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PubMed1973

Relatedness among contractile and membrane proteins: evidence for evolution from common ancestral genes.

A statistical method for quantifying the relatedness among proteins was used to perform 2926 paired comparisons of amino-acid composition among 77 contractile and membrane-associated proteins from diverse species and sources. Relatedness of amino-acid compositions correlates with homology of amino-acid sequence. A high degree of relatedness was detected among K(+)-dependent membrane ATPase of Streptococcus faecalis, coupling factors F(1) and CF(1) from mitochondria and chloroplasts, outer fiber protein of cilia, ciliary dynein, tubulin, various actins, and myosin subfragment S-1. Heavy meromyosin and tropomyosin were related to each other but not to the first group of proteins. Differences in the degree of methylation may account for some differences in physiological function. Because of their diverse sources, the high degree of relatedness among these proteins is more compatible with evolution from common ancestral genes than with convergent evolution. Squid axon filarin, molluscan paramyosin, and bacterial flagellins appear to be unrelated either to each other or to any of the other proteins studied. Existence of persistent homologies among so many diverse proteins implies conservation of genetic information during evolution by utilization of codons for preferred amino-acid sequences in various proteins.

Actins↗

PubMed1973

The amino-acid sequence of lac repressor.

The amino-acid sequence of lac repressor from Escherichia coli has been determined. The sequence contains 347 residues in the subunit single peptide chain. It shows no similarities with the sequences of histones or the known part of beta-galactosidase.

Amino Acid Sequence↗

PubMed1974

The 5'-terminal nucleotide sequence of galactose messenger ribonucleic acid of Escherichia coli.

The 5'-terminal sequence of mRNA from the galactose operon of E. coli has been determined. gal RNA is synthesized in vitro by means of a kinetically controlled purified transcription system and is isolated by a two-step RNA.DNA hybridization scheme. The following sequence of the first 77 nucleotides has been deduced by analysis of oligonucleotides produced by digestion with T(1), pancreatic, and carboxymethylated pancreatic ribonucleases: ppA-U-A-C-C-A-U-A-A-G-C-C-U-A-A-U-G-G-A-G-C-G-A-A-U-U-A-U-G-A-G-A-G-U-U-C-U-G-G-U-U-A-C-C-G-G-U-G-G-U-A-G-C-G-G-U-U-A-C-A-U-U-G-G-A-A-G-U-C-A-U-A-C-C-U-G-U. Residues 27-77 correspond to the amino terminal 17 amino acids of UDPgalactose 4-epimerase (EC 5.1.3.2), the protein specified by the promoter-proximal structural gene of the operon. A self-complementary sequence occurs near the 5' terminus; 12 of 15 nucleotides between residues 4 and 18 are symmetrically located about position 11. The sequence of residues 22-33 closely resembles part of the lactose operator sequence reported previously [Gilbert, W. & Maxam, A. (1973) Proc. Nat. Acad. Sci. USA 70, 3581-3584].

Amino Acid Sequence↗

PubMed1994

Molecular cloning of MSSP-2, a c-myc gene single-strand binding protein: characterization of binding specificity and DNA replication activity.

We have previously reported the human cDNA encoding MSSP-1, a sequence-specific double- and single-stranded DNA binding protein [Negishi, Nishita, Saëgusa, Kakizaki, Galli, Kihara, Tamai, Miyajima, Iguchi-Ariga and Ariga (1994) Oncogene, 9, 1133-1143]. MSSP-1 binds to a DNA replication origin/transcriptional enhancer of the human c-myc gene and has turned out to be identical with Scr2, a human protein which complements the defect of cdc2 kinase in S.pombe [Kataoka and Nojima (1994) Nucleic Acid Res., 22, 2687-2693]. We have cloned the cDNA for MSSP-2, another member of the MSSP family of proteins. The MSSP-2 cDNA shares highly homologous sequences with MSSP-1 cDNA, except for the insertion of 48 bp coding 16 amino acids near the C-terminus. Like MSSP-1, MSSP-2 has RNP-1 consensus sequences. The results of the experiments using bacterially expressed MSSP-2, and its deletion mutants, as histidine fusion proteins suggested that the binding specificity of MSSP-2 to double- and single-stranded DNA is the same as that of MSSP-1, and that the RNP consensus sequences are required for the DNA binding of the protein. MSSP-2 stimulated the DNA replication of an SV40-derived plasmid containing the binding sequence for MSSP-1 or -2. MSSP-2 is hence suggested to play an important role in regulation of DNA replication.

Base Sequence↗

PubMed1980

DNA sequence of the E. coli trpR gene and prediction of the amino acid sequence of Trp repressor.

A DNA sequence of 1041 base pairs from a BamHI fragment containing the E. coli trpR gene has been determined. With this sequence and other experimental evidence, the primary structure (88 amino acids) of the Trp repressor can be predicted. Additional features of the DNA sequences include a 22 base pair region upstream from the proposed structural gene which exhibits striking homology with the trp operator, thus implying that expression of the trpR gene may be under autogenous regulation.

Amino Acid Sequence↗

PubMed1975

Multiple gene loci for a single species of glycine transfer ribonucleic acid.

The study of suppressors of tryptophan synthase A protein missense mutations in Escherichia coli has led to the establishment of two nonadjacent genetic loci (gly V and gly W) specifying identical nucleotide sequences for a single isoaccepting species of glycine transfer ribonucleic acid (tRNA GLY 3 GGU/C). In one case, suppression of the missense mutation trpA78 was due to a mutation in a structural gene (gly W) for tRNA Gly 3 GGU/C. This mutation resulted in a base change in the anticodon and modification of an A residue adjacent to the 3' side of the anticodon, leading to the production of a tRNA Gly 3 UGU/C species. The resulting glyW51 (SU UGU/C) allele was mapped by interrupted mating and was located at approximately 37 min on the Escherichia coli genetic map. Other suppressor mutations affecting the primary sequence of tRNA Gly GGU/C and giving rise to the Ins and SU+A58 phenotypes were positioned at 86 min (glyV). Several independently arising missense suppressor mutations resulting in the SU+A78 phenotypes were isolated and mapped at these two genetic loci (glyV and glyW). The ratio of appearance of suppressor mutations at glyV and glyW suggests that there are three of four tRNAGly3 GGU/C structural gene copies at the glyV locus to one copy at the glyW locus. Structural genes for tRNA ly isoacceptors are now known at four distinct locations on the Escherichia coli chromosome: glyT (77 MIN), TRNA Gly 2 GGA/G; gly U (55 min), tRNAGly-1 minus; and gly V (86 MIN) AND GLYW (37 min), tRNAGly 3 GGU/C.

Alleles↗

PubMed2002

Group I self-splicing intron in the recA gene of Bacillus anthracis.

Self-splicing introns are rarely found in bacteria and bacteriophages. They are classified into group I and II according to their structural features and splicing mechanisms. While the group I introns are occasionally found in protein-coding regions of phage genomes and in several tRNA genes of cyanobacteria and proteobacteria, they had not been found in protein-coding regions of bacterial genomes. Here we report a group I intron in the recA gene of Bacillus anthracis which was initially found by DNA sequencing as an intervening sequence (IVS). By using reverse transcriptase PCR, the IVS was shown to be removable from the recA precursor mRNA for RecA that was being translated in E. coli. The splicing was visualized in vitro with labeled free GTP, indicating that it is a group I intron, which is also implied by its predicted secondary structure. The RecA protein of B. anthracis expressed in E. coli was functional in its ability to complement a recA defect. When recA-negative E. coli cells were irradiated with UV, the Bacillus RecA reduced the UV susceptibility of the recA mutant, regardless of the presence of intron.

Bacillus anthracis↗

PubMed1989

Selection of new biological activities from random nucleotide sequences: evolutionary and practical considerations.

Recent advances in the selection of biologically active DNA sequences from random populations are reviewed. Within the framework of evolution, forces are considered that have precluded the testing of all possible DNA sequences, purely with regard to their functionality as genetic regulatory elements or protein coding sequences. Examples are drawn from cassette mutagenesis of enzyme active sites, protein domain replacement by fusion with random genomic digests, and the selection of bacterial promoters from random DNA. Efforts to derive new activities are examined, and the likelihood of future success is evaluated.

Base Sequence↗

PubMed1993

Parathyroid hormone represses alpha 1(I) collagen promoter activity in cultured calvariae from neonatal transgenic mice.

We examined the effect of PTH on the activity of alpha 1(I) collagen promoter fusion genes in cultured calvariae from transgenic mice. The parent construct, ColCAT 3.6, contains 3520 basepairs of 5' rat alpha 1(I) collagen DNA, 115 basepairs of untranslated alpha 1(I) collagen-coding DNA, and the bacterial chloramphenicol acetyltransferase reporter gene, while the 5'-deletion ColCAT 2.3 contains 2296 kilobases of rat alpha 1(I) collagen promoter sequence. Transgenic mouse lines harboring these collagen promoter fusion genes were developed using the oocyte microinjection technique, and for each construct, three different lines of mice were tested. Calvariae from 6- to 8-day-old transgenic mice were cultured for 48 h with or without bovine PTH-(1-34). ColCAT 3.6 and ColCAT 2.3 were expressed at comparable levels in calvariae and were inhibited by PTH. There were parallel decreases in the incorporation of [3H]proline into collagen and levels of the endogenous alpha 1(I) collagen mRNA and transgene mRNA. Forskolin at 10 microM mimicked the inhibitory effect of PTH on promoter activity in ColCAT 3.6 and ColCAT 2.3 calvariae. A RNase protection assay showed that the transgene was initiated correctly from the transgene promoter. These data show that PTH and cAMP can repress collagen promoter activity in calvariae from transgenic mice, suggesting that the alpha 1(I) collagen promoter may contain cis elements down-stream of -2.3 kilobases that mediate PTH and cAMP repression of collagen gene expression in bone. Cultured bone explants from transgenic mice can be used as a model to study hormonal regulation of alpha 1(I) collagen promoter constructs.

Animals↗

PubMed2006

Modeling the 3D structure of wheat subtilisin/chymotrypsin inhibitor (WSCI). Probing the reactive site with two susceptible proteinases by time-course analysis and molecular dynamics simulations.

Comparative modeling and time-course hydrolysis experiments have been applied to investigate two enzyme-inhibitor complexes formed between the wheat subtilisin-chymotrypsin inhibitor (WSCI) and two susceptible proteinases. WSCI represents the first case of a wheat protein inhibitor active against animal chymotrypsins and bacterial subtilisins. The model was created using as template structure that of the CI-2A inhibitor from barley (PDB code: 2CI2), which shares 87% sequence identity with WSCI. Under these conditions of high similarity, the comparative modeling approach can be successfully applied. We predicted the WSCI 3D model and used it to investigate enzyme-inhibitor complex systems. Experimental observations indicated that chymotrypsin, but not subtilisin, in addition to cleavage at the primary reactive site Met48-Glu49, is able to hydrolyze a second peptide bond between Phe58 and Val59. Here, we report on cleavage of the peptide bond at the inhibitor's reactive site (Met48-Glu49) determined using time-course hydrolysis experiments; the same event was investigated for both subtilisin/WSCI and chymotrypsin/WSCI complexes using molecular dynamics simulations. The molecular details of the initial inhibitor-enzyme interactions, as well as of the changes observed during the simulations, allow us to speculate on the different fates of the two WSCI-proteinase complexes.

Amino Acid Sequence↗

PubMed1991

Characterization of DNA fragment from Chlamydia psittaci avian strain which shows high homology with hypB gene of Chlamydia.

A study was performed to characterize DNA fragment No. 17 of C. psittaci strain P-1041 which encoded 42 KD beta-galactosidase fusion protein with type-specific antigenicity. Sequence determination identified a partial open reading frame that spanned about 1,200b. p. nucleotides. Screening the literatures for the nucleotide and deduced amino acid sequences revealed extensive similarity between the DNA fragment of P-1041 and two chlamydial hypB genes. This DNA showed 91.5% homology with C. psittaci GPIC hypB gene in nucleotide sequence and 96.4% homology in deduced amino acid sequence. The hypB gene of C. trachomatis serovar A and the P-1041 DNA fragment showed 81.2% and 91.3% homology in nucleotide and amino acid sequences, respectively. Dot enzyme-linked immunosorbent assay, for the products of deleted DNA fragments defined the coding region for type-specific antigenic polypeptide. In addition, the P-1041 DNA fragment carried a sequence highly homologous (greater than 49%) with other bacterial and plant genes called chaperonin which responds to various stress in cells. From these results, the P-1041 DNA fragment was found to be a part of hypB gene and to encode the region critical for type-specific antigenicity.

Amino Acid Sequence↗

PubMed2005

Bacterial insertion sequence IS256 as a potential molecular marker to discriminate invasive strains from commensal strains of Staphylococcus epidermidis.

The skin commensal Staphylococcus epidermidis has become one of the most important causative agents of nosocomial infections associated with medical devices. Differentiation between invasive S. epidermidis and its commensal counterpart is crucial for clinical decision making. The ica gene locus, which codes for production of polysaccharide intercellular adhesion (PIA), represents a frequently suggested molecular marker for infectivity. Our data demonstrated that production of PIA was not significantly increased among clinical strains, which may explain the controversial results obtained previously on the correlation of ica presence with origin from infection. Therefore, in this study, we attempted to identify novel genes discriminating between invasive and commensal strains based on the comparison of genome sequences. Our results indicated that the bacterial insertion sequence element IS256 occurred significantly more frequently in strains of clinical origin. Importantly, IS256 might thus constitute a molecular marker to discriminate invasive strains from commensal strains of S. epidermidis.

Carrier State↗

PubMed1999

Symbiosis of Aeromonas veronii biovar sobria and Hirudo medicinalis, the medicinal leech: a novel model for digestive tract associations.

Hirudo medicinalis, the medicinal leech, is applied postoperatively in modern medicine. Infections by Aeromonas occur in up to 20% of patients unless a preemptive antibiotic treatment is administered. The associated infections demonstrate the need for a better understanding of the digestive tract flora of H. medicinalis. Early studies reported the presence of a single bacterial species in the digestive tract and suggested that these bacteria were endosymbionts contributing to the digestion of blood. In this study, we cultivated bacteria from the digestive tract and characterized them biochemically. The biochemical test results identified the isolates as Aeromonas veronii biovar sobria. This species identification was supported by sequence comparison of a variable region of the genes coding for 16S rRNA. In a colonization assay, a rifampin-resistant derivative of a symbiotic isolate was fed in a blood meal to H. medicinalis. The strain colonized the digestive tract rapidly and reached a concentration similar to that of the native bacterial flora. For the first 12 h, the in vivo doubling time was 1.2 h at 23 degreesC. After 12 h, at a density of 5 x 10(7) CFU/ml, the increase in viable counts ceased, suggesting a dramatic reduction in the bacterial growth rate. Two human fecal isolates, identified as Aeromonas hydrophila and A. veronii biovar sobria, were also able to colonize the digestive tract. These data demonstrate that the main culturable bacterium in the crop of H. medicinalis is A. veronii biovar sobria and that the medicinal leech can be used as a model for digestive tract association of Aeromonas species.

Aeromonas↗

PubMed1990

Cloning and nucleotide sequence of cDNA for Ki antigen, a highly conserved nuclear protein detected with sera from patients with systemic lupus erythematosus.

Patients with systemic lupus erythematosus (SLE) produce autoantibodies against a variety of nuclear antigens including Ki antigen. Although anti-Ki autoantibodies were found in a significant number of SLE patients, the nature of Ki antigen is poorly characterized. By using anti-Ki serum as a probe we have cloned a bovine cDNA directing the synthesis in Escherichia coli of a polypeptide immunologically indistinguishable from the authentic Ki antigen. A homologous human cDNA was also cloned and its nucleotide sequence predicted the entire primary structure of a novel nuclear protein with a molecular weight of 29 508 and with highly hydrophilic and weakly acidic character. The gene is highly conserved not only in the coding region but also in the 3'-untranslated region. The bacterially produced Ki antigen would be valuable for diagnosis of SLE.

Amino Acid Sequence↗

PubMed1993

The structure of the chloramphenicol resistance gene on a transferable R plasmid from the fish pathogen, Pasteurella piscicida.

The chloramphenicol resistance gene (pp-cat) was cloned from a transferable R plasmid of Pasteurella piscicida, pSP9351, and the sequence of the gene was determined. Subcloning and deletion analysis localized the resistance gene, pp-cat, to within a 2.3 kb HincII-BamHI fragment. The fragment as a probe hybridized with the type I chloramphenicol acetyltransferase (CAT) gene and did not hybridize to CAT types II, III, and CAT-VA. The fragment hybridized to transferable R plasmids encoded with resistance to chloramphenicol, which were detected from P. piscicida isolated in different years. Nucleotide sequences of the coding and flanking regions of pp-cat (2031 bp) identified an open reading frame coding type I CAT of a molecular mass of about 25,000 Da. Comparison analysis of the sequences outside the cat open reading frame showed also that pp-cat has homology, in part, with the gene that coding for the endonuclease EcoRII and those that flank the cat gene derived from the Acinetobacter baumannii chromosome.

Animals↗

PubMed1979

Human growth hormone: complementary DNA cloning and expression in bacteria.

The nucleotide sequence of a DNA complementary to human growth hormone messenger RNA was cloned; it contains 29 nucleotides in its 5' untranslated region, the 651 nucleotides coding for the prehormone, and the entire 3' untranslated region (108 nucleotides). The data reported predict the previously unknown sequence of the signal peptide of human growth hormone and, by comparison with the previously determined sequences of rat growth hormone and human chorionic somatomammotropin, strengthens the hypothesis that these genes evolved by gene duplication from a common ancestral sequence. The human growth hormone gene sequences have been linked in phase to a fragment of the trp D gene of Escherichia coli in a plasmid vehicle, and a fusion protein is synthesized at high level (approximately 3 percent of bacterial protein) under the control of the regulatory region of the trp operon. This fusion protein (70 percent of whose amino acids are coded for by the human growth hormone gene) reacts specifically with antibodies to human growth hormone and is stable in E. coli.

Amino Acid Sequence↗

PubMedDate not supplied

A gene encoding a host-specific elicitor protein of Phytophthora parasitica.

Extracellular elicitor proteins (elicitins) from Phytophthora species induce local and distal defense responses specifically in plants of the Solanaceae and Cruciferae. Based on elicitin amino acid sequences, elicitin-coding sequences from P. parasitica were amplified by the polymerase chain reaction. A genomic clone containing a complete elicitin gene, parA1, was isolated and sequenced. Elicitin was confirmed to be encoded as a precursor protein containing a 20-amino acid signal peptide that is processed before secretion. Bacterial expression of the cloned elicitin gene as a translational fusion protein containing glutathione S-transferase yielded a biologically active protein capable of inducing a hypersensitive response in tobacco, suggesting that fungus-specific postranslational modifications of elicitin are not required for its activity. Southern blot analysis indicated that elicitin genes occur as a multigene family (at least two to 10 copies) in P. parasitica, P. capsici, P. citricola, P. citrophthora, P. cryptogea, P. drechsleri, P. megasperma, and P. palmivora. Some isolates of P. parasitica that did not produce elicitins still contained elicitin-coding sequences but did not accumulate elicitin mRNA.

Algal Proteins↗

PubMed2002

Establishment and application of bicistronic classical swine fever virus genomes for foreign gene expression and complementation of E2 deletion mutants.

Bicistronic genomes of the classical swine fever virus (CSFV) strain Alfort/187 (A187) were established by insertion of a second cistron consisting of an internal ribosome entry site of the encephalomyocarditis virus and a coding sequence in the 3' untranslated region of the genome. Introduction of the selectable marker gene for neomycin phosphotransferase into the second cistron of the CSFV replicon A187 Delta E2-CAT allowed the establishment of porcine SK-6 cell lines constitutively expressing the respective bicistronic replicon RNA. In cells transfected with RNA representing the full-length viral genome and containing the gene coding for bacterial enhanced green fluorescence protein (EGFP) in the second cistron infectious bicistronic virus was synthesized. Expression of EGFP in cells infected with this virus indicated the potential of CSFV as a viral vector. Finally, after insertion of the sequence encoding the signal peptide of the CSFV E2 protein followed either by the E2 or the E2-p7 sequence into the replicon A187 Delta E2 which carries an in frame deletion of 465 nucleotides in the E2 gene, infectious viruses vA187 Delta E2-IRES-sigE2 and vA187 Delta E2-IRES-sigE2p7, respectively, were obtained. This shows that E2 deletion mutants can be complemented by expression of E2 from a separate cistron.

Animals↗