

Amino Acid Sequence↗

PubMed1998

Molecular cloning and expression analysis of the Rhodobacter capsulatus sodB gene, encoding an iron superoxide dismutase.

Genetic complementation of a sodA sodB Escherichia coli mutant strain was used to clone Rhodobacter capsulatus genes involved in detoxification of superoxide radicals. After sequence analysis, 1 of the 16 identical clones obtained by this selection procedure was shown to contain an open reading frame with sequence similarity to that coding for Fe-containing superoxide dismutases (SodB). The R. capsulatus sodB gene was expressed in E. coli, and the nature of the metal ligand was confirmed by inhibitor sensitivity assays with lysates from both bacterial species. Activity staining of cleared Rhodobacter lysates resolved by polyacrylamide gel electrophoresis indicated that SodB was the only superoxide dismutase present in this phototrophic organism. The sodB gene was expressed at low levels in R. capsulatus cells grown under anaerobic or semiaerobic conditions, but expression was strongly induced upon exposure of the bacteria to air or to methyl viologen. Attempts to construct a sodB mutant in this organism by allelic exchange of the chromosomal copy of the gene with a suicide plasmid containing a mutated sodB gene were unsuccessful, strongly suggesting that the encoded superoxide dismutase is essential for viability of R. capsulatus in aerobic cultures.

Aerobiosis↗

PubMed1994

Molecular cloning and characterization of a novel human sperm antigen (HE2) specifically expressed in the proximal epididymis.

A novel human cDNA encoding a small secretory glycopeptide specifically expressed within the caput and proximal corpus regions of the human epididymis, HE2, has been cloned and sequenced. Southern hybridization analysis showed it to be derived from a single-copy gene. A subclone containing the coding region of the mature peptide was used to generate the putatively encoded peptide as a 10,000 M(r) recombinant fusion protein in a bacterial expression system. This recombinant HE2 protein, as well as an oligopeptide deduced from the open-reading frame, was used to raise polyclonal antisera. Immunohistochemistry and immunofluorescence showed that the predicted HE2 peptide is present in the epididymal epithelium and on the sperm surface, where it adopts a typical antigenic pattern with a subacrosomal equatorial distribution on the sperm head.

Amino Acid Sequence↗

PubMed1995

Cloning and nucleotide sequence of the bglA gene from Erwinia herbicola and expression of beta-glucosidase activity in Escherichia coli.

Genomic DNA fragments encoding beta-glucosidase activity from the wild-type strain WD4 of Erwinia herbicola were cloned into Escherichia coli. Two clones containing a common fragment encoded a polypeptide of 58,000 Da. Cloned beta-glucosidase, expressed in E. coli, showed activity against natural beta-glucoside sugars except for cellobiose. An open reading frame of 1442 bp termed bglA was identified by nucleotide sequencing and it coded for a protein of 480 amino acids (M(r) 53,896) which showed significant homology with beta-glucosidases from glycosyl hydrolase family 1.

Cloning, Molecular↗

PubMed1997

Epitope analysis of the CS3 fimbrial subunit of human enterotoxigenic Escherichia coli and the construction of novel CS3::ST and CS3::LT-B immunogens.

Enterotoxigenic E. coli (ETEC) are the major cause of traveler's diarrhoea and the CS3 fimbriae/fibrillae are expressed by most strains bearing the colonization factor CFA/II. The cstAH gene cluster determining CS3 biosynthesis has been previously cloned and sequenced and it has been shown that cstH encodes the major fimbrial subunit and cstA-G encode an assembly cassette. In the work described here we have sought to define the surface exposed domains on CS3 and to manipulate them so that CS3 can be used as a means of expressing foreign antigenic determinants on the bacterial surface. Using a panel of 21 monoclonal antibodies, which we have used in western blotting, immunofluorescence microscopy and colony blotting, together with computer predictions, we have identified three domains within CstH. Two of these sites were permissive for insertion and we have introduced, in-frame, either an epitope from the B subunit of LT (heat labile toxin) or the entire coding sequence of mature ST (heat stable toxin) to construct hybrid proteins. These proteins could be assembled into hybrid fimbriae which could be recognized by antibodies to both CS3 and the foreign epitope as shown by immunofluorescence microscopy and colony blotting. The immunogenicity of the constructs has been evaluated following both oral and intraperitoneal immunization of mice with the attenuated Salmonella typhimurium strain G30 harbouring the hybrid cst operons. Although plasmid stability is currently a problem, these experiments showed that antibodies to both the carrier and the foreign epitope were generated.

Amino Acid Sequence↗

PubMed1998

Ribosomes inhibit an RNase E cleavage which induces the decay of the rpsO mRNA of Escherichia coli.

The hypothesis generally proposed to explain the stabilizing effect of translation on many bacterial mRNAs is that ribosomes mask endoribonuclease sites which control the mRNA decay rate. We present the first demonstration that ribosomes interfere with a particular RNase E processing event responsible for mRNA decay. These experiments used an rpsO mRNA deleted of the translational operator where ribosomal protein S15 autoregulates its synthesis. We demonstrate that ribosomes inhibit the RNase E cleavage, 10 nucleotides downstream of the rpsO coding sequence, responsible for triggering the exonucleolytic decay of the message mediated by polynucleotide phosphorylase. Early termination codons and insertions which increase the length of ribosome-free mRNA between the UAA termination codon and this RNase E site destabilize the translated mRNA and facilitate RNase E cleavage, suggesting that ribosomes sterically inhibit RNase E access to the processing site. Accordingly, a mutation which reduces the distance between these two sites stabilizes the mRNA. Moreover, an experiment showing that a 10 nucleotide insertion which destabilizes the untranslated mRNA does not affect mRNA stability when it is inserted in the coding sequence of a translated mRNA demonstrates that ribosomes can mask an RNA feature, 10-20 nucleotides upstream of the processing site, which contributes to the RNase E cleavage efficiency.

Base Sequence↗

PubMed1990

Endotoxin-responsive sequences control cachectin/tumor necrosis factor biosynthesis at the translational level.

The biosynthesis of cachectin/TNF is largely regulated at a post-transcriptional level. Bacterial endotoxin, which strongly induces cachectin/TNF production, thus seems to elicit at least some of its effects by altering the macrophage cytoplasmic milieu. It has previously been shown that the 3'-untranslated TTATTTAT element present in numerous cytokines and proto-oncogenes is capable of repressing the translation of mRNA molecules in which it is represented. Using constructs in which the CAT coding sequence is followed by varying segments of the cachectin/TNF 3'-untranslated region, we now demonstrate that downstream sequences present in the cachectin/TNF mRNA are sufficient to mediate greater than 200-fold induction of CAT synthesis in response to activation by endotoxin. Induction of CAT activity is not attributable to a change in cytoplasmic mRNA concentration, but to a marked enhancement of translational efficiency. The response to endotoxin represents "derepression," and is conferred chiefly by the translationally repressive TTATTTAT element, acting in concert with essential flanking sequences.

Base Sequence↗

PubMed1997

Genomic organization, cDNA sequence, bacterial expression, and purification of human seryl-tRNA synthase.

In this paper, we report the cDNA sequence and deduced primary sequence for human cytosolic seryl-tRNA synthetase, and its expression in Escherichia coli. Two human brain cDNA clones of different origin, containing overlapping fragments coding for human seryl-tRNA synthetase were sequenced: HFBDN14 (fetal brain clone); and IB48 (infant brain clone). For both clones the 5' region of the cDNA was missing. This 5' region was obtained via PCR methods using a human brain 5' RACE-Ready cDNA library. The complete cDNA sequence allowed us to define primers to isolate and characterize the intron/exon structure of the serS gene, consisting of 10 introns and 11 exons. The introns' sizes range from 283 bp to more than 3000 bp and the size of the exons from 71 bp to 222 bp. The availability of the gene structure of the human enzyme could help to clarify some aspects of the molecular evolution of class-II aminoacyl-tRNA synthetases. The human seryl-tRNA synthetase has been expressed in E. coli, purified (95% pure as determined by SDS/PAGE) and kinetic parameters have been measured for its substrate tRNA. The human seryl-tRNA synthetase sequence (514 amino acid residues) shows significant sequence identity with seryl-tRNA synthetases from E. coli (25%), Saccharomyces cerevisiae (40%), Arabidopsis thaliana (41%) and Caenorhabditis elegans (60%). The partial sequences from published mammalian seryl-tRNA synthetases are very similar to the human enzyme (94% and 92% identity for mouse and Chinese hamster seryl-tRNA synthetase, respectively). Human seryl-tRNA synthetase, similar to several other class-I and class-II human aminoacyl-tRNA synthetases, is clearly related to its bacterial counterparts, independent of an additional C-terminal domain and a N-terminal insertion identified in the human enzyme. In functional studies, the enzyme aminoacylates calf liver tRNA and prokaryotic E. coli tRNA.

Amino Acid Sequence↗

PubMed1988

Site-directed mutagenesis with the ptsH gene of Bacillus subtilis. Isolation and characterization of heat-stable proteins altered at the ATP-dependent regulatory phosphorylation site.

The codon for Ser-46 of the ptsH gene of Bacillus subtilis was modified by site-directed mutagenesis to the codons for Ala, Thr, Tyr, and Asp. The mutant genes were overexpressed, three of the corresponding proteins were purified to homogeneity with the exception for the Asp derivative, which could not be detected, although the gene had the desired nucleotide sequence. The phosphotransferase activity of the altered proteins was determined to be 20-35% of wild type activity, which correlates well with the slow phosphorylation of heat-stable protein (HPr) by enzyme I and phosphoenolpyruvate. The ATP-dependent HPr kinase, which previously was shown to be involved in the regulation of carbohydrate uptake of Gram-positive bacteria by covalent phosphorylation of Ser-46 of HPr, is entirely inactive toward the OH group of Thr-46 and Tyr-46 proteins. In addition, we constructed a strain of B. subtilis, where the altered gene coding for the Ala-46 derivative of HPr was introduced into the bacterial chromosome. The physiological properties of this mutant are described.

Adenosine Triphosphate↗

PubMed1993

Cloning and characterization of human colon glyoxalase-I.

Glyoxalase-I cDNA clones were isolated from a human colon cDNA library using polyclonal antibodies raised against the protein purified from human colon tissue. Positive clones were purified, subcloned, and their nucleotide sequence determined. The glyoxalase-I cDNA encodes a 184-amino acid protein with a predicted molecular weight of 20,774, corresponding to the monomeric subunit weight of the purified protein from human colon glyoxalase-I. The human enzyme showed 51% homology at the nucleotide level and 42% at the amino acid level with bacterial glyoxalase-I. Transfection of COS-1 cells with the 622-base pair cDNA containing the entire coding region cloned into a pMT2 vector produced an immunoreactive protein and an approximate 180-fold increase in glyoxalase-I enzyme activity as determined with methylglyoxal as a substrate. Transfection of a truncated cDNA lacking 94 base pairs of the 5'-coding sequence also produced an approximately 15-kDa immunoreactive protein, but with no detectable increase in enzyme activity. Northern analysis of the RNA showed an approximately 12-fold increase of the 2.2-kilobase glyoxalase-I transcript in carcinoma when compared to normal colon tissue from the same patient. Examination of colon carcinomas for the amplification of the glyoxalase-I gene by Southern blot analysis revealed no change in gene copy number. These results suggest induction of the glyoxalase-I gene expression in colon carcinomas.

Amino Acid Sequence↗

PubMed2002

Fast algorithms for large-scale genome alignment and comparison.

We describe a suffix-tree algorithm that can align the entire genome sequences of eukaryotic and prokaryotic organisms with minimal use of computer time and memory. The new system, MUMmer 2, runs three times faster while using one-third as much memory as the original MUMmer system. It has been used successfully to align the entire human and mouse genomes to each other, and to align numerous smaller eukaryotic and prokaryotic genomes. A new module permits the alignment of multiple DNA sequence fragments, which has proven valuable in the comparison of incomplete genome sequences. We also describe a method to align more distantly related genomes by detecting protein sequence homology. This extension to MUMmer aligns two genomes after translating the sequence in all six reading frames, extracts all matching protein sequences and then clusters together matches. This method has been applied to both incomplete and complete genome sequences in order to detect regions of conserved synteny, in which multiple proteins from one organism are found in the same order and orientation in another. The system code is being made freely available by the authors.

Algorithms↗