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PubMed1991

Molecular organization of the minimal replicon of novel, narrow-host-range, lactococcal plasmid pCI305.

Plasmid pCI305 is an 8.7-kb, narrow-host-range, cryptic plasmid originating from Lactococcus lactis subsp. lactis UC317. The nucleotide sequence of the pCI305 replication region was determined. A single open reading frame of 1158 bp was identified in the trans-active domain repB. The size of the predicted repB protein (46 kDa) is in close agreement with the size of the repB product visualized in vivo in Escherichia coli when repB was placed under control of the inducible phi T7 RNA polymerase promoter. In vivo substitution of the native repB promoter sequence with a Tn5-derived promoter sequence was demonstrated. repA, a 344-bp cis-acting region which is the probable pCI305 replication origin region, was noncoding, was AT-rich, and possessed a unique set of inverted and direct repeat sequences. No significant homology between repA or repB and other gram-positive replication regions was evident. Combined with the absence of a detectable single-stranded DNA intermediate during replication, these results indicate that the pCI305 replication region differs markedly from most gram-positive replicons examined to date. The presence on other lactococcal plasmids of replication regions related to that of pCI305 was demonstrated.

Amino Acid Sequence↗
PubMed1999

Characterization and cloning of celR, a transcriptional regulator of cellulase genes from Thermomonospora fusca.

CelR, a protein that regulates transcription of cellulase genes in Thermomonospora fusca (Actinomycetaceae) was purified to homogeneity. A 6-kilobase NotI-SacI fragment of T. fusca DNA containing the celR gene was cloned into Esherichia coli and sequenced. The celR gene encodes a 340-residue polypeptide that is highly homologous to members of the GalR-LacI family of bacterial transcriptional regulators. CelR specifically binds to a 14-base pair inverted repeat, which has sequence similarity to the binding sites of other family members. This site is present in regions upstream of all six cellulase genes in T. fusca. The binding of CelR to the celE promoter is inhibited specifically by low concentrations of cellobiose (0.2-0.5 mM), the major end product of cellulases. The other sugars tested did not affect binding at equivalent or 50-fold higher concentrations. The results suggest that CelR may act as a repressor, and that the mechanism of induction involves a direct interaction of CelR with cellobiose.

Actinomycetaceae↗
PubMed2001

Evidence for a rolling-circle mechanism of phage DNA synthesis from both replicative and integrated forms of CTXphi.

The genes encoding cholera toxin, the principal virulence factor of Vibrio cholerae, are part of the circular single-stranded DNA genome of CTXphi. In toxigenic V. cholerae strains, the CTXphi genome is typically found in integrated arrays of tandemly arranged CTX prophages. Infected cells that lack a chromosomal integration site harbour the CTXphi genome as a plasmid (pCTX). We studied the replication of pCTX and found several indications that this plasmid replicates via a rolling-circle (RC) mechanism. The initiation and termination sites for pCTX plus-strand DNA synthesis were mapped to a 22 bp sequence that contains inverted repeats and a nonanucleotide motif found in the plus-strand origins of several RC replicons. Furthermore, similar to other RC replicons, replication of plasmids containing duplicated pCTX origins resulted in the deletion of sequences between the two origins and the formation of a single chimeric origin. Our previous work revealed that CTX prophage arrays give rise to hybrid CTX virions that contain sequences derived from two adjacent prophages. We now report that the boundaries between the sequences contributed to virions by the upstream and the downstream prophages in an array correspond to the site at which synthesis of plus-strand pCTX DNA is initiated and terminated. These data support the model that plus-strand CTXphi DNA is generated from chromosomal prophages via a novel process analogous to RC replication.

Bacteriophages↗
PubMed1983

A new class of site-specific endodeoxyribonucleases. Endo.Sce I isolated from a eukaryote, Saccharomyces cerevisiae.

We had found that yeasts had intracellular endodeoxyribonucleases that cut phage DNA into a set of double-stranded fragments with discrete chain lengths. We purified one of them to apparent homogeneity from Saccharomyces cerevisiae and designated it Endo.Sce I. Sequence analysis around 5 cleavage sites in plasmid DNA and phage DNA revealed that Endo.Sce I cuts a defined phosphodiester bond in each strand of double helix at the cleavage sites and produces free cohesive ends consisting of 4 nucleotides protruding at 3'-termini. However, unlike in the case of prokaryotic type II-restriction endonucleases, (i) Endo.Sce I seems to consist of two nonidentical subunits, (ii) no common palindrome or consensus sequence including more than 5 base pairs is detected at or near these cleavage sites, and (iii) Endo.Sce I can cut the DNA isolated from the cells that produced Endo.Sce I. All of the 5 cleavage sites are included in inverted repeats, but these inverted repeats are variable in size, nucleotide sequence, and distance between repeating units. An inverted repeat itself is not a structure recognized by Endo.Sce I. This study shows that Endo.Sce I is the first example of eukaryotic site-specific endonuclease and has properties, as described above, which distinguish it from prokaryotic restriction endonucleases.

Base Sequence↗
PubMed1997

Identification and DNA sequence of the mobilization region of the 5-nitroimidazole resistance plasmid pIP421 from Bacteroides fragilis.

The nucleotide sequence of the DNA mobilization region of the 5-nitroimidazole resistance plasmid pIP421, from strain BF-F239 of Bacteroides fragilis, was determined. It contains a putative origin of transfer (oriT) including three sets of inverted repeats and two sequences reminiscent of specific integration host factor binding sites. The product of the mobilization gene mob421 (42.2 kDa) is a member of the Bacteroides mobilization protein family, which includes the MobA of pBI143, NBUs, and Tn4555. Sequence similarity suggests that it has both oriT binding and nicking activities. The transfer frequency of pIP421 in a B. fragilis donor strain possessing a Tc(r) or Tc(r) Em(r)-like conjugative transposon was significantly enhanced by tetracycline. Moreover, the mobilization region of pIP421 confers the ability to be mobilized from Escherichia coli by an IncP plasmid.

Amino Acid Sequence↗
PubMed1984

Involvement of retrovirus reverse transcriptase-associated RNase H in the initiation of strong-stop (+) DNA synthesis and the generation of the long terminal repeat.

Reconstructed enzymatic reactions containing purified reverse transcriptase and defined analog substrates which mimic those purported to be natural substances for reverse transcription in vivo were employed to delineate the mechanism of strong-stop (+) DNA synthesis. Our analysis of this system has indicated that strong-stop (+) DNA synthesis is initiated after the introduction of a nick in the viral RNA genome between a polypurine sequence and an inverted repeat that represents the end of the long terminal repeat. Since inhibitors of the reverse transcriptase-associated RNase H activity prevent the introduction of the nick and the synthesis of strong-stop (+) DNA synthesis, it appears that this particular reverse transcriptase-associated enzymatic activity is responsible for the initiation of strong-stop (+) DNA. Our data also indicated that the RNase H activity creates a second nick in the viral RNA genome 11 nucleotides upstream from the strong-stop (+) DNA initiation site since the strong-stop (+) DNA synthesized in these reactions is covalently linked to an oligoribonucleotide 11 residues in length. Nucleotide sequence analysis of the oligoribonucleotide primer molecule indicated that a single homogenous oligomer was associated with strong-stop (+) DNA exhibiting the sequence rArGrGrGrArGrGrGrGrGrA. The oligoribonucleotide primer can be removed from strong-stop (+) DNA by the purified reverse transcriptase, which creates a nick at the junction between the primer and strong-stop (+) DNA. These data demonstrate that the initiation of strong-stop (+) DNA synthesis is mediated by RNase H and that the site of initiation is exactly at the end of the long terminal repeat, providing evidence for yet another function of this reverse transcriptase-associated enzymatic activity in the synthesis of retrovirus DNA.

Animals↗
PubMed1986

Structural and functional studies of insertion element IS200.

The nucleotide sequence of the insertion element IS200 has been determined partially, including the junctions between the element and the host chromosome at the insertion site. At most, two bases (A-A) are found repeated at the junctions and could be duplications of host sequences generated by the insertion of the element. No obvious sequence repeats, either direct or inverted, have been detected between the sequences just within the two ends of the element. The element is an extremely strong block to host transcription across the insertion site. A sequence similar to known transcription termination signals was found just within the element near the right end. Removal of less than 50 base-pairs at the right end of the element abolishes the transcription block. The putative terminator sequence is located within this 50 base-pair region. Genetic studies suggest that the element contains a promoter located more than 93 base-pairs from its left end. The proposed promoter and terminator are in proper orientation to form an internal transcription unit.

Autoradiography↗
PubMed1996

[The sequences of intron 50 and 51 of DMD gene and the deletion mechanism of 51st exon].

OBJECTIVE: To investigate the relationship between the structural characters of 50th and 51st introns and deletion mechanisms of 51st exon of DMD gene. METHODS: We cloned the 3.1 Kb-Hind I human genome DNA fragment containing the 50 and 51 introns of DMD gene, sequenced the fragment completely, and analysed the character of the sequences with computer. RESULTS: The length of the fragment was 3179, including the entire 51st exon and partial 50th and 51st intron sequences. There were 36 short tandom repeats, 22 direct and inverted repeats, and 8 homologous sequences in the 50th and 51st introns. A TATA box was found in the intron 50 and it may be another new promotor of DMD gene. CONCLUSION: The hypothesis, which the recombination of repeat sequences may result in the deletion of 51st exon, is put forward.

Base Sequence↗
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↗
PubMed1997

Nucleotide sequence analysis of the junctions of the terminal inverted repeats of the Streptomyces lividans linear chromosome.

The junctions of the Streptomyces lividans chromosomal terminal inverted repeats (TIRs) were isolated from cosmid clones as 6.2 kb PstI and 2.9 kb BamHI fragments, respectively. The fragments were completely sequenced. In each of the fragments just one open reading frame could be identified. One putative gene product showed significant similarities to a sensor and the other to a transcriptional regulator protein of prokaryotic two-component signalling systems. Next to one TIR numerous long direct repeats were found within a region of about 400 bp.

Amino Acid Sequence↗
PubMed2005

GIL16, a new gram-positive tectiviral phage related to the Bacillus thuringiensis GIL01 and the Bacillus cereus pBClin15 elements.

One of the most notable characteristics of Tectiviridae resides in their double-layer coats: the double-stranded DNA is located within a flexible lipoprotein vesicle covered by a rigid protein capsid. Despite their apparent rarity, tectiviruses have an extremely wide distribution compared to other phage groups. Members of this family have been found to infect gram-negative (PRD1 and relatives) as well as gram-positive (Bam35, GIL01, AP50, and phiNS11) hosts. Several reports have shown that tectiviruses infecting gram-negative bacteria are closely related, whereas no information is currently available on the genetic relationship among those infecting gram-positive bacteria. The present study reports the sequence of GIL16, a new isolate originating from Bacillus thuringiensis, and a genetic comparison of this isolate with the tectiviral bacteriophages Bam35 and GIL01, which originated from B. thuringiensis serovars Alesti and Israelensis, respectively. In contrast to PRD1 and its relatives, these are temperate bacteriophages existing as autonomous linear prophages within the host cell. Mutations in a particular motif in both the GIL01 and GIL16 phages are also shown to correlate with a switch to the lytic cycle. Interestingly, both bacterial viruses displayed narrow, yet slightly different, host spectrums. We also explore the hypothesis that pBClin15, a linear plasmid hosted by the Bacillus cereus reference strain ATCC 14579, is also a prophage. Sequencing of its inverted repeats at both extremities and a comparison with GIL01 and GIL16 emphasize its relationship to the Tectiviridae.

Bacillus cereus↗
PubMed1992

Regulation of expression of the interleukin 6 gene: structure and function of the transcription factor NF-IL6.

The interleukin 6 (IL-6) promoter is rapidly and transiently activated by other cytokines, including IL-1 and tumour necrosis factor (TNF), as well as by phorbol esters and cyclic AMP agonists. Studies using promoter mutants suggested that an IL-1-responsive element mapped within the -180 to -123 region of the IL-6 promoter. A nuclear factor (NF-IL6) that recognized a unique sequence containing an inverted repeat, ACATTGCACAATCT, was identified within the region. Direct cloning of the human NF-IL6 revealed its similarity to C/EBP, a liver- and adipose tissue-specific transcription factor. C/EBP and NF-IL6 recognize the same nucleotide sequence, but exhibit distinct patterns of expression. NF-IL6 is expressed at a low level in normal tissues, but is rapidly and drastically induced by bacterial lipopolysaccharide (LPS) or inflammatory cytokines such as IL-1, TNF and IL-6. Recently, NF-IL6 has been shown to be identical to IL-6DBP, the DNA-binding protein which is responsible for IL-6-mediated induction of several acute-phase proteins. Evidence that NF-IL6 DNA-binding activity is increased after IL-6 stimulation without increased NF-IL6 protein synthesis demonstrates the importance of post-translational modification. There are some results indicating that phosphorylation is involved in transcriptional and binding activities of NF-IL6. Taken together, these findings indicate that NF-IL6 may be an important transcription factor on the signal transduction pathways of IL-1 and IL-6.

Acute-Phase Proteins↗
PubMed1991

The kalilo linear senescence-inducing plasmid of Neurospora is an invertron and encodes DNA and RNA polymerases.

The nucleotide sequence of kalilo, a linear plasmid that induces senescence in Neurospora by integrating into the mitochondrial chromosome, reveals structural and genetic features germane to the unique properties of this element. Prominent features include: (1) very long perfect terminal inverted repeats of nucleotide sequences which are devoid of obvious genetic functions, but are unusually GC-rich near both ends of the linear DNA; (2) small imperfect palindromes that are situated at the termini of the plasmid and are cognate with the active sites for plasmid integration into mtDNA; (3) two large, non-overlapping open-reading frames, ORF-1 and ORF-2, which are located on opposite strands of the plasmid and potentially encode RNA and DNA polymerases, respectively, and (4) a set of imperfect palindromes that coincide with similar structures that have been detected at more or less identical locations in the nucleotide sequences of other linear mitochondrial plasmids. The nucleotide sequence does not reveal a distinct gene that codes for the protein that is attached to the ends of the plasmid. However, a 335-amino acid, cryptic, N-terminal domain of the putative DNA polymerase might function as the terminal protein. Although the plasmid has been co-purified with nuclei and mitochondria, its nucleotide composition and codon usage indicate that it is a mitochondrial genetic element.

Amino Acid Sequence↗
PubMed1979

Characterization of the genome of the free-living nematode Panagrellus silusiae: absence of short period interspersion.

The complexity of the DNA of the free-living nematode Panagrellus silusiae has been examined. Reassociation kinetics of pressure-sheared fragments (approximately 290 nucleotides) in 0.18 M Na+ at 60 degrees C showed the presence of foldback, repetitive, and unique DNA sequence elements. The three classes comprise 9.3%, 26.1%, and 61.3% of the total DNA, respectively. The mean length of the foldback duplex DNA after digestion with S1 nuclease is about 185 nucleotides. There are about 1.8 x10(4) inverted repeats per genome. Sequence arrangement was deduced from (1) renaturation kinetic profiles of long and short fragments on hydroxylapatite; (2) the pattern of renaturation of tracer DNA, labeled in vitro with 125I, of various sizes after incubation with excess short fragments; and (3) thermal denaturation behavior of DNA that had been reassociated to various C0t values. It was found that DNA fragments of the repetitive fraction that are, at least, 2000 nucleotides in length are virtually free of unique sequences. Moreover, it is estimated that the repeated segments in this species could extend for 10,000 nucleotide pairs. Thus, Panagrellus DNA lacks the pattern of extensive short period interspersion that is typified by the DNA of Xenopus.

Animals↗
PubMed2004

Detection of template strand switching during initiation and termination of DNA replication of porcine circovirus.

Nucleotide substitution mutagenesis was conducted to investigate the importance of the inverted repeats (palindrome) at the origin of DNA replication (Ori) of porcine circovirus type 1 (PCV1). Viral genomes with engineered mutations on either arm or both arms of the palindrome were not impaired in protein synthesis and yielded infectious progeny viruses with restored or new palindromes. Thus, a flanking palindrome at the Ori was not essential for initiation of DNA replication, but one was generated inevitably at termination. Among the 26 viruses recovered, 16 showed evidence of template strand switching, from minus-strand genome DNA to palindromic strand DNA, during biosynthesis of the Ori. Here I propose a novel rolling-circle "melting-pot" model for PCV1 DNA replication. In this model, the replicator Rep protein complex binds, destabilizes, and nicks the Ori sequence to initiate leading-strand DNA synthesis. All four strands of the destabilized inverted repeats exist in a "melted" configuration, and the minus-strand viral genome and a palindromic strand are available as templates, simultaneously, during initiation or termination of DNA replication. Inherent in this model is a "gene correction" or "terminal repeat correction" mechanism that can restore mutilated inverted-repeat sequences to a palindrome at the Ori of circular DNAs or at the termini of circularized linear DNAs. Potentially, the melted state of the inverted repeats increases the rate of noncomplementary or illegitimate nucleotide incorporation into the palindrome. Thus, this melting-pot model provides insight into the mechanisms of DNA replication, gene correction, and illegitimate recombination at the Ori of PCV1, and it may be applicable to the replication of other circular DNA molecules.

Animals↗
PubMed1983

Genes and pseudogenes for mouse U1 and U2 small nuclear RNAs.

Regions of the mouse genome homologous to U1 and U2 small nuclear RNAs have been isolated. The U1 regions are identified as pseudogenes on the basis of: 1) insertions, deletions, and substitutions in the sequence of the DNA as compared with that of U1 RNA; 2) absence of complementary RNA in mouse tissues; and 3) lack of expression upon microinjection in Xenopus oocyte nuclei. A U2 region is identified as a gene on the basis of near identity of sequence with U2 RNA and capacity to direct the synthesis of U2-like RNA in Xenopus oocyte nuclei. Both U1 and U2 sequences are repeated in the mouse genome, the U2 sequences analyzed here in an inverted repeat arrangement.

Animals↗
PubMed1991

Diversification of the rice Waxy gene by insertion of mobile DNA elements into introns.

The waxy (wx) gene of Oryza glaberrima was cloned, and its nucleotide sequence was determined. A waxy mutant of O. glaberrima showing a glutinous phenotype was found to contain a substitution mutation generating a termination codon in the coding region of the wx gene. The Wx sequence of O. glaberrima was different from that of Oryza sativa by substitutions and insertions/deletions, among which only a few substitutions occurred in several exons not to severely alter the amino acid sequence of the Wx protein. The most striking difference observed in introns was a 139-bp deletion (or insertion) in intron 10 of O. glaberrima (or O. sativa). In O. sativa, 125 bp of the 139-bp sequence was flanked by direct repeats of a 14-bp sequence. A sequence homologous to the 125-bp sequence was found in the region preceding exon 2; this sequence was also flanked by direct repeats of another 14-bp sequence. This result and the observation that the 125-bp sequence was interspersed in rice genomes indicate that they are SINEs (short interspersed elements) in the plant system. We also identified a DNA sequence with long terminal inverted repeats in intron 13 of both O. glaberrima and O. sativa. This sequence was present in multiple copies in rice genomes, suggesting that it is a transposable element. These results obtained suggest that mobile DNA elements have diversified the rice Waxy gene by inserting into introns, each of which may originally have a length of about 100 bp.

Amino Acid Sequence↗
PubMed1994

The complete DNA sequence and the genetic organization of the short unique region (US) of the bovine herpesvirus type 1 (ST strain).

The DNA sequence of bovine herpesvirus type 1 (BHV-1) ST strain (BHV-1.2 subtype) entire unique short (US) region and part of adjacent flanking sequences of the inverted repeats was determined. The BHV-1 ST US region is 9745 bp in size and has a 70.5% G + C content. Eight potential open reading frames (ORFs) longer than 100 amino acids and designated ORF1 to ORF8 were identified on this sequence. Seven of these had counterparts in the US of herpes simplex type 1 (HSV-1), pseudorabies virus (PRV), and equine herpesvirus type 1 (EHV-1). BHV-1 ORF2 encodes a protein homologous to HSV-1 US2; ORF3 putative protein exhibits homology with HSV-1 US3 (serine-threonine protein kinase); ORF4 encodes a putative glycoprotein homologous to PRV gG (gX) and EHV-1 gG (gX); ORFs 5,6, and 7 encode respectively HSV-1 glycoproteins gD, gl, and gE homologues; and ORF8 codes for a putative homologue of HSV-1 US9. Although BHV-1 ST strain apparently lacks a HSV-1 US1 homologue, the genetic content and the genomic organization of its US region was found to be similar to the general organization already described for HSV-1, EHV-1, PRV, and avian herpesviruses HVT and MDV US regions. In fact, the BHV-1 US region genomic organization is the closest to the general consensus determined from the comparison of known alphaherpesvirus US regions.

Amino Acid Sequence↗