A combined bacterial and liver test system for detection and classification of carcinogens as mutagens.
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Animals↗
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Results for “bacterial coding sequence”
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Molecular cloning and nucleotide sequence of the gene coding photosensitive nitrile hydratase.
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Amino Acid Sequence↗
Escherichia coli purB gene: cloning, nucleotide sequence, and regulation by purR.
Escherichia coli purB encodes adenylosuccinate lyase (ASL), the enzyme that catalyzes step 8 in the pathway for de novo synthesis of IMP and also the final reaction in the two-step sequence from IMP to AMP. Gene purB was cloned and found to encode an ASL protein of 435 amino acids having a calculated molecular weight of 49,225. E. coli ASL is homologous to the corresponding enzymes from Bacillus subtilis and chickens and also to fumarase from B. subtilis. Gene phoP is 232 bp downstream of purB. Gene purB is regulated threefold by the purine pool and purR. Transcriptional regulation of purB involves binding of the purine repressor to the 16-bp conserved pur regulon operator. The purB operator is 224 bp downstream of the transcription start site and overlaps codons 62 to 67 in the protein-coding sequence.
Adenosine Monophosphate↗
Bacterial synthesis of herpes simplex virus types 1 and 2 glycoprotein D antigens.
We have used elements of the E. coli lactose (lac) operon to produce a collection of herpes simplex virus types 1 and 2 glycoprotein D (gD-1 and gD-2) antigens. Our approach employed recombinant DNA techniques to construct plasmids with various segments of the gD-1 and gD-2 coding sequences fused to the lacZ gene. Such hybrid genes were expressed in a regulated manner in E. coli by joining them to the lac promoter-operator region. Efficient translation of these hybrid genes was facilitated by incorporating a coding sequence specifying a short peptide leader (lambda cro) in the plasmid expression vectors resulting in synthesis of chimeric Cro-gD-beta-galactosidase proteins. In addition, insertion of synthetic translation terminators at the junction of gD and lacZ enabled us to produce specific truncated gD polypeptide sequences unfused to beta-galactosidase. The gD antigens produced in E. coli were not glycosylated and were generally recovered as dense insoluble aggregates. Proteins containing portions of gD-1 or gD-2 were analyzed by immunoprecipitation using anti-HSV rabbit serum and a number of monoclonal antibodies recognizing different epitopes of gD-1. Initial animal studies were done with antigens that reacted with neutralizing antisera or monoclonal antibodies. When these bacterially produced proteins were injected into rabbits, antibodies were produced that specifically immunoprecipitated authentic gD polypeptides and neutralized the infectivity of both virus types. These studies suggest that gene fusion techniques can be used to produce immunogenic proteins in large quantity. These polypeptides are not only useful in analyses of gene structure and function, but also can provide novel diagnostic reagents and well-defined pure antigens for vaccine development.
Amino Acid Sequence↗
Computer aided identification of a potential 5'-3' exonuclease gene encoded by Escherichia coli.
The predicted amino acid sequence of a previously overlooked open reading frame (ORF) from E. coli has been subjected to sequence analysis. The ORF, sequenced independently by two research groups, appears to encode a protein of 251 amino acids residues (25 kDa). The protein sequence shares a high degree of similarity with the amino acid termini of E. coli DNA polymerase I and other bacterial DNA polymerases (and phage-encoded enzymes) known to possess 5'-3' exonuclease activity. The newly identified E. coli gene, designated exo, is positioned downstream of signals characteristic of an efficient translation initiation sequence. Codon bias analysis indicates that the ORF has good protein coding potential. These observations indicate that the gene is likely to be expressed. The gene is 60% identical with the 5'-region of the E. coli DNA PolI gene (polA) over a 260 base-pair region. These two genes may have arisen by duplication of an ancestral gene or polA may have arisen by a recombination event involving exo. A comparison of the predicted secondary structures of 5'-3' exonucleases revealed the presence of several conserved regions of secondary structure, including a potential helix-turn-helix motif.
Amino Acid Sequence↗
Functional variation of MC1R alleles from red-haired individuals.
Red hair in humans is associated with variant alleles of the alphaMSH receptor gene, MC1R. Loss of MC1R function in other mammals results in red or yellow hair pigmentation. We show that a mouse bacterial artificial chromosome (BAC) which contains Mc1r will efficiently rescue loss of Mc1r in transgenic mice, and that overexpression of the receptor suppresses the effect of the endogenous antagonist, agouti protein. We engineered the BAC to replace the mouse coding region with the human MC1R sequence and used this in the transgenic assay. The human receptor also efficiently rescued Mc1r deficiency, and in addition, appeared to be completely resistant to the effects of agouti, suggesting agouti protein may not play a role in human pigmentary variation. Three human variant alleles account for 60% of all cases of red hair. We engineered each of these in turn into the BAC and find that they have reduced, but not completely absent, function in transgenic mice. Comparison of the phenotypes of alphaMSH-deficient mice and humans in conjunction with this data suggests that red hair may not be the null phenotype of MC1R.
Alleles↗
Characterization of a protocatechuate catabolic gene cluster from Rhodococcus opacus 1CP: evidence for a merged enzyme with 4-carboxymuconolactone-decarboxylating and 3-oxoadipate enol-lactone-hydrolyzing activity.
The catechol and protocatechuate branches of the 3-oxoadipate pathway, which are important for the bacterial degradation of aromatic compounds, converge at the common intermediate 3-oxoadipate enol-lactone. A 3-oxoadipate enol-lactone-hydrolyzing enzyme, purified from benzoate-grown cells of Rhodococcus opacus (erythropolis) 1CP, was found to have a larger molecular mass under denaturing conditions than the corresponding enzymes previously purified from gamma-proteobacteria. Sequencing of the N terminus and of tryptic peptides allowed cloning of the gene coding for the 3-oxoadipate enol-lactone hydrolase by using PCR with degenerate primers. Sequencing showed that the gene belongs to a protocatechuate catabolic gene cluster. Most interestingly, the hydrolase gene, usually termed pcaD, was fused to a second gene, usually termed pcaC, which encodes the enzyme catalyzing the preceding reaction, i.e., 4-carboxymuconolactone decarboxylase. The two enzymatic activities could not be separated chromatographically. At least six genes of protocatechuate catabolism appear to be transcribed in the same direction and in the following order: pcaH and pcaG, coding for the subunits of protocatechuate 3,4-dioxygenase, as shown by N-terminal sequencing of the subunits of the purified protein; a gene termed pcaB due to the homology of its gene product to 3-carboxy-cis,cis-muconate cycloisomerases; pcaL, the fused gene coding for PcaD and PcaC activities; pcaR, presumably coding for a regulator of the IclR-family; and a gene designated pcaF because its product resembles 3-oxoadipyl coenzyme A (3-oxoadipyl-CoA) thiolases. The presumed pcaI, coding for a subunit of succinyl-CoA:3-oxoadipate CoA-transferase, was found to be transcribed divergently from pcaH.
Amino Acid Sequence↗
Origins of minigene-dependent growth inhibition in bacterial cells.
The expression of very short open reading frames in Escherichia coli can lead to the inhibition of translation and an arrest in cell growth. Inhibition occurs because peptidyl-tRNA hydrolase fails to recycle sufficiently rapidly peptidyl-tRNA released from ribosomes at the stop signal in competition with normal termination, causing starvation for essential species of tRNA. Previous studies have shown that the last sense codon, the strength of the Shine-Dalgarno sequence and the nature and context of the stop codon affect the toxicity associated with mini-gene expression. Here, several important parameters are studied as a function of the length of the mini-gene coding sequence. The rate of peptidyl-tRNA drop-off catalysed by translation factors decreases dramatically for peptides longer than a hexamer. The probability that ribosomes recycle without dissociation of the mini-gene mRNA varies strongly with the length of the coding sequence. The peptidyl-tRNA hydrolase rap mutant, unlike the wild-type enzyme, is highly sensitive to the length and sequence of the peptide. Together, these parameters explain the length dependence of mini-gene toxicity.
Bacterial Proteins↗
Sequence of the protease of human subgroup E adenovirus type 4.
A fragment of DNA containing the protease gene and 3' and 5' flanking regions of human adenovirus type 4 (Ad4) has been cloned and sequenced. The gene is located between 59 and 62 map units and codes for a protein of 201 amino acids with a calculated Mr of 22,758. The Ad4 protease has a 72% amino acid homology with the Ad2 protease, the divergence being concentrated in the middle of the molecule. Comparison with other mammalian and bacterial proteases failed to reveal any significant homology and in particular a putative active site. The adenoviral proteases may therefore represent a novel class of enzymes.
Adenoviruses, Human↗
Sequence analysis of 0.5 Mb of the rabbit germline immunoglobulin heavy chain locus.
A bacterial artificial chromosome (BAC) library was created using partially digested rabbit chromosomal DNA. Four BAC clones spanning about 0.5 Mb of the rabbit immunoglobulin (Ig) heavy chain locus were isolated and sequenced. Three of the BAC clones were partially overlapping. Thirty-four V elements, 11 D elements, DQ52, six J elements and the coding regions of Cmicro, Cgamma, C and four Calpha genes were identified and characterized. Additionally, the sequence of a fosmid clone spanning Calpha13 and 30 kb 3'enhancer region was determined. The organization of the locus and the potential function of newly identified functional and structural elements are discussed.
Amino Acid Sequence↗
The human glucocerebrosidase gene and pseudogene: structure and evolution.
We report the sequence of the entire human gene encoding beta-glucocerebrosidase and that of the associated pseudogene. The gene contains 11 exons extending from base pair 355 to base pair 7232 in the overall sequence. The gene promoter contains TATA- and CAT-like boxes upstream of the major 5' end of the glucocerebrosidase RNA. The two TATA boxes lie between nucleotides (-23)-(-27) and (-33)-(-39) and the two possible CAT boxes reside between nucleotides (-90)-(-94) and (-96)-(-99) in relation to the major 5' end of the mRNA. The functionality of the promoter region was monitored by coupling it to the bacterial gene coding for chloramphenicol acetyltransferase (CAT) and assaying the expression of the enzyme in cells transfected with this vector. The glucocerebrosidase promoter not only directs synthesis of the bacterial enzyme but also exhibits the same pattern of tissue-specific expression as that of the endogenous gene. An apparently tightly linked pseudogene is approximately 96% homologous to the functional gene. However, introns 2, 4, 6, and 7 have large "deletions" consisting of Alu sequences 313, 626, 320, and 277 bp in length, respectively. It is entirely possible that the ancestral gene lacks these sequences and that they have been inserted into the introns of the functioning gene. There is also a 55-bp deletion from a part of exon 9 flanked by a short inverted repeat. The sequence data should facilitate development of methods for diagnosis of Gaucher disease at the molecular level.
Base Sequence↗
A sugar-specific porin, ScrY, is involved in sucrose uptake in enteric bacteria.
During the molecular analysis of a plasmid-coded sucrose metabolic pathway of enteric bacteria, a gene, scrY, was found whose product, ScrY, had all the properties of a bacterial porin (Schmid et al., 1988). Loss of this protein (Mr 58 kDa), localized in the outer membrane, led, as shown here, to an increase in the apparent Km for sucrose transport in whole cells from 10 microM in wild-type cells to 300 microM in mutant cells. This contrasts with the Km for sucrose phosphorylation as measured in membrane vesicles from mutant and wild-type cells, which remained unchanged at about 10 microM, and reflects the activity of the sucrose-specific Enzymell of the phosphoenolpyruvate-dependent carbohydrate:phosphotransferase system (PTS) responsible for uptake through the inner membrane. Furthermore, the presence of ScrY restored growth on maltodextrins in cells devoid of LamB, thus complementing the lack of this maltoporin. The amino acid sequence deduced from the DNA sequence was determined for the plasmid-coded and the ScrY porin coded in the chromosome of Klebsiella pneumoniae. Both show high identity (86%) to each other, and to the channel domain of LamB, further corroborating the conclusion that they constitute porins.
Amino Acid Sequence↗
Polyadenylation of mRNA in prokaryotes.
The 3'-ends of both prokaryotic and eukaryotic mRNA are polyadenylated, but the poly(A) tracts of prokaryotic mRNA are generally shorter, ranging from 15 to 60 adenylate residues and associated with only 2-60% of the molecules of a given mRNA species. The sites of polyadenylation of bacterial mRNA are diverse and include the 3'-ends of primary transcripts, the sites of endonucleolytic processing in the 3' untranslated and intercistronic regions, and sites within the coding regions of mRNA degradation products. The diversity of polyadenylation sites suggests that mRNA polyadenylation in prokaryotes is a relatively indiscriminate process that can occur at all mRNA's 3'-ends and does not require specific consensus sequences as in eukaryotes. Two poly(A) polymerases have been identified in Escherichia coli. They are encoded by unlinked genes, neither of which is essential for growth, suggesting significant functional overlap. Polyadenylation promotes the degradation of a regulatory RNA that inhibits the replication of bacterial plasmids and may play a similar role in the degradation of mRNA. However, under certain conditions, poly(A) tracts may lead to mRNA stabilization. Their ability to bind S1 ribosomal protein suggests that poly(A) tracts may also play a role in mRNA translation.
Archaea↗
Delimiting a rice wide-compatibility gene S5n to a 50 kb region.
Wide-compatibility (WC) is one of the most important traits in rice, which can overcome the fertility barrier in the indica/japonica hybrids, and hence to make it possible to utilize the higher yield potential of inter-subspecific hybrids. The S5n gene located on chromosome 6 has been previously reported to be responsible for the wide-compatibility in rice. Here we report the precise location of the S5n gene. In the first-pass mapping, the S5n gene was restricted within a 200 kb region by using a population of 242 isogenic lines in combination with high-density markers developed in the S5 region. In the fine mapping, the S5 region was further saturated with newly developed markers and more isogenic lines (549 in total) were investigated. Eventually, the S5n gene was mapped within a 50 kb region delimited by the left marker J13 and the right marker J17. One BAC clone screened from the BAC library of the WC rice variety 02428 covered the whole S5 region. Sequence analysis of the 50 kb region revealed two candidate genes, coding an aspartyl protease and a hypothetical protein. This result would greatly accelerate both cloning and marker-assisted selection of this important S5n gene.
Chromosome Mapping↗
Nucleotide sequence and analysis of the plant-inducible locus pinF from Agrobacterium tumefaciens.
Several loci on the tumor-inducing plasmid from Agrobacterium tumefaciens were transcriptionally activated in the presence of wounded plant tissue or extracts. The inducible virulence loci were required for efficient tumor formation. In contrast, the plant-inducible locus pinF was not observed to be absolutely essential for virulence. Mutants in pinF showed an attenuated virulence on a variety of dicotyledonous hosts, and this attenuation became more pronounced with decreasing numbers of bacterial cells in the inoculum. The DNA sequence of a 5.5-kilobase region which included the pinF locus from the octopine-type tumor-inducing plasmid A6 was determined. Four open reading frames consistent with the observed transcription of pinF were observed. Two of the open reading frames, pinF1 and pinF2, coded for polypeptides with relative molecular weights of 47,519 (pinF1) and 46,740 (pinF2). A comparison of the amino acid sequences of pinF1 and pinF2 indicated that they were similar to each other and to known polypeptide sequences for cytochrome P-450 enzymes.
Amino Acid Sequence↗
KRC transcripts: identification of an unusual alternative splicing event.
Mouse KRC is a large zinc finger protein that binds to the kappaB motif of gene transcription and to the recognition signal sequences for the somatic recombination of the immunoglobulin and T-cell receptor gene segments. The mouse KRC gene is more than 70 kilobases (kb) in size, and contains at least seven exons, with the largest transcript being approximately 9.5 kb. Multiple differentially spliced transcripts of KRC were identified in thymus and brain, which would result in the production of multiple KRC protein isoforms with different N-termini and number of DNA binding domains. Alternative splicing events leading to the production of these multiple transcripts have been elucidated. Of particular interest are the exclusions in some transcripts of sequences from a gigantic exon of 5487 base pairs (bp), or from an exon of 176 bp. Both potentially deleted exons code for zinc finger motifs that are essential components of the N-terminal and C-terminal DNA binding domains, respectively. Another intriguing phenomenon found in some KRC transcripts is the skipping of a 459 bp fragment within the gigantic exon that would code for the N-terminal DNA binding domain. Bacterial fusion proteins derived from this fragment bind specifically to KRC target DNAs. Apparently, distinct alternative splicing events could eliminate the N-terminal DNA binding domain of KRC.
Alternative Splicing↗
Gene and protein characterization of the human glutathione S-transferase kappa and evidence for a peroxisomal localization.
Kappa class glutathione S-transferase (GST) cDNA sequences have been identified in rat, mouse, and human. In the present study, we determined the structure and chromosomal location of the human GST Kappa 1 (hGSTK1) gene, characterized the protein, and demonstrated its subcellular localization. The human gene spans approximately 5 kb, has 8 exons, and maps onto chromosome 7q34. The 5'-flanking region lacks TATA or CCAAT boxes, but there is an initiator element overlapping the transcription start site. hGSTK1 amino acid sequence showed homology to bacterial 2-hydroxychromene-2-carboxylate isomerase, an enzyme involved in naphthalene degradation pathway. hGSTK1 mRNA was expressed in all of the organs examined. Subcellular fractionation of HepG2 cells showed that the protein was located in peroxisomes and mitochondria and was not detectable in cytoplasm. The peroxisomal localization was confirmed by transfection of HepG2 cells with a plasmid coding a green fluorescent protein fused inframe to the N terminus of hGSTK1. The C terminus of hGSTK1 was essential for localization of the protein to peroxisomes, and the C-terminal sequence Ala-Arg-Leu represents a peroxisome targeting signal. This is the first time that a human GST has been found in peroxisomes, suggesting a new function for this family of enzymes.
Amino Acid Sequence↗
Reannotation of Shewanella oneidensis genome.
As more and more complete bacterial genome sequences become available, the genome annotation of previously sequenced genomes may become quickly outdated. This is primarily due to the discovery and functional characterization of new genes. We have reannotated the recently published genome of Shewanella oneidensis with the following results: 51 new genes have been identified, and functional annotation has been added to the 97 genes, including 15 new and 82 existing ones with previously unassigned function. The identification of new genes was achieved by predicting the protein coding regions using the HMM-based program GeneMark.hmm. Subsequent comparison of the predicted gene products to the non-redundant protein database using BLAST and the COG (Clusters of Orthologous Groups) database using COGNITOR provided for the functional annotation.
Algorithms↗