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PubMed1987

In vitro sodium bisulfite mutagenesis of restriction endonuclease recognition sites.

Sodium bisulfite treatment of single-stranded DNA deaminates exposed cytosine residues to form uracil, resulting in cytosine-to-thymidine transition mutations following DNA replication. We have used this reaction in vitro to destroy the recognition sequences for the restriction endonucleases HindIII and XmaI in the aminoglycoside 3'-phosphotransferase I coding region of plasmid pUC4K. This procedure should be applicable to the mutation of any recognition sequence of restriction endonucleases which generate cytosine-containing single-stranded ends. The possibility of mutagenesis of restriction sites to generate stop codons in coding regions is discussed.

Base Sequence↗

PubMed1989

In vivo selected promoter and ribosome binding site up-mutations: demonstration that the Escherichia coli bla promoter and a Shine-Dalgarno region with low complementarity to the 16 S ribosomal RNA function in Bacillus subtilis.

We have constructed a plasmid, pQS1, in which a mouse dihydrofolate reductase (5,6,7,8-tetrahydrofolate:NADP:oxidoreductase; EC 1.5.1.3; DHFR) cDNA is inserted in the unique PstI site of a gram-positive/gram-negative shuttle vector derived from pBR322. The cDNA is expressed under the control of the bla promoter, which, like most gram-negative bacterial genes, is considered not to be expressed in Bacillus subtilis, and its coding sequence is translated from a polycistronic message. We have selected in vivo and studied, in Escherichia coli and B. subtilis, expression mutants with promoter and ribosome binding site sequence mutations. One promoter mutation changes the third nucleotide of the -35 region from a C to a G. As expected, this substitution results in increased transcriptional activity in E. coli. In B. subtilis, this mutation induces the accumulation not only of a low but significant amount of dhfr mRNA but also of DHFR, demonstrating that binding strengths with a free energy as low as -9.4 kcal/mol are sufficient to promote ribosome binding in B. subtilis. The association of the promoter mutation (C-G) with a mutation which creates a strong B. subtilis ribosome binding site (-21 kcal/mol) results in the accumulation of a large amount of dhfr mRNA. This demonstrates the importance of having an efficient ribosome binding site in the evaluation of promoter function: for example, with this strong ribosome binding site we can show that the wild-type bla promoter is recognized by the B. subtilis transcription machinery.

Animals↗

PubMed1988

Nucleotide sequences of the Escherichia coli nagE and nagB genes: the structural genes for the N-acetylglucosamine transport protein of the bacterial phosphoenolpyruvate: sugar phosphotransferase system and for glucosamine-6-phosphate deaminase.

The genes coding for the enzymes of N-acetylglucosamine (GlcNAc) uptake and metabolism (nagA, nagB, and nagE) are located next to glutaminyl-tRNA synthetase gene (glnS) in the Escherichia coli genome. We determined the nucleotide sequence of the nagE (ptsN) gene, encoding the GlcNAc-specific enzyme II (NagE) of the phosphoenolpyruvate: sugar phosphotransferase system, and the sequence of the putative nagB gene, for glucosamine-6-phosphate deaminase. S1 mapping identified the mRNA transcript for nagE, indicating that nagE might be a sole constituent of the nagE operon, and divergent transcripts which are probably of the nagB, nagA genes. An evaluation of the hydrophobic and hydrophilic properties of NagE shows characteristics of a membrane protein. Also, NagE shows homologies to lactose permease and to the glucose-specific transport protein (enzyme IIGlc), and the glucose-specific phosphoryl carrier protein (enzyme IIIGlc). The latter two homologies are particularly interesting since no enzyme III-like protein for GlcNAc transport has been reported and enzyme IINag is of similar size as the combined enzymes IIGlc plus IIIGlc. This supports the idea that these two transport and phosphorylation systems may have evolved from a common ancestral gene.

Aldose-Ketose Isomerases↗

PubMed1989

Identification of the promoter sequences involved in the interleukin-6 dependent expression of the rat alpha 2-macroglobulin gene.

The 5'-region of the rat alpha 2-macroglobulin gene has been characterized. A 5.6 kb Sal I - Xba I fragment containing the first 4 exons of the alpha 2-macroglobulin gene and 1.3 kb of its 5'-flanking region was sequenced. The putative transcriptional start site was determined by RNase protection and primer extension analysis. TATA- and CAAT-box equivalent sequences were found. A potential glucocorticoid receptor binding site was located on the antisense strand. DNA sequences containing the 5'-flanking region of the rat alpha 2-macroglobulin gene were linked to the gene coding for the bacterial chloramphenicol acetyltransferase and introduced into Hep G2 cells. In these transfected Hep G2 cells CAT activity could be induced by recombinant human interleukin-6. Deletion analyses have shown that the sequences between -852 and -777 as well as between -404 and -165 relative to the cap site, contain regulatory elements involved in the interleukin-6 dependent induction of the alpha 2-macroglobulin gene.

Amino Acid Sequence↗

PubMed1987

Ontogenic expression of a CyI actin fusion gene injected into sea urchin eggs.

The 5' terminus of the CyI actin gene transcription unit of Strongylocentrotus purpuratus was located by primer extension and other procedures, and the flanking upstream region was partially sequenced and mapped. A fusion gene was constructed containing about 2.5 kb of 5' flanking sequence, the transcribed leader sequence, and the first few codons of the CyI gene ligated to the bacterial gene coding for chloramphenicol acetyl transferase (CAT). This was micro-injected into the cytoplasm of S. purpuratus eggs, and CAT enzyme activity was measured at various stages of embryonic development. CAT synthesis was activated between 10 and 14 h postfertilization, the same time at which newly synthesized transcripts of the endogenous CyI gene first appear. The exogenous CyI.CAT fusion DNA replicated actively during cleavage, as observed previously for other DNAs injected into sea urchin egg cytoplasm. Thus the absence of CAT activity prior to 10 h postfertilization could not be due to insufficient CyI.CAT genes. The amounts of CAT enzyme produced by embryos bearing CyI.CAT deletions that lack various regions of the CyI sequence were measured. As little as 254 nucleotides of upstream CyI sequence suffice for correct temporal activation of the fusion construct, although the level of CAT enzyme produced in embryos bearing any deletion retaining less than 850 nucleotides of upstream sequence was significantly lowered compared to controls bearing the complete CyI.CAT fusion construct.

Actins↗

PubMed2002

%(G+C) variation and prediction by a model of bacterial gene transfer and codon adaptation.

The %(G + C) of bacterial genomes ranges from 25% in Mycoplasma to 75% in Micrococcus. Our model for horizontal gene flow enabled a theoretical study of the adaptation of relative codon frequency to match the pattern of the tRNA set of a new host. This study explored the dynamic relationship of %(G + C) to vectors of relative codon frequency (F(gamma)), relative amino acid coding frequency (F(alpha)), and absolute codon frequency (F(|gamma|)) in chromosomes of nine, fully sequenced bacterial genomes that varied widely in %(G + C). At constant F(alpha), the theoretical maximum average range possible was %(G + C) = 37.4 +/- 0.9%. In simulations of F(gamma) adaptation to a new host following hypothetical gene transfer, we modeled %(G + C) as a function of F(gamma) and F(alpha). The simulation revealed that %(G + C) is dependent on F(gamma) and F(alpha) in an explicit relationship described in this paper. We conclude that (1) F(gamma) and F(alpha) determine %(G + C), and (2) the degree of adaptation of %(G + C) in a transferred gene depends upon the degree of F(gamma) equilibration and the similarity of F(alpha) of the transferred gene to that of the new host.

Base Sequence↗

PubMed1995

Induction of a cellular immune response to a defined T-cell epitope as an insert in the flagellin of a live vaccine strain of Salmonella.

Attenuated strains of Salmonella have been used as vaccines to deliver heterologous antigens mainly to generate a humoral immune response. However, little is known about their ability to induce a cell-mediated immune response to the T-cell epitopes of another infectious agent or how optimally to deliver these epitopes to the host immune system. In order to study this question, a well defined MHC class II-restricted epitope (residues 88-103) from moth cytochrome C (MCC) was inserted into the central hypervariable domain of the flagellin of an attenuated strain of Salmonella dublin. The resulting flagellin was exported to the bacterial surface and polymerized into flagellar filaments that contained multiple copies of the MCC epitope. When flanked by Lys-Lys cathepsin B cleavage sites to facilitate its proteolytic release within the endosomal compartment of antigen-presenting cells, the MCC-chimeric flagellin epitope was efficiently processed in vitro by mouse peritoneal macrophages and presented to 2B4 T-hybridoma cells (specific for the MCC epitope 88-103). Stable expression of the epitope and a higher immune response was obtained in H-2k mice by integrating the chimeric flagellin gene into the chromosome of the vaccine strain. Bacteria with MCC-chimeric flagellins that were expressed from a stable chromosomal locus and flanked by cathepsin B cleavage sites were cleared more rapidly from the livers and spleens of transgenic mice with T-cell receptor (TCR) alpha and beta chains specific for the MCC epitope than were bacteria lacking the epitope. Antigen processing and presentation of class II-restricted epitopes expressed as chimeric proteins by attenuated bacterial vaccine vectors may be facilitated by the presence of endosomal protease cleavage sites on each side of the epitope and by chromosomal integration of the coding sequence.

Amino Acid Sequence↗

PubMed2004

Isolation and expression of the genes coding for the membrane bound transglycosylase B (MltB) and the transferrin binding protein B (TbpB) of the salmon pathogen Piscirickettsia salmonis.

We have isolated and sequenced the genes encoding the membrane bound transglycosylase B (MltB) and the transferring binding protein B (TbpB) of the salmon pathogen Piscirickettsia salmonis. The results of the sequence revealed two open reading frames that encode proteins with calculated molecular weights of 38,830 and 85,140. The deduced aminoacid sequences of both proteins show a significant homology to the respective protein from phylogenetically related microorganisms. Partial sequences coding the amino and carboxyl regions of MltB and a sequence of 761 base pairs encoding the amino region of TbpB have been expressed in E. coli. The strong humoral response elicited by these proteins in mouse confirmed the immunogenic properties of the recombinant proteins. A similar response was elicited by both proteins when injected intraperitoneally in Atlantic salmon. The present data indicates that these proteins are good candidates to be used in formulations to study the protective immunity of salmon to infection by P. salnonis.

Animals↗

PubMed1998

The chloroplast division protein FtsZ is encoded by a nucleomorph gene in cryptomonads.

Guillardia theta is a cryptomonad alga, whose phototrophic symbiont was acquired by secondary endocytobiosis. The nucleomorph, the vestigial nucleus of the eukaryotic endosymbiont, harbors three linear chromosomes with a total coding capacity of 515 kb. Sequencing of the nucleomorph genome reveals that it encodes an ORF homologous to the bacterial cell division protein FtsZ, supporting the hypothesis that FtsZ is common in chloroplasts. We show that the nucleomorph-encoded ftsZ gene is transcribed. The transcript is polyadenylated and therefore shows features typical of eukaryotic transcripts. However, 3' processing of nucleomorph mRNA is inaccurate. Transcripts of nucleomorph genes in G. theta overlap with neighboring UTRs and coding regions. We demonstrate that the reading frame encoding NmFtsZ is not interrupted by introns. Subcellular localization of the protein reveals that FtsZ is localized exclusively in the chloroplast of G. theta, demonstrating that FtsZ is imported into the organelle.

Amino Acid Sequence↗

PubMed1996

Construction of a human cytochrome P450 1A1: rat NADPH-cytochrome P450 reductase fusion protein cDNA and expression in Escherichia coli, purification, and catalytic properties of the enzyme in bacterial cells and after purification.

A plasmid (pCW) was modified to code for a fusion protein consisting of the complete sequence of human cytochrome P450 (P450) 1A1 (with only the second amino acid changed) in the N-terminal portion connected by a Ser-Thr linker to the portion of rat NADPH-P450 reductase beginning at amino acid 57. This plasmid was used to express the fusion protein in Escherichia coli DH5alpha cells and the protein was purified from detergent-solubilized bacterial membranes using DEAE and 2',5'-ADP agarose chromatography. The purified fusion protein catalyzed benzo[a]pyrene 3-hydroxylation, 7-ethoxyresorufin O-deethylation, and zoxazolamine 6-hydroxylation. Catalytic activity was not increased in the presence of added NADPH-P450 reductase, cytochrome b5, or phospholipid. The fusion protein could also transfer electrons to cytochromes c and b5 but not P450 lA2. The same oxidation products of benzo[a]pyrene were formed with the purified fusion protein and the fusion protein functioning in bacterial cells. The catalytic activity of the human P450 1A1 fusion protein toward several substrates is markedly less than that of a similar fusion protein constructed with rat P450 1A1, in line with the reported differences in catalytic activities of the rat and human P450 1A1 enzymes. The purified fusion protein also oxidized (+)- and (-)-benzo[a]pyrene 7,8-dihydrodiols and eight aryl and heterocyclic amines to genotoxic products, in the absence of added NADPH-P450 reductase. The demonstration of catalytic activities of the human fusion protein within bacterial cells suggests the prospect of utilizing such cellular systems for production of human P450 metabolites.

Animals↗

PubMed1994

Two nuclear-coded subunits of mitochondrial complex I are similar to different domains of a bacterial formate hydrogenlyase subunit.

A computer comparison of protein sequences revealed similarity between the 30.4 kDa subunit of complex I from the fungus Neurospora crassa and the ORF5 subunit of formate hydrogenlyase from Escherichia coli. The ORF5 protein was previously known to be homologous to the 49 kDa component of the mitochondrial enzyme. We show that the 30.4 kDa corresponds to the N-terminal part while the 49 kDa subunit corresponds to the C-terminal portion of the bacterial protein. Thus, this bacterial protein represents a fusion of the two mitochondrial polypeptides suggesting that the two complex I genes arose from a single ancestor. Our results indicate that the 30.4 kDa and 49 kDa subunits are part of a structural and functional unit in complex I.

Amino Acid Sequence↗

PubMed1988

Mapping and sequencing of the dihydrofolate reductase gene (DFR1) of Saccharomyces cerevisiae.

The dihydrofolate reductase gene (DFR1) from Saccharomyces cerevisiae has been mapped and sequenced. The gene was isolated on an 8.8-kb BamHI fragment from a yeast genomic library by screening of Escherichia coli transformants for resistance to trimethoprim. A 1.8-kb SalI-BamHI fragment which was able to confer methotrexate resistance in yeast also complemented an E. coli DHFR-deficient (folA) mutant. Nucleotide sequence analysis revealed that the yeast DFR1 gene encoded a polypeptide with a predicted Mr of 24230. The deduced sequence of 211 amino acid residues showed considerable homology with DHFRs from both bacterial and animal sources. The codon bias index of the DFR1 coding region is 0.0083, which indicates a random pattern of codon usage. The upstream region contains two consensus sequences required for binding of the yeast's positive regulatory factor, GCN4, suggesting that the DFR1 gene might be subject to the amino acid general control. Several potential 'TATA' boxes are located in the sequence 5' to the gene. Located in the 3' flanking region are homologies with several canonical sequences thought to be required for efficient transcription termination in yeast. We also mapped the DFR1 gene to a position 1.4 cM proximal to the MET7 locus on chromosome XV.

Amino Acid Sequence↗

PubMed1993

Heat induction of sigma 32 synthesis mediated by mRNA secondary structure: a primary step of the heat shock response in Escherichia coli.

Induction of heat shock proteins following transfer of E. coli cells from 30 degrees C to 42 degrees C depends on rapid accumulation of sigma 32, a minor sigma factor specifically required for transcription of heat shock genes. The synthesis of sigma 32 is induced by enhancing translation of its mRNA transcribed from the rpoH (htpR) gene. We previously showed that the translational control of rpoH-lacZ gene fusion is mediated by two cis-acting rpoH coding regions presumably involving mRNA secondary structure. To further examine this model, we constructed and analyzed a set of gene fusions carrying base substitution(s) or internal deletions within rpoH, including constitutive mutations predicted to destroy the mRNA secondary structure and compensatory second-site mutations that may restore the secondary structure. The results demonstrate that base pairings between the translation initiation region of some 20 nucleotides and part of the internal complementary sequences are critical for maintaining repression during steady-state growth and for modulating heat-induced synthesis of sigma 32-beta-galactosidase fusion protein upon temperature upshift. Furthermore, some of the compensatory mutations resulted in super-repressed (non-inducible) phenotypes, suggesting that the heat induction depends on a specific nucleotide sequence(s) as well as the mRNA secondary structure within the 5'-proximal regulatory segment of rpoH coding region.

Bacterial Proteins↗

PubMed2006

Naturally occurring adenines within mRNA coding sequences affect ribosome binding and expression in Escherichia coli.

Translation initiation requires the precise positioning of a ribosome at the start codon. The major signals of bacterial mRNA that direct the ribosome to a translational start site are the Shine-Dalgarno (SD) sequence within the untranslated leader and the start codon. Evidence for the presence of many non-SD-led genes in prokaryotes provides a motive for studying additional interactions between ribosomes and mRNA that contribute to translation initiation. A high incidence of adenines has been reported downstream of the start codon for many Escherichia coli genes, and addition of downstream adenine-rich sequences increases expression from several genes in E. coli. Here we describe site-directed mutagenesis of the E. coli aroL, pncB, and cysJ coding sequences that was used to assess the contribution of naturally occurring adenines to in vivo expression and in vitro ribosome binding from mRNAs with different SD-containing untranslated leaders. Base substitutions that decreased the downstream adenines by one or two nucleotides decreased expression significantly from aroL-, pncB-, and cysJ-lacZ fusions; mutations that increased downstream adenines by one or two nucleotides increased expression significantly from aroL- and cysJ-lacZ fusions. Using primer extension inhibition (toeprint) and filter binding assays to measure ribosome binding, the changes in in vivo expression correlated closely with changes in in vitro ribosome binding strength. Our data are consistent with a model in which downstream adenines influence expression through their effects on the mRNA-ribosome association rate and the amount of ternary complex formed. This work provides evidence that adenine-rich sequence motifs might serve as a general enhancer of E. coli translation.

Adenine↗

PubMed1985

A change in the genetic code in Mycoplasma capricolum.

Mycoplasma capricolum was previously found to use UGA instead of UGG as its codon for tryptophan and to contain 75% A + T in its DNA. The codon change could have been due to mutational pressure to replace C + G by A + T, resulting in the replacement of UGA stop codons by UAA, change of the anticodon in tryptophan tRNA from CCA to UCA, and replacement of UGG tryptophan codons by UGA. None of these changes should have been deleterious.

Anticodon↗

PubMed1998

PPF-1, a post-floral-specific gene expressed in short-day-grown G2 pea, may be important for its never-senescing phenotype.

We cloned a developmentally regulated gene from a cDNA library constructed from short-day (SD) grown G2 pea tissue using cDNA representational difference analysis (cDNA RDA) and named it PPF-1 for the first Pisum sativum post-floral-specific gene. Sequence comparisons with various databases revealed that PPF-1 shares a substantial homology only at the deduced amino-acid level with the Bacillus subtilis gene SP3J, which is required for maintaining vegetative growth, and with other genes coding for bacterial inner membrane proteins. All five potential hydrophobic regions from the bacterial proteins were maintained in the PPF-1 sequence. A series of Northern blots showed that this gene was only expressed after floral initiation and was limited to the apical buds, with non-detectable levels in roots, stems and mature leaves. Under SD conditions, when G2 pea displays an unlimited growth habit, PPF-1 expression was sustained at a relatively high level long after floral initiation. Under long-day (LD) conditions, when G2 pea undergoes an apical senescence similar to wild-type plants with genotype sn hr, PPF-1 was only expressed very briefly after flower initiation. Interestingly, in day-neutral, wild-type Alaska pea, the PPF-1 level was hardly detectable under any growth conditions. Treatment of LD-grown G2 pea with gibberellin A3 (GA3) was able to stimulate PPF-1 expression unless it was applied at a very late growth stage, at which time the process of apical senescence cannot be reversed.

Amino Acid Sequence↗

PubMed1985

Sequence of the serotype-specific glycoprotein of the human rotavirus Wa strain and comparison with other human rotavirus serotypes.

Complementary DNA was synthesized from the double-stranded RNA of the Wa strain of human rotavirus and inserted into the bacterial plasmid pBR322. Clones which contained the gene that codes for the viral glycoprotein (VP7) were identified and the nucleotide sequence was determined. The gene was 1062 base pairs in length with an open reading frame which coded for 326 amino acids. Two potential glycosylation sites were found as well as two hydrophobic regions at the N-terminus of the polypeptide. The untranslated regions at the 5' and 3' ends were 48 base pairs and 33 base pairs long, respectively. Only one nucleotide at position 493 differed from the sequence of the Wa VP7 gene described by Richardson et al. (1984, J. Virol. 51, 860-862). A strong prokaryotic promoter sequence was also found between residues 434 and 462. A comparison of the amino acid sequence of the Wa strain (serotype 1) to the Hu/5 strain of human rotavirus (serotype 2) and SA11, the simian rotavirus (serotype 3), revealed a high degree of homology (79.1% and 83.1%, respectively) between the serotypes, suggesting that rotavirus serotypes are stable. The hydrophilic regions of VP7 of the three serotypes were identified and compared for homology. Four of these regions showed variation between serotypes.

Amino Acid Sequence↗

PubMed1980

Expression of cloned beta-endorphin gene sequences by Escherichia coli.

DNA coding for the opiate peptide beta-endorphin has been cloned into bacterial plasmids in such a way as to direct the synthesis of a hybrid beta-galactosidase/beta-endorphin protein. This hybrid protein can readily be cleaved in vitro to release biologically active beta-endorphin.

Animals↗