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PubMed1992

The mitochondrial genomes of two nematodes, Caenorhabditis elegans and Ascaris suum.

The nucleotide sequences of the mitochondrial DNA (mtDNA) molecules of two nematodes, Caenorhabditis elegans [13,794 nucleotide pairs (ntp)], and Ascaris suum (14,284 ntp) are presented and compared. Each molecule contains the genes for two ribosomal RNAs (s-rRNA and l-rRNA), 22 transfer RNAs (tRNAs) and 12 proteins, all of which are transcribed in the same direction. The protein genes are the same as 12 of the 13 protein genes found in other metazoan mtDNAs: Cyt b, cytochrome b; COI-III, cytochrome c oxidase subunits I-III; ATPase6, Fo ATPase subunit 6; ND1-6 and 4L, NADH dehydrogenase subunits 1-6 and 4L: a gene for ATPase subunit 8, common to other metazoan mtDNAs, has not been identified in nematode mtDNAs. The C. elegans and A. suum mtDNA molecules both include an apparently noncoding sequence that contains runs of AT dinucleotides, and direct and inverted repeats (the AT region: 466 and 886 ntp, respectively). A second, apparently noncoding sequence in the C. elegans and A. suum mtDNA molecules (109 and 117 ntp, respectively) includes a single, hairpin-forming structure. There are only 38 and 89 other intergenic nucleotides in the C. elegans and A. suum mtDNAs, and no introns. Gene arrangements are identical in the C. elegans and A. suum mtDNA molecules except that the AT regions have different relative locations. However, the arrangement of genes in the two nematode mtDNAs differs extensively from gene arrangements in all other sequenced metazoan mtDNAs. Unusual features regarding nematode mitochondrial tRNA genes and mitochondrial protein gene initiation codons, previously described by us, are reviewed. In the C. elegans and A. suum mt-genetic codes, AGA and AGG specify serine, TGA specifies tryptophan and ATA specifies methionine. From considerations of amino acid and nucleotide sequence similarities it appears likely that the C. elegans and A. suum ancestral lines diverged close to the time of divergence of the cow and human ancestral lines, about 80 million years ago.

Amino Acid Sequence↗
PubMed1995

IS292: a novel insertion element from Agrobacterium.

A new insertion sequence, IS292, located in the 6b gene of a nopaline-type Agrobacterium strain (X88-292) isolated from poplar was identified and sequenced. IS292 is 2494 bp long, has 21 bp inverted terminal repeats with two mismatches, and generates 10 bp direct repeats upon integration. No sequence similarity was found between IS292 and other insertion elements associated with Agrobacterium, but it shows strong similarity with ISR/1 from Rhizobium leguminosarum bv. viciae. The occurrence of IS292-like sequences in various Agrobacterium isolates, especially different Agrobacterium strains isolated from the same biotope, was demonstrated by DNA hybridization.

Amino Acid Sequence↗
PubMed1993

Analysis of the Ros repressor of Agrobacterium virC and virD operons: molecular intercommunication between plasmid and chromosomal genes.

The virulence genes of the Agrobacterium tumefaciens Ti plasmid are regulated both positively and negatively. The products of the genes of the virC and virD operons play an important role in host specificity and T-DNA processing. These operons are transcribed in opposite directions and therefore bear diametrically oriented promoters. These promoters are positively regulated by the VirG protein, which is believed to be activated through phosphorylation by a histidine kinase encoded by the virA gene. The virC and virD operons are also regulated by a 15.5-kDa repressor protein encoded by the ros chromosomal gene. A mutation in ros causes the constitutive expression of virC and virD in the complete absence of the VirG protein. It appears, therefore, that the Ros repressor interacts with the regulatory region of these operons. The Ros repressor is shown here to bind to an upstream sequence (Ros box) comprising 40 bp bearing a 9-bp inverted repeat, TATATTTCA/TGTAATATA, in the promoter region of these operons. The affinity for this sequence is specific and tenacious, since the addition of at least a 20,000-fold excess of competitor DNA failed to remove the Ros protein coding sequence from the Ros box. DNase I footprint analysis showed that the Ros box overlaps the binding site of VirG (Vir box). This result suggests that virC and virD transcription is modulated by Ros and VirG proteins.

Agrobacterium tumefaciens↗
PubMed1998

Dictyostelium discoideum nuclear plasmid Ddp6 is a new member of the Ddp2 plasmid family.

Ddp6 is a high-copy number, circular plasmid found in the nucleus of the simple eukaryote Dictyostelium discoideum wild isolate NC47.2. The complete nucleotide sequence, 5257 bp, shows that Ddp6 has a structure similar to that of other members of the Ddp2 plasmid family: a single long 2.8-kb open reading frame (rep gene) and an inverted repeat containing a pair of 654-bp elements. A single constitutively expressed 3.3-kb transcript of the rep gene was detected in RNA prepared from vegetative and developmental cells. Maintenance assays revealed that sequences within the inverted repeat and the intact Ddp6 ORF are essential for maintenance of the plasmid. Mutation of the inverted repeat, or of the rep gene, lowered the plasmid copy number but did not affect autonomous replication of shuttle-vector constructs. Comparisons of the predicted protein products of the rep genes of five members of the Ddp2 plasmid family identified a set of ten conserved features distributed throughout the peptides.

Amino Acid Sequence↗
PubMed1998

Chimeric amplicons containing the c-myc gene in HL60 cells.

The major amplicon present in HL60 cells is chimeric in nature being composed of 70 kb of DNA sequence derived from the MYC locus linked to 80 kb of novel DNA sequence derived from a non contiguous region located telomeric to the c-myc gene at 8q24 (Feo et al., 1996). Here we show by fluorescence in situ hybridization (FISH) that these coamplified sequences, MCR (Myc Coamplified Region), are derived from a locus located 3-4 Mb telomeric to the c-myc gene in the q24.2-24.3 region of chromosome 8. Genomic cloning and Southern blot analysis indicate the arrangement of chimeric amplicons are in tandem arrays. Analysis of the DNA sequences at the juncture of the MYC locus and the MCR suggest that these non syntenic regions were joined by nonhomologous recombination events. Visualization of the organization of the amplified DNA by fiber-FISH analysis illustrates we have cloned the complete amplicon. This is the first complete mammalian amplicon to be cloned and have its structure visualized. In addition to the major class of tandemly repeated amplicons, a second class of amplicons was detected by fiber-FISH in which the extent of the MCR component is about twice the size of the MCR component in the major amplicon. These longer amplicons most likely contain inverted repeats of MCR and MYC region sequences. Whether the amplicons contain mixtures of these two types of structures or separate amplicons only contain one type of structure has not yet been resolved. Properties of the MCR sequences responsible for retention in the chimeric HL60 amplicons upon long term passage are discussed.

Base Sequence↗
PubMed1984

Nucleotide sequence analysis of the long terminal repeat of integrated bovine leukemia provirus DNA and of adjacent viral and host sequences.

The nucleotide sequence of the 3' long terminal repeat and adjacent viral and host sequences was determined for a bovine leukemia provirus cloned from a bovine tumor. The long terminal repeat was found to comprise 535 nucleotides and to harbor at both ends an imperfect inverted repeat of 7 bases. Promoter-like sequences (Hogness box and CAT box), an mRNA capping site, and a core enhancer-related sequence were tentatively located. No kinship was detected between this bovine leukemia proviral fragment and other retroviral long terminal repeats, including that of human T-cell leukemia virus.

Animals↗
PubMed2000

The degenerate DNA transposon Pat and repeat-induced point mutation (RIP) in Podospora anserina.

A degenerate DNA transposon, Pat, was identified in the genomes of various wild-type strains of the filamentous fungus Podospora anserina. In these strains, the number (approximately 20-25 copies per genome) and location of Pat sequences appear to be conserved. Two copies of Pat, one complete and one partial, were cloned and characterized. The sequence of the complete element is 1856 bp long and contains imperfect inverted terminal repeats (ITRs) of 53 bp. The target site duplication comprises the sequence TA. The amino acid sequence derived from one reading frame of Pat shows significant homology to members of the Fot1 family of transposons. However, this reading frame is interrupted by numerous stop codons. Since no transcripts of Pat were identified in different P. anserina strains grown under standard conditions and under increased stress, we conclude that none of the copies of Pat is active in the strains analyzed, under the environmental conditions investigated. Comparison of the sequences of the two cloned Pat sequences revealed 89% (589/747 nucleotides) identity. Most of the differences (82%, 129/158) can be attributed to transitions preferentially at CpA:TpG and CpT:ApG dinucleotides. The dinucleotide ratios in Pat are similar to those in a Neurospora crassa transposon which was subject to repeat-induced mutation (RIP), but differ significantly from those found in single-copy genes of P. anserina and in fungal DNA transposons not modified by this mechanism. Molecular analysis of the progeny of a cross between the wild-type strain and a transgenic strain in which a nuclear gene was duplicated by transformation yielded the first clear evidence that a RIP-like process is active in P. anserina.

Amino Acid Sequence↗
PubMed2001

Characterization of cis-acting sequences involved in canine adenovirus packaging.

The cis-acting packaging domain in adenovirus serotype 5 (Ad5) is a series of redundant, albeit not functionally equivalent, "A-repeats" made up of the consensus sequence 5'-TTTGN(8)CG-3'. A-repeats may bind trans-acting factors that direct packaging of the adenovirus genome into the preformed capsid. To try to understand this basic mechanism, we examined the packaging domain from a nonhuman adenovirus. We delimited the canine adenovirus type 2 (CAV-2) packaging domain to within 156 bp via a conditional mutation based on the Cre/loxP excision. Using an insertion, deletion, and substitution strategy, we generated packaging-defective CAV-2 vectors. Our results demonstrate that, like Ad5, CAV-2 cis-acting packaging sequences are located near the left inverted terminal repeat and are redundant, but not functionally equivalent. However, the bipartite motif found in Ad5 is present only once in CAV-2 and deletion of it caused only a minor variation in the packaging efficiency. We have identified at least four functional cis-acting packaging sequences in CAV-2. The CAV-2 vectors that we generated were not replication-defective in an E1-transcomplementing cell line and as heat stable as the parental vectors that did not contain mutations.

Adenoviridae↗
PubMed1981

Mobile dispersed genetic element MDG1 of Drosophila melanogaster: nucleotide sequence of long terminal repeats.

Long terminal repeats (LTRs) of two members of mdg1 family were sequenced. In the both cases, they are represented by perfect direct repeats 442 and 444 bp in length. Sixteen nucleotides in the LTRs of two different mdg1 elements are different. Each LTR contains slightly mismatched 16-nucleotide inverted repeats located at the ends of the LTR. Six base pairs closest to the termini of LTR form perfect inverted repeats. On the gene-distal sides of LTRs, short 4-nucleotide direct repeats are located, probably representing the duplication of a target DNA sequence arising from insertion of mdg. They are different in the two cases analyzed. Just as the other analyzed eukaryotic transposable elements, mdg1 starts with TGT and ends with ACA. Within the both strands of LTR, the sequences similar to Hogness box (a putative signal for RNA initiation, or a selector) and AATAAA blocks (putative polyadenylation signals) are present. The LTR of mdg1 contains many short direct and inverted repetitive sequences. These include a 10-nucleotide sequence forming a perfect direct repeat with the first ten nucleotides of the LTR. A region of LTR about 70 bp long is represented by simple repetitive sequences (TAT).

Animals↗
PubMed1992

A maize cryptic Ac-homologous sequence derived from an Activator transposable element does not transpose.

Sequences sharing homology to the transposable element Activator (Ac) are prevalent in the maize genome. A cryptic Ac-like DNA, cAc-11, was isolated from the maize inbred line 4Co63 and sequenced. Cryptic Ac-11 has over 90% homology to known Ac sequences and contains an 11 bp inverted terminal repeat flanked by an 8 bp target site duplication, which are characteristics of Ac and Dissociation (Ds) transposable elements. Unlike the active Ac element, which encodes a transposase, the corresponding sequence in cAc-11 has no significant open reading frame. A 44 bp tandem repeat was found at one end of cAc-11, which might be a result of aberrant transposition. The sequence data suggest that cAc-11 may represent a remnant of an Ac or a Ds element. Sequences homologous to cAc-11 can be detected in many maize inbred lines. In contrast to canonical Ac elements, cAc-11 DNA in the maize genome is hypermethylated and does not transpose even in the presence of an active Ac element.

Base Sequence↗
PubMed1991

Transcription inhibition of the somatic-type phosphoglycerate kinase 1 gene in vitro by a testis-specific factor that recognizes a sequence similar to the binding site for Ets oncoproteins.

To elucidate the mechanism by which transcription of the somatic-type phosphoglycerate kinase 1 gene is inactivated during mammalian spermatogenesis, we examined the presence of specific transcription inhibitor(s) in the testis by a cell-free transcription system. Transcription of the mouse phosphoglycerate kinase 1 gene using nuclear extracts of the rat liver was significantly inhibited by the addition of testis extracts, whereas brain extracts had little effect. Transcription inhibition required the binding of a testis-specific factor, designated TIN-1, to the region between positions -268 and -259 relative to transcription initiation site at +1. This region had the sequence 5'-AGGAAGTTCC-3' that includes an inverted repeat of the binding motif, 5'-GGAA-3', for the oncoprotein Ets. A UV-crosslinking experiment revealed that 43- and 45-kDa polypeptides present in testis extracts bind to that sequence. These results suggest that a testis-specific transcription inhibitor TIN-1 inactivates the phosphoglycerate kinase 1 gene in the mammalian spermatogenic pathway.

Animals↗
PubMed2003

An active DNA transposon family in rice.

The publication of draft sequences for the two subspecies of Oryza sativa (rice), japonica (cv. Nipponbare) and indica (cv. 93-11), provides a unique opportunity to study the dynamics of transposable elements in this important crop plant. Here we report the use of these sequences in a computational approach to identify the first active DNA transposons from rice and the first active miniature inverted-repeat transposable element (MITE) from any organism. A sequence classified as a Tourist-like MITE of 430 base pairs, called miniature Ping (mPing), was present in about 70 copies in Nipponbare and in about 14 copies in 93-11. These mPing elements, which are all nearly identical, transpose actively in an indica cell-culture line. Database searches identified a family of related transposase-encoding elements (called Pong), which also transpose actively in the same cells. Virtually all new insertions of mPing and Pong elements were into low-copy regions of the rice genome. Since the domestication of rice mPing MITEs have been amplified preferentially in cultivars adapted to environmental extremes-a situation that is reminiscent of the genomic shock theory for transposon activation.

Amino Acid Sequence↗
PubMed1976

Electron microscopy of analysis of circular repetitive mitochondrial DNA molecules from genetically characterized rho- mutants of Saccharomyces cerevisiae.

1. We have studied mtDNA purified from nine p- petite mutants in which most of the wild type sequence has been deleted but the genetic markers conferring resistance to erythromycin of oligomycin or paromomycin have been retained. 2. All mtDNA contained numerous circular molecules. The size distribution of the circles conformed to a multimeric series which was characteristic for each mutant. We conclude that any one region of the wild type mtDNA molecule, when maintained in a p- clone, while other regions are deleted, can give rise to a multimeric series of circles. 3. In tandem straight repetitive mtDNAs the circles contain odd and even number of unit sequence repeats. In palindrome repetitive mtDNAs the circles contain mostly even number of unit sequence repeats. Thus, one straight or two inverted repeats constitute the monomeric unit of circularization. 4. We found that the frequency distribution of circles follows on a number basis a simple rule: frequency of numeric circles = 1/n frequency of monomeric circles, for n = 2, 3 and 4. Thus, on a mass basis each class represents the same fraction of total mtDNA and the mitochondrial genome has the same probability to constitute one monomeric circle or to be a part of n-meric circle. We interpret this finding that in vivo all molecules are circular. 5. Four mutants displayed a single multimeric series of circles ranging from 0.3 mum to 2.4 mum monomer circle length. Five mutants displayed multiple different multimeric series. In the latter case, the longest unit sequence repeat length was equal to the sum of the two shorter unit sequence repeat lengths. Sorting out, recombination and internal deletions of circular repetitive p- mtDNA molecules are discussed.

Centrifugation, Density Gradient↗
PubMed2005

Duplications between direct repeats stabilized by DNA secondary structure occur preferentially in the leading strand during DNA replication.

To ascertain a leading or lagging strand preference for duplication mutations, several short DNA sequences, i.e. mutation inserts, were designed that should demonstrate an asymmetric propensity for duplication mutations in the two complementary DNA strands during replication. The design of the mutation insert involved a 7-bp quasi inverted repeat that forms a remarkably stable hairpin in one DNA strand, but not the other. The inverted repeat is asymmetrically placed between flanking direct repeats. This sequence was cloned into a modified chloramphenicol acetyltransferase (CAT) gene containing a -1 frameshift mutation. Duplication of the mutation insert restores the reading frame of the CAT gene resulting in a chloramphenicol resistant phenotype. The mutation insert showed greater than a 200-fold preference for duplication mutations during leading strand, compared with lagging strand, replication. This result suggests that misalignment stabilized by DNA secondary structure, leading to duplication between direct repeats, occurred preferentially during leading strand synthesis.

Base Sequence↗
PubMed1990

A palindromic mutation of the linear killer plasmid k2 of yeast.

Production of the killer toxin in Kluyveromyces lactis is dependent on the presence of two linear DNA plasmids, k1 and k2. We isolated a non-killer mutant, VM5, with a modified plasmid composition. It had lost k1, but conserved k2, and acquired, in addition, three new DNA species. The new species were found to be rearranged derivatives of the k2 plasmid. One of them, pVM5-1, was made of the left terminal 4720 bp sequence of k2, including the inverted terminal repeat, and was organized as a large palindromic dimer molecule. The second, pVM5-2, was made of one strand of the pVM5-1 palindrome, folded into a hairpin structure. Like normal k2, pVM5-1 and 2 were present in a high copy number. The third species, pVM5-x, of variable size, was also a deletion product of k2, but not palindromic, and did not contain the terminal repeat. Genetic analysis showed that the presence of the palindromic derivatives appeared to destabilize the normal k2 genome, leading to gradual accumulation of plasmid-less cells.

Base Sequence↗
PubMed1997

Plasmid pIP501 encoded transcriptional repressor CopR binds asymmetrically at two consecutive major grooves of the DNA.

Replication of the streptococcal plasmid pIP501 is regulated by the CopR protein and an antisense-RNA (RNAIII). CopR acts as transcriptional repressor at the essential repR promoter pII by binding to inverted repeat IR1 upstream of pII. To further characterize the interaction of CopR with its target, footprinting studies were performed. Methylation interference identified three guanine bases (G240, G242 and G251) in the top strand and two (G252 and G254) in the bottom strand contacted by CopR in the major groove of the DNA. Missing base interference revealed the contribution of the bases in the neighbourhood of these guanine bases to the specific DNA-protein contacts. Phosphate residues essential for CopR binding were determined by ethylation interference. The recognition sequence was localized at the centre of inverted repeat IR1. CopR contacts two consecutive major grooves (site I and II) on the same face of the DNA. Although the two sites share a common sequence motif, neighbouring bases are contacted differently. DNA fragments carrying single mutations in site I or II were analysed by band shift assays. Gel filtration and native gel electrophoresis demonstrated that CopR exists only as a dimer. A sigmoidal binding curve of CopR to its DNA target was observed and allowed the determination of the apparent dissociation constant K(D). The significance of the relatively high apparent K(D) for the role of CopR in pIP501 copy number regulation is discussed.

Alkylation↗
PubMed1997

Genetic characterization of insertion sequence ISJP4 on plasmid pJP4 from Ralstonia eutropha JMP134.

Directly adjacent to the (tfdT-) tfdCDEF gene cluster for chlorocatechol breakdown on plasmid pJP4 of Ralstonia eutropha (formerly Alcaligenes eutrophus) JMP134, we identified a 0.9-kb DNA element, designated ISJP4, with the typical features of a bacterial insertion sequence. ISJP4 occurs as a single complete copy on plasmid pJP4. About 9 kb away from this copy, in the tfdA-tfdS intergenic region, we found a 71-bp duplication of the ISJP4 right-hand extremity. In addition, we discovered a complete copy of ISJP4 on the chromosome of the R. eutropha JMP134 strain that we use routinely in our laboratory. We suppose that this copy resulted from a recent transposition of the plasmid-borne ISJP4, since it was shown to be lacking from the chromosomes of R. eutropha JMP222 and JMP289, two previously pJP4-cured derivatives of JMP134. By comparing both complete copies and their flanking regions, we could establish that element ISJP4 has a size of 915 bp and is bordered by 18-bp inverted repeats with one mismatch. Based on sequence similarity of its coding regions, ISJP4 could be classified into the IS5 group of the IS4 family of bacterial insertion sequences, where it is mostly related to IS402 of Burkholderia cepacia. A TAA direct repeat, presumably resulting from a duplication of the target site, flanked the chromosomal copy of ISJP4. We could demonstrate that a piece of DNA that is flanked by two complete copies of ISJP4 can be transposed. Even more so, one complete ISJP4 plus its tfdA-tfdS intergenic remnant were sufficient to mediate transposition of intervening DNA. A possible role of ISJP4 in the formation of the tfd pathway genes will be discussed.

Alcaligenes↗
PubMed2003

Sequence analysis of the complete genome of rice black-streaked dwarf virus isolated from maize with rough dwarf disease.

The complete nucleotide sequences of 10 genomic segments (S1-S10) from an isolate of rice black-streaked dwarf virus causing rough dwarf disease on maize (RBSDV-Hbm) in China were determined, a total of 29,142 base pairs (bp). Each segment possessed the genus-specific termini with conserved nucleotide sequences of (+) 5'-AAGUUUUU......CAGCUNNNGUC-3' and a perfect or imperfect inverted repeat of seven to eleven nucleotides immediately adjacent to the terminal conserved sequence. While the coding strand of most RBSDV-Hbm segments contained one open reading frame (ORF), there were two non-overlapping ORFs in S7 and S9, and one small overlapping ORF downstream of the major ORF in S5. Homology comparisons suggest that S1 encodes a RNA-dependent RNA polymerase (RdRp), with 63.5% and 32.6% identity to the putative RdRp encoded by Fiji disease virus (FDV) and Nilaparvata lugens reovirus (NLRV), respectively. The proteins encoded by S2, S3, and S4 showed various degrees of similarity to those encoded by the corresponding segments of FDV or NLRV. In S5 and S6, low identities were found to those of FDV only, but not to NLRV. Sequence analyses showed that RBSDV-Hbm had the most similarities in the genome organizations and the coding assignments with a RBSDV isolated from rice in China, in which each pair of the corresponding segments shared sequence identities of 93.8-98.9% and 93.5-100% at nucleotide or amino acid levels, respectively. In addition, phylogenetic analyses suggested that RBSDV-Hbm had the closest evolutionary relationship to RBSDV in Fijivirus.

Conserved Sequence↗

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