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PubMed1994

Extensive sequence divergence in the 3' inverted repeat of the chloroplast rbcL gene in non-flowering land plants and algae.

A stem-loop region is present at the 3' terminus of the chloroplast rbcL mRNA in all taxa surveyed to date. In spinach, this structure has been shown by others to be involved in modulating transcript stability and correct 3' terminus processing, and is a conserved feature of other flowering plant rbcL mRNAs. In Chlamydomonas reinhardtii, an analogous structure has been shown by others to serve as a transcription terminator. Our sequencing data have shown that this region is highly divergent in several non-flowering land plants, as evidenced by representatives from the ferns, conifers, 'fern-allies' and liverworts. To extend our analysis, a computer-assisted survey of the stem-loop region of the 3' flanking region of published chloroplast rbcL genes was undertaken. The flowering plant rbcL inverted repeats (IR) were remarkably conserved in sequence, allowing for precise multiple alignments of both monocot and dicot sequences within a single matrix. Surprisingly, sequences obtained from non-flowering land plants, algae, photosynthetic protists and photosynthetic prokaryotes were extremely variant, in terms of both sequence composition and thermodynamic parameters.

Amino Acid Sequence↗
PubMed2000

The protelomerase of temperate Escherichia coli phage N15 has cleaving-joining activity.

Escherichia coli phage N15 encodes the slightly acidic, 630-residue protein of 72.2 kDa called protelomerase (TelN). TelN is a component of the N15 replication system proposed to be involved in the generation of the linear prophage DNA. This linear DNA molecule has covalently closed ends. The reaction converting circular plasmids into linear molecules was catalyzed in vitro. We demonstrate that the product of telN functions as the protelomerase in the absence of other N15-encoded factors. Purified TelN processes circular and linear plasmid DNA containing the proposed target site telRL to produce linear double-stranded DNA with covalently closed ends. The 56-bp telRL target site consists of a central telO palindrome of 22 bp and two 14-bp flanking sequences comprising inverted repeats. telO is separated from these repeats by 3 bp on each side. The telRL sequence is sufficient for TelN-mediated processing. The ends of the DNA molecules generated in vitro have the same configuration as do those observed in vivo. TelN exerts its activity as cleaving-joining enzyme in a concerted action.

Base Sequence↗
PubMed1994

Cloning, sequence, and footprint analysis of two promoter/operators from Corynebacterium diphtheriae that are regulated by the diphtheria toxin repressor (DtxR) and iron.

DtxR is an iron-dependent sequence-specific DNA-binding protein that binds to the tox operator, an inverted-repeat nucleotide sequence located upstream from the diphtheria toxin gene. In this study, two additional iron-regulated promoter/operator sequences (IRP1 and IRP2) that are controlled by DtxR were cloned from the chromosome of Corynebacterium diphtheriae and characterized. Operon fusions to lacZ were used to analyze expression from IRP1 and IRP2 in Escherichia coli. Transcription from both promoters was strongly repressed in high-iron medium in the presence of the cloned dtxR gene; however, transcription in the absence of dtxR was 50- to 100-fold greater, regardless of the iron concentration. Purified DtxR altered the electrophoretic mobility of DNA fragments carrying IRP1 or IRP2, and the nucleotide sequences of the two promoter/operator regions indicated that they are both homologous with the tox operator. DtxR protected an approximately 30-bp region on both IRP1 and IRP2 from DNase I digestion. A 19-bp consensus DtxR-binding site was derived from a comparison of the various DtxR-regulated operator/promoter sequences. Footprinting experiments using hydroxyl radicals and dimethyl sulfate demonstrated that DtxR interacted with these operators in a symmetrical manner, probably as a dimer or multimer. The deduced amino acid sequence of an open reading frame (ORF1) located downstream from IRP1 was homologous with a family of periplasmic proteins involved in iron transport in gram-negative bacteria and with the ferrichrome receptor, FhuD, from Bacillus subtilis. These findings suggest that ORF1 encodes a membrane-associated lipoprotein that may serve as the receptor for a ferric-siderophore complex in C. diphtheriae.

Amino Acid Sequence↗
PubMed2005

Cis and trans-acting effects on a mutational hotspot involving a replication template switch.

A natural mutational hotspot in the thyA gene of Escherichia coli accounts for over half of the mutations that inactivate this gene, which can be selected by resistance to the antibiotic trimethoprim. This T to A transversion, at base 131 of the coding sequence, occurs within a 17 bp quasi-palindromic sequence. To clarify the mechanism of mutagenesis, we examine here cis and trans-acting factors affecting thyA131 mutational hotspot activity at its natural location on the E.coli chromosome. Confirming a template-switch mechanism for mutagenesis, an alteration that strengthens base-pairing between the inverted repeat DNA sequences surrounding the hotspot stimulated mutagenesis and, conversely, mutations that weakened pairing reduced hotspot activity. In addition, consistent with the idea that the hotspot mutation is templated from DNA synthesis from mispaired strands of the inverted repeats, co-mutation of multiple sites within the quasipalindrome was observed as predicted from the DNA sequence of the corresponding repeat. Surprisingly, inversion of the thyA operon on the chromosome did not abolish thyA131 hotspot mutagenesis, indicating that mutagenesis at this site occurs during both leading and lagging-strand synthesis. Loss of the SOS-induced DNA polymerases PolII, PolIV, and PolV, caused a marked increase in the hotspot mutation rate, indicating a heretofore unknown and redundant antimutagenic effect of these repair polymerases. Hotspot mutagenesis did not require the PriA replication restart factor and hence must not require fork reassembly after the template-switch reaction. Deficiency in the two major 3' single-strand DNA exonucleases, ExoI and ExoVII, stimulated hotspot mutagenesis 30-fold and extended the mutagenic tract, indicating that these exonucleases normally abort a large fraction of premutagenic events. The high frequency of quasipalindrome-associated mutations suggests that template-switching occurs readily during chromosomal replication.

Base Pairing↗
PubMed1985

Junction sequences generated by 'one-ended transposition'.

In the presence of the cognate transposase, plasmids containing a single inverted repeat (IR) sequence of Tn21 or of Tn1721 can fuse efficiently with other plasmids ('one-ended transposition'). The junctions across the sequences of donor and recipient DNA in recombinants generated by this process have been determined. These show that the segment of donor DNA starts precisely at the IR sequence (it is variable at the other end), and is flanked by a direct repeat of host DNA (usually 5bp) that was present only once in the original host sequence. These are characteristics of recombinants generated by transposition of Tn21 and Tn1721 themselves, suggesting that the mechanism of one-ended transposition is very similar to that of the corresponding entire elements.

Base Sequence↗
PubMed1991

Molecular characterization of the class II multiresistance transposable element Tn1403 from Pseudomonas aeruginosa.

Transposon Tn1403 is a 19.9-kb multiresistance class II transposable element originally found on the RPL11 plasmid from a clinical isolate of Pseudomonas aeruginosa. It encodes resistance to ampicillin (PSE-1 beta-lactamase), streptomycin and spectinomycin (aadA and aphC), and chloramphenicol (cat). It has structural homology with the tnpM and tnpI sequences of Tn21 and inverted repeats and res and tnpR sequences of Tn501, but it has no structural homology nor functional complementation with the resolvase gene of Tn21 or Tn3. Sequence analysis revealed long inverted repeats at each extremity of Tn1403 containing 38-bp inverted repeats that were 97.4% similar to those of Tn1721 and 5-bp direct repeats. Transposition assays showed a low frequency of transposition (3.5 x 10(-6)) compared with that of Tn3 (3.3 x 10(-3)) and no resolution of cointegrates.

Base Sequence↗
PubMed1999

The fortuitous cloning of retroelement-like sequences from wheat and rye as by-products of a specific polymerase chain reaction.

Cloning of by-products of a specific PCR reaction, directed to the Em genes of wheat and rye, has resulted in the identification of ten sequences with homology to the known Tyl-copia-like retroelements WIS 2-1A from wheat and BARE-1 from barley. These sequences were amplified by only one of the primers due to the presence of an inverted repeat. Nine sequences are ca. 740 bp long and contain part of the left LTR, the adjacent primer-binding site and part of the leader sequence, whereas one shorter sequence (535 bp) consists of part of the leader sequence only. The dendrogram, constructed from the multiple sequence alignment, classified the isolated sequences into two narrowly related groups that belong to the WIS-2 family of cereal retroelements.

Base Sequence↗
PubMed1991

Mitochondrial-like DNA sequences flanked by direct and inverted repeats in the nuclear genome of Toxoplasma gondii.

In the course of our genetic studies on Toxoplasma gondii, it was discovered that one cosmid hybridized to a repetitive element. The hybridization pattern observed for the enzyme BglII indicated that this cosmid hybridized to a large number of discrete, but related elements. Four BglII fragments were subcloned from the cosmid, and each was shown to hybridize with all the others, as well as to numerous dispersed sequences in genomic DNA. Three subclones were sequenced in their entirety, and shown to contain fragments of the genes for cytochrome oxidase subunit I and apocytochrome b, complete and functional copies of which have been found in only mitochondrial genomes. All the subcloned fragments were bounded at both ends by a 91 base-pair sequence, which contains a site for BglII. This 91 base-pair sequence could be found as either a direct or inverted repeat. It was determined that the BglII elements are arrayed downstream from a single copy nuclear gene. Comparison of genomic and cosmid DNAs confirmed that the cosmid faithfully reflects the nuclear genome. Although the mitochondrial genome of Toxoplasma has not been characterized, these nuclear mitochondrial-like sequences appear to be internally rearranged with respect to known, functional mitochondrial genomes, and with respect to each other. The finding of short repeated sequences flanking these elements may be a clue to the mechanism of their dissemination.

Amino Acid Sequence↗
PubMed2000

The terminal protein of a linear mitochondrial plasmid is encoded in the N-terminus of the DNA polymerase gene in white-rot fungus Pleurotus ostreatus.

The gene structure and expression of the linear mitochondrial plasmids of the white-rot fungus Pleurotus ostreatus, pMLP1 and pMLP2, were analyzed. Cleavage by proteinase K and exonucleases indicated that the 5' ends of pMLP1 and pMLP2 DNAs were associated with terminal proteins. Nucleotide sequencing of the entire pMLP1 DNA revealed that it consists of 9,879 bp with terminal inverted repeat (TIR) sequences of 381 bp. The end sequence of TIR in pMLP1 is 3'-CCCCC-5', similar to those of Escherichia coli phage PRDI. The pMLP1 plasmid harbors two long open reading frames (ORFI and ORF2) and at least one minor ORF (mORF1). The deduced product of ORF1 is homologous to RNA polymerases of yeast mitochondria and several bacteriophages, whereas that of ORF2 is homologous to the protein-primed DNA polymerases of family B type. The mORF1 encodes a highly basic protein, most likely a TIR-binding protein, with no apparent sequence homology in the database. Expression of the predicted gene products from pMLP1 in mitochondria was demonstrated by Western blot analysis using antibodies against various expressed regions of pMLP1 ORFs. A plasmid-free strain, generated by curing with ethidium bromide, did not express any of these gene products. Terminal proteins of 70 kDa (TP1) and 73 kDa (TP2) were identified from pMLP1 and pMLP2, respectively. Western blot analysis indicated that TP1 was generated from the N-terminal half of the full-length product of ORF2 encoding a putative DNA polymerase.

Amino Acid Sequence↗
PubMed1993

Sequence and transcript analysis of the nitrogenase structural gene operon (nifHDK) of Rhodobacter capsulatus: evidence for intramolecular processing of nifHDK mRNA.

Northern blot analysis of RNA prepared from cells of Rhodobacter capsulatus derepressed for nitrogenase (N2ase) synthesis, using a 6.0-kb DNA probe containing the entire nifHDK operon, revealed the presence of at least six hybridizing species of the estimated sizes, 4.4, 3.5, 2.7, 1.3, 0.9 and 0.38 kb. No hybridization was detected with RNA prepared from cells grown in the presence of an excess of NH4+, which represses N2ase synthesis. Hybridization with gene-specific probes revealed that the 4.4-kb species hybridized with all three probes, and presumably corresponded to the full-length nifHDK transcript, whereas the 3.5-kb species hybridized with nifD and nifK only, and the 2.7-kb transcript hybridized with nifH and nifD. The 1.3 and 0.9-kb species hybridized with all three probes, but appeared to hybridize most strongly with nifH. In contrast, the 0.38-kb species hybridized with none of the gene-specific probes, and was also detected in RNA from cells of strain RcM1, which contains a chromosomal deletion of the nifHDK operon. This species probably corresponds to the transcript of a gene, named fdxD, which was found to be located just upstream from the nifHDK operon. Nucleotide (nt) sequencing of the nifH-D and nifD-K intergenic regions revealed the presence of inverted repeat (IR) sequences potentially capable of forming stable stem-loop structures in mRNA. Primer extension analysis of the nifDK-homologous species showed that the 5' end was located one or two nt downstream from the IR sequence between nifH and nifD, suggesting that the putative stem-loop structure may be a target for intramolecular processing of the nifHDK mRNA.

Amino Acid Sequence↗
PubMed2000

Sequence analysis of porcine adenovirus type 3 E1 region, pIX and pIVa2 genes, and two novel open reading frames.

The porcine adenovirus type 3 (PAd3) genome between map units 0 and 13.7 was sequenced and compared with similar regions of other adenoviruses. This region consists of the left inverted terminal repeat sequences involved in DNA packaging, the entire early region 1 (E1) and the protein IX (pIX) transcription unit. The lower strand contains the C-terminal end of IVa2 of the E2A transcriptional unit and two novel open reading frames (ORFs). The E1 transcription unit consists of ORFs for proteins homologous to the E1A, E1B-17k and E1B-55k of both human adenovirus type 5 (HAd5) and bovine adenovirus type 3 (BAd3). The predicted PAd3 pIX demonstrated homology with the N-terminal portion of the pIXs of HAd5 and BAd3. On the lower strand, immediately after the putative IVa2 ORF, there are two unique ORFs of 208 and 203 amino acid residues that showed homology with Epstein-Barr virus nuclear antigens and other cellular transcription factors.

Adenovirus E1A Proteins↗
PubMed2005

[The effect of short hairpin RNA on hTERT expression].

AIM: To study the effect of short hairpin RNA (shRNA) on hTERT expression. METHODS: Oligonucleotides encoding shRNA against hTERT was cloned into a mammalian shRNA expression vector pUC18U6 to form pUC18U6ht which was transfected into HepG2 cells by using liposome. HepG2 cells transfected by pUC18U6 and pUC18U6GFPsir which expressed shRNA against green fluorescent protein were used as controls. hTERT mRNA in the transfected cells was quantified by using real-time fluorescent RT-PCR. RESULTS: Compared with the controls, the short hairpin RNA against hTERT decreased the hTERT mRNA level by 49% (P<0.05). CONCLUSION: hTERT expression was reduced by the shRNA.

Animals↗
PubMed1989

Identification and nucleotide sequence of attachment site of the Cflt filamentous phage from Xanthomonas campestris pv. citri.

It has been reported that the attachment site on the phage attP is located from 69.2 to 73.8 min on Cflt RF DNA. KpnI and PstI were used, which cut respectively at 67.2 and 72.6 min of Cflt RF DNA. A 0.54 kb fragment containing attP was obtained. For isolation of the right (attR) and left (attL) junctions of prophage and host chromosomal DNA, lysogen DNA was digested with HindIII and used to prepare a recombinant plasmid library. With Cflt RF DNA as a probe, three types of recombinant plasmids respectively containing inserts of 1.7, 4.3 and 2.8 kb DNA fragments were obtained. Since 1.7 kb represents the internal HindIII fragment of Cflt, 2.8 kb were from the junctional regions of prophage and host chromosomal DNA. Further analysis of restriction fragment patterns suggested that the 2.8 kb fragment contains attR, while the 4.3 kb fragment contains attL. To isolate the attachment site on the host bacteria (attB), recombinant plasmids constructed from HindIII DNA fragments of uninfected cells are screened by using molecular probes prepared from the host DNA sequences immediately adjacent to the prophage in the lysogenic host chromosomal DNA. A 1.45 kb fragment was obtained. Further analysis with HindIII site on this fragment established that it represents attB. The DNA nucleotide sequences of attP, attB attR and attL were determined. All of them contain a common 15 nucleotide core with the sequence of 5'-TATACATTATGCGAA-3'. The common core region of the recombination site shows two inverted-repeat DNA sequence.

Attachment Sites, Microbiological↗
PubMed1989

Mu transposable elements are structurally diverse and distributed throughout the genus Zea.

The Robertson's Mutator stock of maize exhibits a high mutation rate due to the transposition of the Mu family of transposable elements. All characterized Mu elements contain similar approximately 200-bp terminal inverted repeats, yet the internal sequences of the elements may be completely unrelated. Non-Mutator stocks of maize have a 20-100-fold lower mutation rate relative to Mutator stocks, yet they contain multiple sequences that hybridize to the Mu terminal inverted repeats. Most of these sequences do not cohybridize to internal regions of previously cloned Mu elements. We have cloned two such sequences from the maize line B37, a non-Mutator inbred line. These sequences, termed Mu4 and Mu5, have an organization characteristic of transposable elements and possess approximately 200-bp Mu terminal inverted repeats that flank internal DNA, which is unrelated to other cloned Mu elements. Mu4 and Mu5 are both flanked by 9-bp direct repeats as has been observed for other Mu elements. However, we have no direct evidence that they have recently transposed because they have not been found in known genes. Although the internal regions of Mu4 and Mu5 are not related by sequence similarity, both elements share an unusual structural feature: the terminal inverted repeats extend more than 100 bp internally from Mu-similar termini. The distribution of these elements in maize lines and related species suggests that Mu elements are an ancient component of the maize genome. Moreover, the structure of the Mu termini and the fact that Mu termini are found flanking different internal sequences leads us to speculate that Mu termini once may have been capable of transposing as independent entities.

Base Sequence↗
PubMed1983

Selective cleavage in the avian retroviral long terminal repeat sequence by the endonuclease associated with the alpha beta form of avian reverse transcriptase.

M13 recombinant DNA clones containing a 350-base sequence derived from the EcoRI fragment of two tandemly linked Rous-associated virus 2 (RAV-2) long terminal repeat (LTR) sequences have been used to map reverse transcriptase-associated endonuclease (RT-endonuclease) cleavage sites by primer extension studies. Under appropriate conditions, the alpha beta form of RT-endonuclease (composed of both the alpha and beta subunits) purified from Avian sarcoma virus (Pr-C and B-77 strains) introduces a specific break in the inverted complementary repeat sequence found at the junction of the LTRs. The cleavage sites occur in the same nucleotide sequence in (-) and (+) DNA strands; together they have the potential of generating a 6-base-pair staggered overlap that spans the junction. This supports the notion that the enzyme is involved in viral DNA integration. Other RT-endonuclease sites were analyzed. A second site, which occurs in the lac region of the M13 vector DNA upstream from the unique EcoRI cloning site, bears no apparent sequence homology to the site at the junction of the LTRs. However, it also lies within an inverted complementary repeat and, as is the case for the site in the LTR, the break occurs to the 5' side of the axis of symmetry. Cleavage at this second site is suppressed when the vector contains the RAV-2 LTR insert. Thus, the viral LTR appears to exert a cis effect that can influence a region over 300 base pairs away.

Avian Sarcoma Viruses↗
PubMed1996

IS431mec-mediated integration of a bleomycin-resistance gene into the chromosome of a methicillin-resistant Staphylococcus aureus strain isolated in Japan.

A methicillin-resistant Staphylococcus aureus (MRSA) strain B-26, isolated clinically in Hiroshima University Hospital, is resistant to bleomycin together with kanamycin. In the present study, we analysed the nucleotide sequence of the 5.1-kb HindIII fragment containing the bleomycin- and kanamycin-resistance genes, which were previously cloned [Bhuiyan et al. (1995) Appl Microbiol Biotechnol 43: 65-69] from the chromosomal DNA of MRSA B-26. The present study found that the DNA sequence contains the duplicated target sequence (GATTAGAT) consisting of 8 bp for transposase and the entire nucleotide sequence of the plasmid pUB110, together with the sequence of inverted repeats (16 bp), designated IR-r and IR-1 in IS431 mec. The 8-bp duplication sequence, produced by the transposable element, was first found by us. We proposed that bleomycin resistance in MRSA B-26 is attributed to the IS431 mec-mediated integration of pUB110 into the chromosome.

Bacterial Proteins↗
PubMed2005

How sequence defines structure: a crystallographic map of DNA structure and conformation.

The fundamental question of how sequence defines conformation is explicitly answered if the structures of all possible sequences of a macromolecule are determined. We present here a crystallographic screen of all permutations of the inverted repeat DNA sequence d(CCnnnN6N7N8GG), where N6, N7, and N8 are any of the four naturally occurring nucleotides. At this point, 63 of the 64 possible permutations have been crystallized from a defined set of solutions. When combined with previous work, we have assembled a data set of 37 single-crystal structures from 29 of the sequences in this motif, representing three structural classes of DNA (B-DNA, A-DNA, and four-stranded Holliday junctions). This data set includes a unique set of amphimorphic sequence, those that crystallize in two different conformations and serve to bridge the three structural phases. We have thus constructed a map of DNA structures that can be walked through in single nucleotide steps. Finally, the resulting data set allows us to dissect in detail the stabilization of and conformational variations within structural classes and identify significant conformational deviations within a particular structural class that result from sequence rather than crystal or crystallization effects.

Base Sequence↗