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PubMed1992

Context rules of rightward overlapping reading.

We have investigated the mechanism and sequence context rules governing ribosome frameshifting promoted by aminoacyl-tRNA limitation. In the case of one shifty sequence, frameshifting promoted by lysyl-tRNA limitation occurs at the sequence AAG C and is due to rightward movement of the ribosome so as to read the AGC triplet overlapping the hungry codon from the right. The frequency of this event is unaffected by sequence elements more than three bases to the left (upstream) or two bases to the right (downstream) of the hungry codon, and only slightly affected by the identity of the base two bases to the right. It is strongly affected by the base immediately to the right of the hungry codon, which becomes the wobble base of the shifted triplet; and by the third base of the hungry codon, even though the two synonyms (AAG and AAA) call for the same aminoacyl-tRNA; and by the identity of the base immediately to the left of the hungry codon. The latter result suggests that the aminoacyl-tRNA in the P site affects the maintenance of reading frame at the adjacent A site of the ribosome. However, the DNA sequence makes it seem unlikely that the P-site tRNA shifts to the right in concert with the A-site tRNA, a mechanism that can account for leftward frameshifting (in the opposite direction) in retroviral translation. The specificity of sequence determinants of leftwing versus rightwing frameshifting is discussed.

Amino Acid Sequence↗
PubMed1992

Sequence and expression in Escherichia coli of the 40-kDa subunit of activator 1 (replication factor C) of HeLa cells.

Activator 1 (A1; also called replication factor C), in conjunction with proliferating-cell nuclear antigen (PCNA), is essential for the elongation of primed DNA templates by DNA polymerases delta and epsilon. A1 contains five distinct subunits of 145, 40, 38, 37, and 36.5 kDa. Here we describe the isolation, sequence, and bacterial expression of a cDNA coding for the 40-kDa subunit. In keeping with the presence of an ATP-binding motif, the bacterially expressed 40-kDa subunit binds ATP. The interaction between the 40-kDa subunit and ATP was reduced by the addition of PCNA. In addition, antibodies raised against the 40-kDa subunit abolished the A1- and PCNA-dependent synthesis of DNA catalyzed by polymerase delta. The putative amino acid sequence of the 40-kDa subunit of A1 revealed significantly homology with the bacteriophage T4 gene 44 protein and, to a lesser degree, with the tau and gamma subunits of Escherichia coli DNA polymerase III holoenzyme.

Adenosine Triphosphate↗
PubMed1988

Sequences contained within the promoter of the human thymidine kinase gene can direct cell-cycle regulation of heterologous fusion genes.

Recent evidence on the transcriptional regulation of the human thymidine kinase (TK) gene raises the possibility that cell-cycle regulatory sequences may be localized within its promoter. A hybrid gene that combines the TK 5' flanking sequence and the coding region of the bacterial neomycin-resistance gene (neo) has been constructed. Upon transfection into a hamster fibroblast cell line K12, the hybrid gene exhibits cell-cycle-dependent expression. Deletion analysis reveals that the region important for cell-cycle regulation is within -441 to -63 nucleotides from the transcriptional initiation site. This region (-441 to -63) also confers cell-cycle regulation to the herpes simplex virus thymidine kinase (HSVtk) promoter, which is not expressed in a cell-cycle manner. We conclude that the -441 to -63 sequence within the human TK promoter is important for cell-cycle-dependent expression.

Base Sequence↗
PubMed1992

Isolation and sequencing of the cDNA coding for spinach 10-formyltetrahydrofolate synthetase. Comparisons with the yeast, mammalian, and bacterial proteins.

The one-carbon metabolism enzymes 10-formyltetrahydrofolate synthetase (EC 6.3.4.3), 5,10-methenyltetrahydrofolate cyclohydrolase (EC 3.5.4.9), and 5,10-methylenetetrahydrofolate dehydrogenase (EC 1.5.1.5) can be found on a single trifunctional protein in the eukaryotes examined. The one exception is in spinach leaves where 10-formyltetrahydrofolate synthetase is monofunctional (Nour, J. M., and Rabinowitz, J. C. (1991) J. Biol. Chem. 266, 18363-18369). In the prokaryotes examined, 10-formyltetrahydrofolate synthetase is either absent or is monofunctional. A cDNA clone encoding spinach leaf 10-formyltetrahydrofolate synthetase was isolated through the use of antibodies to the purified enzyme. This clone had an open reading frame of 1914 base pairs and encoded for a protein containing 636 amino acids with a calculated M(r) of 67,727. The percentage identity between spinach 10-formyltetrahydrofolate synthetase and the synthetase domains in the four trifunctional eukaryotic enzymes and the two monofunctional prokaryotic enzymes that have been cloned and sequenced was: 64.9% human, 63.8% rat, 55.6% yeast cytoplasm, 53.8% yeast mitochondria, 47.8% Clostridium acidi-urici, and 47.9% Clostridium thermoaceticum. Clearly the spinach monofunctional protein had greatest homology with the mammalian proteins. The spinach protein is longer than the two other monofunctional prokaryotic proteins. Possible reasons for this are presented. The codon usage and the putative translation initiation sites are examined and compared with other spinach proteins.

Amino Acid Sequence↗
PubMed1986

Molecular cloning and sequencing of the region of the rubella virus genome coding for glycoprotein E1.

The sequence of the 1600 3' terminal nucleotides of the RNA of rubella virus was determined from cDNA synthesized from both virion and intracellular RNA using reverse transcriptase and an oligodeoxythymidine primer and cloned into a bacterial plasmid vector. This sequence contained the complete coding sequence for virion envelope protein E1 and a 57 nucleotide nontranslated region between the stop codon for E1 and the poly A tract. The predicted size for E1 was 481 amino acids and within this sequence were three potential N-linked glycosylation sites and a putative trans-membrane domain near the carboxy terminus. Immediately preceding the E1 coding region was a putative signal sequence. No homology was found at either the amino acid or nucleotide level between the region of the rubella virus genome sequenced and corresponding regions of the genomes of the alphaviruses, the other genus of the family Togaviridae for which sequence information has been obtained.

Amino Acid Sequence↗
PubMed1985

Efficient expression of the Escherichia coli leuB gene in yeast.

Efficient expression of the Escherichia coli leuB (beta-isopropylmalate dehydrogenase) gene occurred in yeast after in vitro DNase digestion and religation of plasmid bound leuB and the yeast HIS3 DNA which placed the 5' end of the yeast HIS3 gene immediately adjacent to the coding region of the E. coli leuB gene. Two structurally distinct classes of gene fusions were constructed, each involved portions of the yeast HIS3 gene which contributed DNA sequences responsible for leuB expression in yeast. The first class involved fusion of the HIS3 coding region to bacterial DNA resulting in the production of a fusion protein with beta-isopropylmalate dehydrogenase activity. The second class consisted of bacterial DNA, including the leuB coding region, fused to the HIS3 promotor region with the absence of any portion of the HIS3 coding region. In both constructions the HIS3 promotor region is required for transcription, however, translation of the class two fusion is initiated at a bacterial DNA coded AUG, and the 5' end of the mRNA coded by the leuB gene mapped predominantly at bacterial DNA sequences. The DNA sequence responsible for the 5' end of the HIS3 mRNAs remain in the class two gene fusions but this did not preclude the initiation of transcription at bacterial DNA sequences. The pattern of mRNA initiation at bacterial DNA suggests that DNA sequences at, or adjacent to, the site of transcription initiation are involved in the determination of the sites of initiation, and perhaps the frequency at which initiation occurs.

3-Isopropylmalate Dehydrogenase↗
PubMed1990

The promoter of the human proliferating cell nuclear antigen (PCNA) gene is bidirectional.

The proliferating cell nuclear antigen (PCNA) gene codes for a protein that is necessary for cellular DNA synthesis and cell cycle progression. A functional promoter has been identified in the 5' flanking region of the human PCNA gene. An abbreviated promoter (from the capsite to the PvuII restriction site at -395) was found to be equally efficient in directing transcription from a linked reporter, whether placed in the correct or reverse orientation in respect to the coding sequence. The reporter used was a cDNA of human thymidine kinase (TK), and the bidirectionality of the promoter was demonstrated by its ability to confer the TK+ phenotype to TK- ts 13 cells and by the amount of specific message in RNA blots. The PvuII promoter placed between two coding sequences (the TK cDNA and the bacterial gene for neoresistance) is capable of driving transcription simultaneously in both directions. Finally, in blots of RNA from human cells, two transcripts could be detected that hybridized to a sense riboprobe from the 5' flanking region of the human PCNA gene. We conclude that the locus for the human PCNA gene contains a bidirectional promoter producing diverging transcripts.

Cell Line↗
PubMed1986

An efficient chloramphenicol-resistance marker for Saccharomyces cerevisiae and Escherichia coli.

Chloramphenicol (Cm) was demonstrated to be a suitable selective agent for the plasmid-mediated transformation of haploid and polyploid strains of Saccharomyces cerevisiae. A yeast/Escherichia coli shuttle Cm-resistance (CmR) marker was constructed by inserting the CAT coding sequence from Tn9, and its associated bacterial ribosome-binding site, between a modified yeast ADC1 promoter and CYC1 terminator. When present on a 2 microns-based replicating plasmid, this marker transformed yeast as efficiently as the auxotrophic markers TRP1 and LEU2. When included in an integrating vector, single-copy transformants were formed as efficiently as with LEU2 and HIS3. Industrial yeast strains were transformed with both the replicating and integrating plasmids. The CmR marker could also efficiently transform E. coli. This versatile and efficient performance is currently unique for a yeast dominant marker.

Acetyltransferases↗
PubMedDate not supplied

[Convergent origin of repeats in genes coding for globular proteins. An analysis of the factors determining the presence of inverted and symmetrical repeats].

The factors, determining the presence of inverted and symmetrical repeats in genes coding for globular proteins, have been analysed. An interesting property of genetical code has been revealed in the analysis of symmetrical repeats: the pairs of symmetrical codons corresponded to pairs of amino acids with mostly similar physical-chemical parameters. This property may explain the presence of symmetrical repeats and palindromes only in genes coding for beta-structural proteins-polypeptides, where amino acids with similar physical-chemical properties occupy symmetrical positions. A stochastic model of evolution of polynucleotide sequences has been used for analysis of inverted repeats. The modelling demonstrated that only limiting of sequences (uneven frequencies of used codons) is enough for arising of nonrandom inverted repeats in genes.

Amino Acid Sequence↗
PubMedDate not supplied

[Functional and evolutionary aspects of the aminoacyl-tRNA synthetases].

A main event in protein bioshynthesis is the esterification of the correct aminoacid to cognate tRNA catalized by the aminoacyl-tRNA synthetases. The central role of this family of enzymes in metabolism is an evidence of their ancient origin. As it is the case in many others molecules involved in protein synthesis, the emergence of the aminoacyl-tRNA synthetases appears to be a problem that is not yet solved in order to understand the origin of the genetic translation. To obtain a comprehensive view of the evolution of the relationship between each one of the twenty aminoacyl-tRNA synthetases from one organism as well as from different sources (eubacteria, archaebacteria and eukaryotes) we review the information collected from the structural and catalytic properties of these enzyme. The results allow us to establish the following relationship between aminoacyl-tRNA synthetases. On one side there is a monofiletic origin for glutamyl, glutamynil and argynil-tRNA synthetases from Escherichia coli and for valyl, leucyl, metionyl, isoleucyl and phenylalanil-tRNA synthetases from eubacterias, archaebacterias and eukaryotes. On the other side there is an evolutionary relationship between aminoacyl-tRNA synthetases of eubacteria and organelles (plastids and mitochondria) and among eukaryotes and archaebacteria.

Amino Acid Sequence↗
PubMed1990

The primary structure of a halorhodopsin from Natronobacterium pharaonis. Structural, functional and evolutionary implications for bacterial rhodopsins and halorhodopsins.

We cloned and sequenced the gene coding for the polypeptide of a halorhodopsin in Natronobacterium pharaonis (named here pharaonis halorhodopsin). Peptide sequencing of cyanogen bromide fragments, and immunoreactions of the protein and synthetic peptides derived from the COOH-terminal gene sequence, confirmed that the open reading frame is the structural gene for the pharaonis halorhodopsin polypeptide. The flanking DNA sequences, as well as those for other bacterial rhodopsins, were compared to previously proposed archaebacterial consensus sequences. In pairwise comparisons of the open reading frame with DNA sequences for bacterio-opsin and halo-opsin from Halobacterium halobium, silent divergences (mutations/nucleotide at codon positions which do not result in amino acid changes) were calculated. These indicate very considerable evolutionary distance between each pair of genes. In spite of this, the three protein sequences show extensive similarities, indicating strong selective pressures. Conserved and conservatively replaced amino acid residues in all three proteins identify general features essential for ion-motive bacterial rhodopsins, responsible for overall structure and chromophore properties. Comparison of the bacteriorhodopsin sequence with those of the two halorhodopsins, on the other hand, identifies features involved in their specific (proton and chloride ion) transport functions.

Amino Acid Sequence↗
PubMed1989

Thyroid hormones regulate rat thyrotropin beta gene promoter activity expressed in GH3 cells.

Thyroid hormones suppress the synthesis of TSH in part by decreasing the rate of alpha and TSH beta gene transcription. Cis-acting DNA sequences present in the rat TSH beta subunit gene that are induced in transcriptional regulation by thyroid hormone have been identified by deletion-mutation and transient expression studies. Plasmid expression vectors were constructed including 2900, 900, 204, 77, 17 base pairs (bp) of 5'-flanking sequence and exon (5'-untranslated sequence, transcriptional start sites) fused to the coding region of the bacterial chloramphenicol acetyltransferase (CAT) gene. The transfected chimaeric plasmids demonstrated expression (with TSH beta DNA sequences in the 5'- to -3'-but not 3'- to -5'-orientation) in both a clonal pituitary cell line, GH3, and primary pituitary cell cultures, both of which are responsive to thyroid hormones. T3 (10(-11) M to 10(-7) M) treatment of transfected cells produced a dose-dependent decrease in CAT expression with a maximal 70% decrease at 10(-8) M. While a decrease in the basal level of expression was noted with progressive removal of both 5'-flanking and intronic sequences adjacent to exon 1, the fold-decrease in response to T3 was equivalent even in the 57 bp construct. In contrast, T3 had no effect on CAT expression directed by the promoter of the herpes simplex virus thymidine kinase gene. Thus, the rat TSH beta gene 5'-flanking region can direct heterologous gene expression in GH3 cells and contains sequences which have properties of a putative cis-active T3 responsive regulatory element(s).2+he

Animals↗
PubMed1985

Cloning and structure of the gene for the subunits of aspartokinase II from Bacillus subtilis.

A library of Bacillus subtilis DNA in lambda Charon 4A (Ferrari, E., Henner, D.J., and Hoch, J.A. (1981) J. Bacteriol. 146, 430-432) was screened by an immunological procedure for DNA sequences encoding aspartokinase II of B. subtilis, an enzyme composed of two nonidentical subunits arranged in an alpha 2 beta 2 structure (Moir, D., and Paulus, H. (1977a) J. Biol. Chem. 252, 4648-4654). A recombinant bacteriophage was identified that harbored an 18-kilobase B. subtilis DNA fragment containing the coding sequences for both aspartokinase subunits. The coding sequence for aspartokinase II was subcloned into bacterial plasmids. In response to transformation with the recombinant plasmids, Escherichia coli produced two polypeptides immunologically related to B. subtilis aspartokinase II with molecular weights (43,000 and 17,000) indistinguishable from those found in enzyme produced in B. subtilis. Peptide mapping by partial proteolysis confirmed the identity of the polypeptides produced by the transformed E. coli cells with the B. subtilis aspartokinase II subunits. The size of the cloned B. subtilis DNA fragment could be reduced to 2.9 kilobases by cleavage with PstI restriction endonuclease without affecting its ability to direct the synthesis of complete aspartokinase II subunits, irrespective of its orientation in the plasmid vector. Further subdivision by cleavage with BamHI restriction endonuclease resulted in the production of truncated aspartokinase subunits, each shortened by the same extent. This suggested that a single DNA sequence encoded both aspartokinase subunits and provided an explanation for the earlier observation that the smaller beta subunit of aspartokinase II was highly homologous or identical with the carboxyl-terminal portion of the alpha subunit (Moir, D., and Paulus, H. (1977b) J. Biol. Chem. 252, 4655-4661). A map of the gene for B. subtilis aspartokinase II is proposed in which the coding sequence for the smaller beta subunit overlaps in the same reading frame the promoter-distal portion of the coding sequence for the alpha subunit.

Antigen-Antibody Complex↗
PubMed1983

Cloning of the Vibrio harveyi luciferase genes: use of a synthetic oligonucleotide probe.

A mixed-sequence synthetic oligonucleotide probe was used to isolate a clone containing the gene encoding the alpha subunit of bacterial luciferase from Vibrio harveyi and part of the gene coding for the beta subunit. DNA sequence analysis has allowed us to determine that the genes are closely linked on the bacterial chromosome and transcribed in the same direction. Comparison of the sequences in the regions preceding the two structural genes has revealed considerable homology and has identified sites that may be involved in the expression of the genes. Identification of a clone from a clone bank of total genomic DNA from this organism shows that mixed probes can be successfully used to isolate a gene of interest from any bacterium provided some protein sequence for the gene product is available.

Amino Acid Sequence↗
PubMed1990

The meta cleavage operon of TOL degradative plasmid pWW0 comprises 13 genes.

The meta-cleavage operon of TOL plasmid pWW0 of Pseudomonas putida encodes a set of enzymes which transform benzoate/toluates to Krebs cycle intermediates via extradiol (meta-) cleavage of (methyl)catechol. The genetic organization of the operon was characterized by cloning of the meta-cleavage genes into an expression vector and identification of their products in Escherichia coli maxicells. This analysis showed that the meta-cleavage operon contains 13 genes whose order and products (in kilodaltons) are xylX(57)-xylY(20)-xylZ(39)-xylL(28)-xylT(1 2)-xylE(36)-xylG(60)-xylF(34)- xylJ(28)-xylQ(42)-xylK(39)-xylI(29)-xylH(4 ). The xylXYZ genes encode three subunits of toluate 1,2-dioxygenase. The xylL, xylE, xylG, xylF, xylJ, xylK, xylI, and xylH genes encode 1,2-dihydroxy-3,5-cyclohexadiene-1-carboxylate dehydrogenase, catechol 2,3-dioxygenase, 2-hydroxymuconic semialdehyde dehydrogenase, 2-hydroxymuconic semialdehyde hydrolase, 2-oxopent-4-enoate hydratase, 4-hydroxy-2-oxovalerate aldolase, 4-oxalocrotonate decarboxylase and 4-oxaloccotonate tautomerase, respectively. The functions of xylT and xylQ are not known at present. The comparison of the coding capacity and the sizes of the products of the meta-cleavage operon genes indicated that most of the DNA between xylX and xylH consists of coding sequences.

Bacterial Proteins↗