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PubMed1994

Sequence of the essential early region of phi C31, a temperate phage of Streptomyces spp. with unusual features in its lytic development.

The temperate phage phi C31 is the most studied bacteriophage infecting Streptomyces spp., and has been used to develop an extensive and widely used series of cloning vectors. The sequence of 10 kb of phi C31 DNA containing most or all of the essential early genes was determined. Among the ORFs, 14 (perhaps 15) appear to be protein-coding, and these have been designated ORF1 to ORF14 and ORFX. Previously mapped transcripts appear to initiate upstream from ORFs 1, 8, 11 and 12, and within ORF3 and ORF12, in each case close to one example of the unusual ('21-mer') sequences that appear to serve as a recognition site for RNA polymerase early in the phi C31 lytic cycle [Ingham et al., Mol. Microbiol. 9 (1993) 1267-1274]. Further copies of the 21-mer are upstream from ORF2 and ORF13. There are four recognisable examples of a conserved inverted repeat sequence motif (CIR) thought to bind phi C31 repressor [Smith and Owen, Mol. Microbiol. 5 (1991) 2833-2844]. Only one CIR is closely associated with a 21-mer sequence, though three are located between known transcription units. Of all 14 ORFs, only one (ORF11) would encode a protein unmistakably resembling other known proteins; its product appears to be a DNA polymerase. Strikingly, two codons, TTA (Leu) and AGG (Arg), are absent from the 14 ORFs.

Amino Acid Sequence↗
PubMed2001

Cloning and sequence of the gene encoding the muscle fatty acid binding protein from the desert locust, Schistocerca gregaria.

Muscle fatty acid binding protein (FABP) is a major cytosolic protein in flight muscle of the desert locust, Schistocerca gregaria. FABP expression varies greatly during development and periods of increased fatty acid utilization, but the molecular mechanisms that regulate its expression are not known. In this study, the gene coding for locust muscle FABP was amplified by PCR and cloned, together with 1.2 kb of upstream sequence. The sequence coding for the 607 bp cDNA is interrupted by two introns of 12.7 and 2.9 kb, inserted in analogous positions as the first and third intron of the mammalian homologues. Both introns contain repetitive sequences also found in other locust genes, and the second intron contains a GT-microsatellite. The promoter sequence includes a canonical TATA box 24 bp upstream of the transcription start site. The upstream sequence contains various potential myocyte enhancer sequences and a 160 bp segment that is repeated three times. In database searches in the genome database of Drosophila melanogaster, a gene with the same gene organization and promoter structure was identified, likely the dipteran homologue of muscle FABP. Upstream of both insect genes, a conserved 19 bp inverted repeat sequence was detected. A similar but reverse palindrome is also present upstream of all mammalian heart FABP genes, possibly representing a novel element involved in muscle FABP expression.

Amino Acid Sequence↗
PubMed1984

Unusual long terminal repeat sequence of a retrovirus transmissible mouse (VL 30) genetic element: identification of functional domains.

We have determined the nucleotide sequence and mapped the transcriptional boundaries in the long terminal repeats (LTRs) and adjacent regions of a retrovirus transmissible virus-like 30S ( VL30 ) mouse genetic element. The 572 base pair LTRs contain transcriptional regulatory sequences and are bounded by short imperfect repeats, with a minus strand tRNAgly primer binding site and a purine rich plus strand primer site flanking each of their inner boundaries. The 3' end of each LTR consists of an extensive 80 base pair redundancy of tRNA primer site and inverted repeat sequences while 41 and 47 base pair imperfect tandem repeats are present between the 5' capping site and the putative polyadenylation signal. Comparison with other retrovirus-like LTR sequences suggests possible modes of recombination that could occur between VL30 and other genetic elements.

Animals↗
PubMed1995

Interaction of EF-C/RFX-1 with the inverted repeat of viral enhancer regions is required for transactivation.

The hepatitis B virus (HBV) and polyomavirus (Py) enhancer regions contain multiple cis-acting elements that contribute to enhancer activity. The EF-C binding site was previously shown to be an important functional component of each enhancer region. EF-C is a ubiquitous binding activity that interacts with an inverted repeat sequence in the HBV and Py enhancer regions. Although the EF-C binding site is required for optimal enhancer function, the EF-C site does not possess intrinsic enhancer activity when assayed in the absence of flanking elements. With both the HBV and Py enhancer regions, EF-C stimulates the activity of adjacent enhancer elements in a synergistic manner. EF-C corresponds to RFX-1, a protein that binds to a conserved and functionally important site in major histocompatibility complex (MHC) class II antigen promoter regions. Interestingly, the RFX-1 binding site in MHC class II promoters only contains an EF-C half-site, maintaining one arm of the inverted repeat in an EF-C binding site. We have investigated the binding of purified EF-C and RFX-1 to sites in the Py and HBV enhancer regions that carry mutations that either disrupt one arm of the EF-C inverted repeat, or alter the spacing between the repeats. Our results show that the interaction of EF-C and RFX-1 with an intact inverted repeat is required for functional activity of these viral enhancer regions. Chemical footprinting and modification interference assays show that the interaction of EF-C and RFX-1 with the DRA MHC class II promoter truly represents half-site interaction, and that this binding is unstable. In contrast, the binding of EF-C and RFX-1 to the viral inverted repeats is stable. These results suggest that an additional activity may be required to stabilize EF-C/RFX-1 interaction with the MHC class II promoter, and that viral enhancer regions have evolved high affinity binding sites to sequester dimeric EF-C/RFX-1.

3T3 Cells↗
PubMed1999

Genetic variation of chlorella viruses: variable regions localized on the CVK2 genomic DNA.

A physical map of the Chlorella virus CVK2 genomic DNA has been constructed based on a cosmid contig covering the entire genomic region. By using Southern blot analysis with 22 gene probes, the gene arrangement along the genome was compared between CVK2 and PBCV-1, the prototypic member of Phycodnaviridae, whose genomic sequence is now available. The major rearrangements were (1) an insertion of a 20-kbp region around the left end of CVK2 DNA, (2) a duplication of the gene for major capsid protein in CVK2 DNA, (3) deletions/insertions of some open reading frames, and (4) divergence in the terminal inverted repeat sequences. Despite these changes, extensive colinearity was revealed between most of the genes along the CVK2 and PBCV-1 genomes. These data imply that the Chlorella virus genome has an overall high degree of genomic stability, encompassing specific islands of rearrangements.

Amino Acid Sequence↗
PubMed2005

Molecular cloning of the gyrA gene and characterization of its mutation in clinical isolates of quinolone-resistant Edwardsiella tarda.

Knowing the entire sequence of the gene encoding the DNA gyrase Subunit A (gyrA) of Edwardsiella tarda could be very useful for confirming the role of gyrA in quinolone resistance. Degenerate primers for the amplification of gyrA were designed from consensus nucleotide sequences of gyrA from 9 different Gram-negative bacteria, including Escherichia coli. With these primers, DNA segments of the predicted size were amplified from the genomic DNA of E. tarda and then the flanking sequences were determined by cassette ligation-mediated polymerase chain reaction. The nucleotide sequence of gyrA was highly homologous to those of other bacterial species, in both the whole open-reading frame and the quinolone-resistance-determining region (QRDR). The 2637-bp gyrA gene encodes a protein of 878 amino acids, preceded by a putative promoter, ribosome binding site and inverted repeated sequences for cruciform structures of DNA. However, the nucleotide sequence of the flanking region did not show any homologies with those of other bacterial DNA gyrase Subunit B genes (gyrB) and suggested the gyrase genes, gyrA and gyrB, are non-continuous on the chromosome of E. tarda. All of the 12 quinolone-resistant isolates examined have an alteration within the QRDR, Ser83 --> Arg, suggesting that, in E. tarda, resistance to quinolones is primarily related to alterations in gyrA. Transformation with the full sequence of E. tarda gyrA bearing the Ser83 --> Arg mutation was able to complement the sequence of the gyrA temperature-sensitive mutation in the E. coli KNK453 strain and to induce increased resistance to quinolone antibiotics at 42 degrees C.

Base Sequence↗
PubMed1986

Phosphoribosylpyrophosphate synthetase of Escherichia coli. Properties of the purified enzyme and primary structure of the prs gene.

Phosphoribosylpyrophosphate (P-Rib-PP) synthetase of Escherichia coli has been purified to near homogeneity from a strain harboring the prs gene, encoding P-Rib-PP synthetase, on a multicopy plasmid. Analysis of the enzyme showed that it required inorganic phosphate for activity and for stability. Magnesium ions were required both as a complex with the substrate ATP and as a free cation. P-Rib-PP synthetase activity was inhibited strongly by ADP. Kinetic analysis indicated multiple sites of action of ADP. In addition apparent substrate inhibition was exerted by ribose 5-phosphate in the presence of ADP. The nucleotide sequence of the E. coli prs gene has been determined and the coding segment established. The deduced amino acid sequence of P-Rib-PP synthetase contained 314 amino acid residues and the molecular weight was calculated as 34,060. The initiation site of transcription was determined. This site was preceded by well conserved -10 and -35 consensus sequences (pdT-dA-dG-dA-dA-dT and pdT-dT-dG-dA-dT-dG, respectively). The transcription initiation site preceded the potential translation initiation site by 302 nucleotides. Transcription terminated approximately 35 nucleotides downstream from the UAA translation stop codon, within a Thy-rich region following an inverted repeat sequence, indicative of an rho-independent transcription terminator.

Amino Acid Sequence↗
PubMed1991

Nucleotide sequence and expression of the algE gene involved in alginate biosynthesis by Pseudomonas aeruginosa.

Alginate (Alg), a random polymer of mannuronic acid and glucuronic acid residues, is synthesized and secreted by Pseudomonas aeruginosa primarily during its infection of the lungs of cystic fibrosis patients. The molecular biology and biochemistry of the enzymatic steps leading to the production of the Alg precursor GDP-mannuronic acid have been elucidated, but the mechanism of polymer formation and export of Alg are not understood. We report the nucleotide sequence of a 2.4-kb DNA fragment containing the algE gene, previously designated alg76, encoding the AlgE protein (Mr 54,361) that is believed to be involved in these late steps of Alg biosynthesis. Expression of algE appears to occur from its own promoter. The promoter region contains several direct and inverted repeat sequences and shares structural similarity with promoters of several other alg genes from P. aeruginosa. In addition, the AlgE protein was overproduced from the tac promoter in P. aeruginosa. N-terminal amino acid sequence analysis showed that the polypeptide contains a signal peptide which is cleaved to form the mature protein during AlgE export from the cell cytoplasm.

Alginates↗
PubMed1991

Structure of the gas vesicle plasmid in Halobacterium halobium: inversion isomers, inverted repeats, and insertion sequences.

Halobacterium-halobium NRC-1 harbors a 200-kb plasmid, pNRC100, which contains a cluster of genes for synthesis of buoyant gas-filled vesicles. Physical mapping of pNRC100 by using pulsed-field gel electrophoresis showed the presence of a large (35 to 38-kb) inverted repeat (IR) sequence. Inversion isomers of pNRC100 were demonstrated by Southern hybridization analysis using two restriction enzymes, AflII and SfiI, that cut asymmetrically within the intervening small single-copy region and the large single-copy region, respectively, but not within the large IRs. No inversion isomers were observed for a deletion derivative of pNRC100 lacking one IR, which suggests that both copies are required for inversion to occur. Additionally, the identities and approximate positions of 17 insertion sequences (IS) in pNRC100 were determined by Southern hybridization and limited nucleotide sequence analysis across the IS element-target site junctions: ISH2, a 0.5-kb element, was found in four copies; ISH3, a 1.4-kb heterogeneous family of elements, was present in seven copies; ISH8, a 1.4-kb element, was found in five copies; and ISH50, a 1.0-kb element, was present in a single copy. The large IRs terminated at an ISH2 element at one end and an ISH3 element at the other end. pNRC100 is similar in structure to chloroplast and mitochondrial genomes, which contain large IRs and other large halobacterial and prokaryotic plasmids that are reservoirs of IS elements but lack the large IRs.

Base Sequence↗
PubMed1986

Structure and gene expression of the E. coli Mn-superoxide dismutase gene.

Superoxide dismutase is an enzyme which converts superoxide O2- to hydrogen peroxide. Using a single synthetic oligonucleotide 33mer, we screened the E. coli DNA library and isolated a clone containing the E. coli manganese-superoxide dismutase gene. We determined the DNA sequence. The analysis of the DNA sequence and in vivo as well as in vitro transcription has shown the following. The DNA sequence suggests two possible promoters. However, only one of them seems active during normal aerobic growth. Purified RNA polymerase initiates in vitro transcription from the same promoter. It is not clear whether the second promoter is functional. It is possible that this promoter could be activated under different growth conditions. There is an inverted repeat sequence which could form a stem-loop structure downstream of the translation stop codon TAA of the Mn-SOD gene. The results of the analysis of in vivo and in vitro RNA have shown that this is the transcription termination signal. Thus, the Mn-SOD gene constitutes a single gene operon. There is an almost perfect 19 base palindrome at the -35 region. The position and the size of the palindrome suggest that this could be a regulatory site.

Amino Acid Sequence↗
PubMed1989

Flanking AT-rich sequences may lower the activation energy of cruciform extrusion in supercoiled DNA.

In the absence of flanking AT-rich segments, cruciform transition energies of DNA palindromic sequences of random base composition are high and mainly dependent upon the base-stacking and -pairing parameters of the palindromic segment. When AT-rich sequences adjoin palindromes, the transition energy of cruciform extrusion is significantly lowered. An inverse relationship exists between the length of the AT-rich stretch and the cruciform transition energy. Long stretches lower the transition energies more than short stretches. At physiological salt and temperature conditions, equilibrium between cruciform extrusion and absorption for the inverted repeat sequences IRS-B and IRS-C of pBR322 derived plasmids is reached in less than five minutes.

Base Sequence↗
PubMed1997

Identification of sequence requirement for the origin of DNA replication in human papillomavirus type 18.

DNA replication of human papillomavirus type 18 is dependent on viral proteins E1 and E2 and the subsequent interaction of these proteins with the viral origin of replication. Using a site-directed mutagenesis analysis, we examined the sequence requirement for the DNA replication of the human papillomavirus type 18. We showed that both the E1BS palindrome and E2BS are the major determinants of the HPV replication efficiency. In particular, abolishing E2 binding sites demonstrated that E2BS makes a significant contribution towards HPV-18 DNA replication. Each part of the 18-bp inverted repeat sequence of the E1BS motif showed a clear functional difference between two regions: nt 13-21 (3' half segment) is evidently more important for replication than nt 4-12 (5' half segment). Besides E1BS and E2BS, cis-acting elements such as the poly-A6 track, perhaps the YY1 binding site, and the TATA box sequence within the origin region exhibited some contributions to optimum replication. In addition, inserting an enhancer region to the minimum origin DNA derivatives increased replication approximately 2-fold compared with the wild type levels and showed some compensational effects on loss of the cis-element within the HPV-18 minimum origin, suggesting that an enhancer region is required for efficient replication of the papillomavirus origin. These results suggest that the formation of an E1-E2-ori complex is important for replication, and other sequences near the E1 and E2 binding sites assist E1-E2-ori-mediated DNA replication in vivo.

Base Sequence↗
PubMed1985

The structure of the major immediate early gene of human cytomegalovirus strain AD169.

The nucleotide sequence of the major immediate early (IE) gene of human cytomegalovirus strain AD169 was determined. The structure of the gene was examined by nuclease mapping and by sequence analysis of a cDNA clone made from IE mRNA. The gene encodes a spliced molecule of 1736 nucleotides, made up of four exon sequences of 121, 88, 185 and 1342 nucleotides. Three introns (827, 114 and 170n) were located near the 5' end of the gene. A single open reading frame starting in the second exon extends for 491 amino acids, corresponding to a protein of molecular weight 64000. The putative promoter region contains several short direct and inverted repeat sequences of 16, 18, 19 and 21 nucleotides, which extend 509n upstream from the transcription start site. The structure of the major IE gene and its protein product are discussed and compared with the corresponding IE gene from the Towne strain of HCMV.

Amino Acid Sequence↗
PubMed1977

Sequence arrangement of tRNA genes on a fragment of Drosophila melanogaster DNA cloned in E. coli.

A plasmid with the vector Col E1 attached to an insert of Drosophila melanogaster DNA carrying four tRNA genes has been cloned in E. coli. Some features of the sequence arrangement and the positions of the tRNA genes have been determined by electron microscopic methods and by restriction endonuclease mapping. tRNA genes were mapped at 1.4, 4.7, 5.9 and 8.6 kb from one of the Drosophila/Col E1 junctions in the Drosophila insert of total length 9.34 kb. There are several secondary structure features consisting of inverted repeat sequences of length about 70-100 nucleotide pairs, some with and some without intervening loops, irregularly distributed on the insert. Cross-hybridization of tRNAs isolated by hybridization to separated restriction fragments indicate that the tRNA genes at 4.7, 5.9 and 8.6 kb are identical and differ from the one at 1.4 kb. Thus the positions of the genes, of the secondary structure features and of the restriction endonuclease sites all indicate that the spacers between the genes are not identical tandem repeats. In situ hybridization with cRNA transcribed from the plasmid showed localization at region 42A of chromosome 2R.

Animals↗
PubMed1988

The Clostridium perfringens chloramphenicol resistance transposon Tn4451 excises precisely in Escherichia coli.

Nucleotide sequence analysis of the Tn4451-deletion derivatives, pJIR47 and pJIR86, which were derived from Escherichia coli and Clostridium perfringens, respectively, showed that the deletion events that led to the formation of these plasmids were identical and precise. The results also showed that the termini of this C. perfringens-derived transposon contained imperfect 12-bp inverted repeat sequences which had some sequence similarity with the termini of Tn3-like transposons.

Base Sequence↗
PubMed1992

Characterization of the tet(M) determinant of Tn916: evidence for regulation by transcription attenuation.

The nucleotide sequence of the tetracycline resistance determinant tet(M), located on conjugative transposon Tn916 of Enterococcus faecalis, was determined and found to encode a 72,486-dalton protein exhibiting a high degree of homology with other tet(M) determinants. A short open reading frame corresponding to a 28-amino-acid peptide and containing a number of inverted repeat sequences was noted immediately upstream of tet(M), suggesting that regulation might occur by a mechanism involving transcriptional attenuation. Transcription analyses found this to indeed be the case, showing that the expression of tet(M) resulted from an extension of a small transcript representing the upstream leader region into the resistance determinant. Exposure of cells to tetracycline resulted in a significant increase in the amount of tet(M) transcription; this increase could be explained on the basis of increased transcriptional read-through from the upstream transcript. A model suggesting how transcriptional attenuation might operate in this system is presented.

Amino Acid Sequence↗
PubMed2005

Genetic analysis of rDNA-ITS2 and RAPD loci in field populations of the malaria vector, Anopheles stephensi (Diptera: Culicidae): implications for the control program in Iran.

Anopheles stephensi is one of the most important malaria vectors in the Middle-East, the Indian subcontinent, the Far-East and is the main malaria vector in south of Iran. This vector is thought to be a single but polytypic species, despite its enormous geographical range. To examine this hypothesis, we analyzed the rDNA-ITS2 and RAPD loci in different populations of An. stephensi from Iran. rDNA-ITS2 region in all sequenced specimens of An. stephensi contained a (CA)7 microsatellite sequence. Construction of phylogenetic tree based on rDNA-ITS2 sequences revealed that there only is a minor polymorphism between the different populations, despite their vast geographical distances. RAPD-PCR could differentiate rural and urban populations of An. stephensi, but it is unclear whether these two samples represent mysorensis and the type form. Further characterization of interested RAPD fragments by cloning; have shown the nature of inverted repeats and the presence of microsatellite region (GT) in both ends near to inverted repeat sequences of primers. These results showed that An. stephensi in Iran could be considered a single species with different biological and ecological forms in different zoogeographical zones. Further studies are needed to demonstrate the relation between RAPD and microsatellite sequences and the differences seen in the field for this species. This data will serve as first report on the sequence of rDNA-ITS2 and a microsatellite-containing RAPD region, which could be used for species-specific diagnosis and differentiation of urban and rural populations in An. stephensi.

Animals↗
PubMed1990

Ribosomal protein S14 transcripts are edited in Oenothera mitochondria.

The gene encoding ribosomal protein S14 (rps14) in Oenothera mitochondria is located upstream of the cytochrome b gene (cob). Sequence analysis of independently derived cDNA clones covering the entire rps14 coding region shows two nucleotides edited from the genomic DNA to the mRNA derived sequences by C to U modifications. A third editing event occurs four nucleotides upstream of the AUG initiation codon and improves a potential ribosome binding site. A CGG codon specifying arginine in a position conserved in evolution between chloroplasts and E. coli as a UGG tryptophan codon is not edited in any of the cDNAs analysed. An inverted repeat 3' of an unidentified open reading frame is located upstream of the rps14 gene. The inverted repeat sequence is highly conserved at analogous regions in other Oenothera mitochondrial loci.

Amino Acid Sequence↗