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PubMed1983

S1 sensitive sites in adenovirus DNA.

S1 nuclease has been used as a probe for regions of DNA secondary structure in supercoiled recombinant plasmids containing adenovirus (Ad) DNA sequences. In the sequences examined two S1 sensitive sites were identified in the left-terminal 16.5% of Ad 12 DNA, one of which aligned approximately with an inverted repeat region in the DNA sequence. In addition an S1 sensitive site was dictated by a potential cruciform structure in the region of the Ad 2 major late promoter. In contrast to the expected cleavage site at the loop of the cruciform, cleavage occurred at the base of the stem in the region of the TATA box. All three S1 sensitive sites identified were more sensitive to S1 than the endogenous sites in the parent plasmids.

Adenoviridae↗
PubMed1984

Molecular cloning and nucleotide sequencing of the nusB gene of E. coli.

The nusB gene of E. coli has been cloned in plasmid pBR322. Using genetic complementation as an assay for the gene, its location in subclones was analyzed, and the nucleotide sequence of this gene and its flanking regions was determined. The coding region consists of 417 base pairs (bp), which specify a protein of 139 amino acids, and the calculated molecular weight of the nusB protein is 15,702. The nusB protein contains 20 acidic and 21 basic amino acids. A significant promoter sequence was not found to be located in the region upstream from the translational initiation codon. The possibility that the nusB gene consists of an operon is discussed. After the coding region, there is a G-C rich inverted repeat sequence followed by a run of Ts, which could be a transcriptional terminator of the nusB gene.

Amino Acid Sequence↗
PubMed1988

Pilin-gene phase variation of Moraxella bovis is caused by an inversion of the pilin genes.

Moraxella bovis Epp63 can express either of two different pilin proteins, called alpha and beta. We have previously cloned and sequenced the beta-pilin gene and now report that DNAs isolated from bacteria expressing alpha pilin have hybridization patterns consistently different from those of bacteria expressing beta pilin. The phase variation between alpha- and beta-pilin gene expression appears to be associated with an inversion of about 2 kilobases of DNA, whose endpoints occur within the coding region of the expressed pilin gene. Comparisons of the beta-pilin gene sequence with those of well-studied bacterial inversion systems revealed a stretch of 58% sequence similarity (21 of 36 base pairs) between the left inverted repeat of the Salmonella typhimurium flagellar hin control region and the amino-terminal portion of the beta-pilin gene.

Bacterial Outer Membrane Proteins↗
PubMed1991

DNA sequences of three beta-1,4-endoglucanase genes from Thermomonospora fusca.

The DNA sequences of the Thermomonospora fusca genes encoding cellulases E2 and E5 and the N-terminal end of E4 were determined. Each sequence contains an identical 14-bp inverted repeat upstream of the initiation codon. There were no significant homologies between the coding regions of the three genes. The E2 gene is 73% identical to the celA gene from Microbispora bispora, but this was the only homology found with other cellulase genes. E2 belongs to a family of cellulases that includes celA from M. bispora, cenA from Cellulomonas fimi, casA from an alkalophilic Streptomyces strain, and cellobiohydrolase II from Trichoderma reesei. E4 shows 44% identity to an avocado cellulase, while E5 belongs to the Bacillus cellulase family. There were strong similarities between the amino acid sequences of the E2 and E5 cellulose binding domains, and these regions also showed homology with C. fimi and Pseudomonas fluorescens cellulose binding domains.

Amino Acid Sequence↗
PubMed1987

B-lymphoma induction by reticuloendotheliosis virus: characterization of a mutated chicken syncytial virus provirus involved in c-myc activation.

Nondefective reticuloendotheliosis virus induces chicken bursal lymphoma in a manner similar to that of avian leukosis virus. The provirus integrates in the c-myc locus and uses a promoter insertion mechanism to activate c-myc expression. We cloned a provirus involved in c-myc activation from a B lymphoma. Detailed structural characterization of this clone, including sequence determination, revealed proviral insertion at 512 base pairs preceding the second c-myc exon. The provirus has a deletion of 80% of the viral genes but retains two intact long terminal repeats (LTRs). A segment of the viral env sequence is present in an inverted orientation. Elevated expression of c-myc, apparently directed by the 3' LTR, was detected. However, despite the presence of an intact 5' LTR, no viral transcripts were detected. Thus, the internal proviral rearrangement can affect 5' LTR transcription or stability of the message or both. This finding is in consonance with the view that proviral deletion plays an important role in the induction of bursal lymphomas by nonacute retroviruses.

Animals↗
PubMed1992

A retroviral sequence of the Chinese hamster ovary cell line.

A partial cDNA (B52) molecule with the characteristics of retroviral sequences was isolated from the Chinese hamster ovary (CHO) K1 cell line. The B52 cDNA contains 1184 nucleotides. The first 452 nucleotides (nt) are 71% homologous to the env gene of Moloney murine leukemia virus (MMLV) and murine endogenous retroviruses. The 139 amino acids predicted from the 452 nt have 82% homology with the carboxy-terminal amino acids of the env protein of MMLV. The remaining 732 nt have several features of a typical retroviral long terminal repeat (LTR). For example, the first 14 nt are identical to the 5' inverted repeat of the retroviral LTRs. The 41-nt sequence at the 3' end is common to the R region of retroviral LTRs. The 732-nt sequence was shown to have promoter activity. The activity is approximately twofold higher than that of the Rous sarcoma virus LTR, and 1.5-fold lower than that of the early promoter of SV40 virus. Two species of mRNA of 5.2 and 2.7 kb in size were readily detected by B52 cDNA in the CHO K1 cells.

Amino Acid Sequence↗
PubMed1993

Distinct dimerization domains provide antagonist pathways for thyroid hormone receptor action.

Transcriptional regulation by thyroid hormone is mediated through its nuclear receptors, which bind to target response elements as homodimers or as heterodimers with proteins such as retinoid X receptors (RXR). Thyroid hormone response elements exhibit remarkable flexibility in that the receptor binding half-sites can be arranged as direct repeats, inverted repeats, or everted repeats. We report that a limited region at the carboxyl-terminal end of the thyroid hormone receptor differentially contributes to the formation of receptor homo- and heterodimers. The functionally inactive thyroid hormone receptor splicing variant alpha 2, which is altered at the juncture of the homo- and heterodimerization domains, cannot form homodimers. However, alpha 2 can form a heterodimer when bound to half-sites arranged as a direct repeat spaced by 4 base pairs (DR4), but not with other arrangements of response element half-sites. The alpha 2-RXR heterodimer strongly inhibits wild type receptor function mediated by the DR4 element, suggesting that the alpha 2 isoform modulates thyroid hormone action by binding as an antagonist to a subset of response elements.

Amino Acid Sequence↗
PubMed2002

Stabilization of a terminal inversion duplication of 8p by telomere capture from 18q.

Terminal inversion duplications of the short arm of chromosome 8 are one of the more common chromosome rearrangements in humans. We report an infant with multiple congenital anomalies, in whom karyotype analysis showed a terminal inversion duplication of 8p including additional material at the distal end of the derivative chromosome, shown to be of chromosome 18q origin. Terminal inversion duplications of 8p are the result of meiotic recombination between inverted olfactory gene receptor repeats in 8p. This recombination generates a dicentric intermediate that breaks during anaphase, and the broken chromosome end is stabilized by telomere healing or telomere capture. The origin of the telomeric region in the majority of constitutional chromosome deletions studied to date was shown to be from telomere healing; the de novo addition of telomeric repeats. In the proband a cytogenetically detectable piece of chromosome 18q was present on the distal end of the derivative 8, suggesting that this chromosome was stabilized by telomere capture of 18q. FISH analyses of additional cases may yield information as to whether telomere capture or telomere-healing events are the predominant mechanism of chromosome stabilization in terminal inversion duplications of 8p.

Adult↗
PubMed1997

Transcriptional analysis of the threonine dehydrogenase gene of Xanthomonas campestris.

The nucleotide sequence has previously been determined for the Xanthomonas campestris pv. campestris gene coding for threonine dehydrogenase (tdh). Flanking this gene are the upstream region possessing promoter activity and the downstream perfect inverted repeat having potential to form a stem-loop structure which resembles a transcription terminator. In addition, Northern blot analysis suggested the transcript of this gene to be monocistronic. In the present study, the essential region for promoter activity was narrowed down to a stretch of 57 bp which still retained 84% of the promoter activity. The first nucleotide to be transcribed is the guanosine at 30 nt upstream from the proposed tdh start codon. The putative terminator exhibited transcriptional termination activity bidirectionally in both Escherichia coli and X. campestris. These observations indicate that the transcriptional structure of X. campestris tdh is different from that of E. coli where tdh and kbl are organized into the tdh operon. Furthermore, the expression of tdh in X. campestris is repressed by leucine, a situation different from that in E. coli where leucine induces the expression of tdh operon.

Alcohol Oxidoreductases↗
PubMed1986

Evidence of sequences resembling avian retrovirus long terminal repeats flanking the trout protamine gene.

Additional TATA boxes are present in the flanking regions of trout protamine genes. Their activity as promoters was assayed using an in vitro transcription system. These additional TATA boxes, together with polyadenylation signals that include the consensus AATAAA and CACTG sequences very close to the promoters, suggest that these sequences may be closely related to retroviral long terminal repeat (LTR) sequences. Other features of retroviral LTRs that are also present are short inverted repeats. The LTR-like sequences flanking the trout protamine gene show significant homology to the avian sarcoma virus LTR over a 40-bp region. The trout protamine gene falls into the relatively rare intronless class of eukaryotic genes. This suggests that the gene could have been derived from a processed gene introduced into the genome by reverse transcription of a mature mRNA. The protamine-mRNA-coding region is flanked by AACA... TGTT sequences, which might represent vestigial traces of past recombination events and whose presence supports the notion that the protamine gene sequence was of foreign origin. Recent attempts in this laboratory to transfer the protamine gene into mouse cells have resulted in a high frequency of deletions similar to those observed with constructs in which a retrovirus was used as a vector to transfect foreign DNA with promoters. The distribution of protamine genes in the animal kingdom is very sporadic, which suggests that protamine genes appeared relatively late in evolution. The nonuniform occurrence of the gene among lower vertebrates may have been the result of its horizontal transmission only to certain species, possibly by infection with retroviruses that acquired it from a different species.

Animals↗
PubMed1981

Molecular mechanisms of nucleotide-sequence rearrangements in cDNA clones of human fibroblast interferon mRNA.

The characterization and nucleotide sequencing of cDNA clones of human fibroblast interferon (HFIF) mRNA (Derynck et al., 1980) revealed two types of structural inversions :(i) an inverted repeat of a 3'-proximal segment at the 5'end, or (ii) an inverted 5'-terminal segment. Based on the nucleotide sequence of these inverted clones, we have deduced molecular mechanisms to explain how the rearrangements could have arisen. We propose that the usual small 3'-terminal hairpin does not form after the reverse transcription produces the first cDNA strand. Instead, base pairing occurs between the 3'-terminal segment and the more distal region of the first cDNA strand, forming a much larger hairpin loop. Second-strand synthesis followed by either nicking or unfolding of the loop structure and continuation of the polymerase reaction would lead to the observed types of inversions. The 5'-truncated clone, pHFIF-1, whose new 5'-terminal sequence diverges from that of the corresponding region in the mRNA, can also be explained by such a mechanism.

Base Sequence↗
PubMed1996

Structural feature of the initiator tRNA gene from Pyrodictium occultum and the thermal stability of its gene product, tRNA(imet).

Pyrodictium occultum is a hyperthermophilic archaeum that grows optimally at 105 degrees C. To study how tRNA molecules in P occulrum are thermally stabilized, we isolated the initiator tRNA gene from the organism using a synthetic DNA probe of 74 bp containing the known nucleotide sequences that are conserved in archaeal initiator tRNAs. A HindIII fragment of 700 bp containing the Pyrodictium initiator tRNA gene was cloned and sequenced by cycle sequencing. The nucleotide sequence revealed that the Pyrodictium initiator tRNA gene has no introns, and that the 3'CCA terminus is encoded. The tRNA gene also contained a unique TATA-like sequence, AAGCTTATAA, which is likely the promoter proposed for archaeal rRNA genes, 450 bp upstream of the 5' end of the tRNA coding region. In the region adjacent to the 3' end of the tRNA coding region, there was a sig G-C base pair inverted repeat followed by a C-rich sequence like the p-independent transcription termination signal of bacterial genes. The Pyrodictium initiator tRNA sequence predicted from the gene sequence contained all of the nucleotide residues A1, A37, U54, A57, U60, and U72, in addition to three G-C base pairs in the anticodon stem region, which are characteristic of archaeal initiator tRNAs. The melting temperature (Tm) of the unmodified initiator tRNA synthesized in vitro using the cloned tRNA gene as a template was 80 degrees C, which is only two degrees lower than that calculated from the G-C content in the stem regions of the tRNA. In contrast, the Tm of the natural initiator tRNA isolated from P occultum was over 100 degrees C. Analysis of digests of purified Pyrodictium initiator tRNA by means of HPLC-mass spectrometry and [32P] post-labeling, indicated that the tRNA contains a variety of modified nucleosides. These results suggest that the extraordinarily high melting temperature of P occultum tRNA(Met)i is due to posttranscriptional modification.

Archaea↗
PubMed2002

PrnA, a Zn2Cys6 activator with a unique DNA recognition mode, requires inducer for in vivo binding.

The PrnA transcriptional activator of Aspergillus nidulans binds as a dimer to CCGG-N-CCGG inverted repeats and to CCGG-6/7N-CCGG direct repeats. The binding specificity of the PrnA Zn cluster differs from that of the Gal4p/Ppr1p/UaY/Put3p group of proteins. Chimeras with UaY, a protein that strictly recognizes a CGG-6N-CCG motif, show that the recognition of the direct repeats necessitates the PrnA dimerization and linker elements, but the recognition of the CCGG-N-CCGG inverted repeats depends crucially on the PrnA Zn binuclear cluster and/or on residues amino-terminal to it. Three high-affinity sites in two different promoters have been visualized by in vivo methylation protection. Proline induction is essential for in vivo binding to these three sites but, as shown previously, not for nuclear entry. Simultaneous repression by ammonium and glucose does not affect in vivo binding to these high-affinity sites. PrnA differs from the isofunctional Saccharomyces cerevisiae protein Put3p, both in its unique binding specificity and in the requirement of induction for in vivo DNA binding.

Aspergillus niger↗
PubMed1984

Analysis of a cloned colicin Ib gene: complete nucleotide sequence and implications for regulation of expression.

The complete nucleotide sequence of a 2,971 base pair EcoRI fragment carrying the structural gene for colicin Ib has been determined. The length of the gene is 1,881 nucleotides which is predicted to produce a protein of 626 amino acids and of molecular weight 71,364. The structural gene is flanked by likely promoter and terminator signals and in between the promoter and the ribosome binding site is an inverted repeat sequence which resembles other sequences known to bind the LexA protein. Further analysis of the 5' flanking sequences revealed a second region which may act either as a second LexA binding site and/or in the binding of cyclic AMP receptor protein. Comparison of the predicted amino acid sequence of colicin Ib with that of colicins A and E1 reveals localised homology. The implications of these similarities in the proteins and of regulation of the colicin Ib structural gene are discussed.

Amino Acid Sequence↗
PubMed1981

A complex repeated DNA sequence within the Drosophila transposable element copia.

A 320 nucleotide repeated DNA sequence within the copia coding element of Drosophila melanogaster has been identified and characterized. This sequence has been localized by DNA-DNA hybridization and electron microscopic analysis of heteroduplexes to the approximate middle of the 5 kb copia coding region. The primary sequence of this repeated DNA has been determined. The sequence is composed of three related subunits, 35-37 nucleotides in length (A, B and C). This 105 nucleotide higher order repeat has apparently been duplicated twice to yield a complex repeated sequence, ABCA'B'C'A"B"C", which exhibits divergence among the individual subunits. This sequence is AT rich, as are the direct terminal repeats which flank the copia coding region, but does not contain any apparent homology with the terminal repeats. This repeated sequence contains three presumptive polyadenylation signals and two 25 nucleotide, imperfectly matched, inverted repeat sequences adjacent to two of the polyadenylation sequences.

Animals↗
PubMed1981

Inserted sequences in bovine satellite DNA's.

The nucleotide sequence of the 1413-base-pair repeat unit of bovine 1.711a satellite DNA (density in cesium chloride, 1.711 grams per cubic centimeter) has been determined. The repeat unit contains two segments consisting of variants of a basic 23-base-pair sequence that is closely related to sequences of bovine 1.706 satellite DNA. A third segment of the repeat unit contains an unrelated 611-base-pair sequence that is not internally repetitive. This segment is flanked by inverted repeats of 8 base pairs and, on one side, by a direct repeat of the terminal sequence. A related segment is present in bovine 1.711b satellite DNA and is inserted into sequences derived from the 1.715 satellite. These nucleotide sequences suggest the timing of some of the stages in the evolution of these complex, closely related satellite DNA's and indicate the mechanisms inherent in their divergence from a common ancestor.

Animals↗
PubMed1993

Characterization of a periplasmic 3':5'-cyclic nucleotide phosphodiesterase gene, cpdP, from the marine symbiotic bacterium Vibrio fischeri.

Vibrio fischeri, a marine bacterium that forms a bioluminescent symbiosis with certain fish and squids, exhibits the unusual attribute of growth on 3':5'-cyclic AMP (cAMP), apparently through the activity of a 3':5'-cyclic nucleotide phosphodiesterase (3':5'-CNP) with exceptionally high activity. The V. fischeri 3':5'-CNP is located in the periplasm, a novel cellular location for this enzyme in bacteria. To gain insight into the physiological function of this enzyme, we cloned the gene (designated cpdP) encoding it from V. fischeri MJ-1. This is the first bacterial 3':5'-CNP gene to be cloned. Sequencing and analysis of the 1.26-kb cpdP locus revealed a single open reading frame specifying a protein of 330 amino acid residues, including a 22-amino-acid leader peptide. The putative cpdP promoter contained a reasonable -10 promoter region (TATTAT) but contained no obvious -35 region; instead, a 12-bp inverted repeat (TTAAATATTTAA) occurred just upstream of this location. A possible rho-independent transcriptional terminator with a calculated free energy of -21.2 kcal.mol-1 (ca. -88.7 kJ.mol-1) followed the CpdP protein coding sequence. The predicted subunit molecular weight of 33,636 for the mature CpdP protein (36,087 less 2,451 for the leader peptide) was consistent with the molecular weight of 34,000 estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The deduced amino acid sequence of the CpdP protein exhibited 30.3% identity with that of the low-affinity 3':5'-CNP (PDE1) of Saccharomyces cerevisiae and 33.6% identity with that of the extracellular 3':5'-CNP of Dictyostelium discoideum. The residue identities clustered in two regions, residues 100 to 146 and 238 to 269, which contained 30 of the 33 amino acids conserved in all three proteins, 4 of which were histidines. A gene replacement mutant of V. fischeri MJ-1 containing a 0.45-kb BglII deletion within the cpdP gene lacked periplasmic 3':5'-CNP activity and did not grow on cAMP, confirming for V. fischeri the relationship among cpdP, synthesis of the periplasmic 3':5'-CNP, and growth on cAMP. The mutant exhibited no obvious sensitivity to high extracellular concentrations of cAMP (5 and 10 mM), suggesting that the enzyme does not play a role in defense against extracellular cAMP.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1993

Nucleotide sequence analysis of the termini and chromosomal locus involved in site-specific integration of the streptococcal conjugative transposon Tn5252.

The 47-kb, broad-host-range, streptococcal conjugative transposon Tn5252 is capable of site-specific integration into the pneumococcal chromosome. We present the nucleotide sequence of the terminal regions of the transposon and its target site in the pneumococcal genome. No inverted repeats were found at the termini of the transposon. A 72-bp region of the target was present on either side following the insertion of Tn5252 and appeared to serve as a signal for its integration and excision. The data suggest that the left copy of the 72-bp segment was a part of the conjugative element, the crossover point of integration was nonrandom within this region, and the mechanism of insertion could resemble that of the site-specific temperate phages.

Base Sequence↗