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DNA binding by the coliphage 186 repressor protein CI.

The cI gene of coliphage 186 maintains lysogeny and confers immunity to 186 infection by repressing the major early promoter, p(R), and the promoter for the late transcription activator gene, p(B). Gel mobility shirt and DNase I footprinting show that CI protein binds to the DNA at p(R) and p(B) and also to sites approximately 300 base pairs upstream and downstream of p(R), called FL and FR. Mutations which cause virulence reduce CI binding to p(R). The biochemical and genetic data identify three CI operators at p(R), two at p(B), and single operators at FL and FR. The operators at the p(B), FL, FR, and central p(R) sites are inverted repeat sequences, separated by 5 base pairs (Type A) or, in the case of p(R), by 4 base pairs (Type A'). A different inverted repeat operator sequence (Type B) is proposed for the binding sites on each side of the central site at p(R). Thus, CI appears to recognize two distinct DNA sequences. CI binds cooperatively to adjacent operators, and binding at p(R) is strongly dependent on these cooperative interactions. A high order CI multimer appears to be the active DNA binding species, even at single operators.

Amino Acid Sequence↗

Cyclic adduct formation at structural perturbations in supercoiled DNA molecules.

Supercoiled DNA can stabilize the existence of micropolymorphic structures, which may be sequence dependent. A good example is cruciform formation from inverted repeat sequences. DNA sequences exhibiting a high degree of two-fold symmetry, for instance, the 13-bp inverted repeat of ColE1, can adopt cruciform geometry when supercoiled, as judged by sensitivity to single-strand-specific nucleases and to resolvase and by topological changes revealed by gel electrophoresis. The present report shows that these structures are also excellent substrates for the local formation of cyclic adducts by bromoacetaldehyde, glyoxal or osmium tetroxide. Individual reagents may show pronounced differences in sequence selectivity, although all appear to react strongly at the ColE1 sequence. The reactions initially occur at the centre of the inverted repeats; this is followed by a conformational change such that the modification propagates outwards from the centre. These compounds constitute a valuable addition to the list of probes available for the investigation of local perturbations in DNA structure.

Acetaldehyde↗

Genetic and DNA sequence analysis of the kanamycin resistance transposon Tn903.

The kanamycin resistance transposon Tn903 consists of a unique region of about 1000 base pairs bounded by a pair of 1050-base-pair inverted repeat sequences. Each repeat contains two Pvu II endonuclease cleavage sites separated by 520 base pairs. We have constructed derivatives of Tn903 in which this 520-base-pair fragment is deleted from one or both repeats. Those derivatives that lack both 520-base-pair fragments cannot transpose, whereas those that lack just one remain transposition proficient. One such transposable derivative, Tn903 delta I, has been selected for further study. We have determined the sequence of the intact inverted repeat. The 18 base pairs at each end are identical and inverted relative to one another, a structure characteristic of insertion sequences. Additional experiments indicate that a single inverted repeat from Tn903 can, in fact, transpose; we propose that this element be called IS903. To correlate the DNA sequence with genetic activities, we have created mutations by inserting a 10-base-pair DNA fragment at several sites within the intact repeat of Tn903 delta 1, and we have examined the effect of such insertions on transposability. The results suggest that IS903 encodes a 307-amino-acid polypeptide (a "transposase") that is absolutely required for transposition of IS903 or Tn903.

Base Sequence↗

Leader peptides of inducible chloramphenicol resistance genes from gram-positive and gram-negative bacteria bind to yeast and Archaea large subunit rRNA.

catA86 is the second gene in a constitutively transcribed, two-gene operon cloned from Bacillus pumilus . The region that intervenes between the upstream gene, termed the leader, and the catA86 coding sequence contains a pair of inverted repeat sequences which cause sequestration of the catA86 ribosome binding site in mRNA secondary structure. As a consequence, the catA86 coding sequence is untranslatable in the absence of inducer. Translation of the catA86 coding sequence is induced by chloramphenicol in Gram-positives and induction requires a function of the leader coding sequence. The leader-encoded peptide has been proposed to instruct its translating ribosome to pause at leader codon 6, enabling chloramphenicol to stall the ribosome at that site. Ribosome stalling causes destabilization of the RNA secondary structure, exposing the catA86 ribosome binding site, allowing activation of its translation. A comparable mechanism of induction by chloramphenicol has been proposed for the regulated cmlA gene from Gram-negative bacteria. The catA86 and cmlA leader-encoded peptides are in vitro inhibitors of peptidyl transferase, which is thought to be the basis for selection of the site of ribosome stalling. Both leader-encoded peptides have been shown to alter the secondary structure of Escherichia coli 23S rRNA in vitro. All peptide-induced changes in rRNA conformation are within domains IV and V, which contains the peptidyl transferase center. Here we demonstrate that the leader peptides alter the conformation of domains IV and V of large subunit rRNA from yeast and a representative of the Archaea. The rRNA target for binding the leader peptides is therefore conserved across kingdoms.

Base Sequence↗

[Isolation and sequence analysis of a mitochondrial DNA fragment associated with CMS in Hong Lian type rice].

A CMS line specific mtDNA band TA1 was isolated by comparing the mtDNAs of CMS line and maintainer line using AFLP technique. The Northern blot analysis of the TA1 mtDNA fragment show that it transcripts only one RNA molecular each in CMS line, maintain line and the first filial generation, but the RNA moleculars are different among each other. By sequencing, the fragment is found to be 202 base pairs in length. It contains code ATG, ATT, AGA and AGG, two small repeated sequences 5'TGTAC3' and 5'ATTATTTT3' and one small inverted-repeated sequence 5'GGGAAACA3'. The results imply that the mtDNA fragment may be a coding region and may be relative to CMS of Hong Lian type rice.

Base Sequence↗

A uniquely conserved regulatory signal is found around the translation initiation site in three different collagen genes.

We have determined the nucleotide sequence of the 5' untranslated region and the sequence encoding the signal peptide for mRNAs of the chick alpha 1 type I and alpha 1 type III collagen. These sequences were obtained by synthesizing the corresponding cDNAs using as primers either a synthetic oligonucleotide to prime alpha 1 type I cDNA or a DNA fragment isolated from a genomic clone coding for alpha 1 type III collagen to prime the cognate cDNA. Both primers were selected so that the resulting cDNAs would be short and would contain sequence information for the 5' untranslated region and the signal peptide of the proteins. The nucleotide sequences of these cDNAs were compared with the corresponding sequence of alpha 2 type I collagen. In each mRNA the 5' untranslated segment is approximately 130 nucleotides and contains two or more AUG triplets preceding the AUG which serves as a translation initiation codon. A sequence of about 50 nucleotides surrounding the translation initiation codon is remarkably conserved in all three mRNAs, whereas the sequences preceding and following this segment diverge markedly. This homologous sequence contains an almost identical inverted repeat sequence which could form a stable stem-loop structure. The initiation codon and the AUG which precedes it are found at the same place within this symmetrical sequence and the distance between them is invariant. The rest of the conserved sequence shows a less perfect symmetry. This conserved sequence has not been found in other genes. Our data suggest that these three and perhaps other collagen genes contain an identical regulatory signal that may play a role in determining the level of expression of these genes by modulating translational efficiency.

Amino Acid Sequence↗

The sequence and organization of Ddp2, a high-copy-number nuclear plasmid of Dictyostelium discoideum.

The complete nucleotide sequence of the plasmid Ddp2 found in the nucleus of the simple eukaryote Dictyostelium discoideum is reported. This 5852-bp plasmid contains a 2661-bp open reading frame (ORF), named the "Rep gene," and 501-bp imperfect inverted repeats. A 1762-bp section of Ddp2, which includes one of the 501-bp repeat sequences, could be deleted without abolishing extrachromosomal replication. Deletion of the second 501-bp repeat, or interruption of the Rep gene, removed the ability to replicate extrachromosomally. We suggest that Ddp2 encodes a protein, "REP," that positively regulates replication initiation, a regulatory mechanism different from that of the yeast 2 mu plasmid which also possesses inverted repeat sequences. Ddp2 has a structure similar to that of plasmid pDG1, found in an unidentified isolate of Dictyostelium, with a similar sized ORF and inverted repeats. A common evolutionary origin is suggested by considerable sequence homology between the ORFs of pDG1 and Ddp2.

Amino Acid Sequence↗

The simian varicella virus and varicella zoster virus genomes are similar in size and structure.

Simian varicella virus (SVV) DNA was purified from viral nucleocapsids and the molecular structure of the SVV genome was determined. SVV DNA was analyzed by agarose gel electrophoresis of BamHI, BglII, EcoRI, and PstI restriction endonuclease digests. SVV and varicella zoster virus (VZV) DNAs were demonstrated to have distinct restriction endonuclease profiles. Summation of the sizes of individual restriction endonuclease fragments indicate the size of SVV DNA is congruent to 121 kilobase pairs (kbp) or congruent to 76.8 megadaltons (Md). Electron microscopy, lambda exonuclease analysis, and Southern blot DNA hybridizations were utilized to determine the molecular structure of the SVV genome and to construct restriction endonuclease maps. The results indicate that SVV DNA consists of a long component (L, congruent to 100 kbp) covalently linked to a short component (S, congruent to 20 kbp) which is composed of a unique short sequence (Us, 5.3 +/- 0.7 kbp) bracketed by inverted repeat sequences (TRs and IRs, congruent to 7.2 kbp). The presence of 0.5 M PstI restriction endonuclease fragments indicates that the S component may invert relative to the L component and that the genome exists in two major isomeric forms. The findings demonstrate that the SVV and VZV genomes are similar in size and structure.

Animals↗

Identification and characterization of the repetitive DNA element in the genome of insect iridescent virus type 6.

The genome of the Chilo iridescent virus (CIV) was analyzed for existence of repetitive DNA sequences by DNA-DNA hybridization using a defined and complete gene library of the viral genome (209 kbp) and by heteroduplex mapping. These experiments revealed the presence of repetitive DNA elements in the CIV genome, which are located in the EcoRI fragment H and in the EcoRI DNA fragment C at the coordinates 0.535 to 0.548 (EcoRI/Pstl DNA fragment, 2.7 kbp) and 0.920 to 0.944 (PvuII CIV DNA fragment L, 5.1 kbp), respectively. The DNA nucleotide sequence (2708 bp) of the EcoRI/Pstl subfragment was determined. The comparative analysis of the DNA sequences of this particular region of the viral genome with the DNA sequences of the PvuII DNA fragment L (5064 bp) revealed the presence of several DNA sequences within the EcoRI/Pstl subfragment of the EcoRI CIV DNA fragment H which show homology to DNA sequences of the PvuII DNA fragment L. For example, a DNA element (box A, 91 bp) is located at nucleotide positions 1981 to 2072 of the EcoRI CIV DNA fragment H which are complementary (greater than 90%) to the nine regions of the PvuII DNA fragment L (L-boxes 1 to 9). Furthermore heteroduplex mapping revealed the existence of a stem-loop structure (stem, 65 +/- 10 bp and loop, 652 +/- 80 bp) at the genome coordinates 0.571 to 0.582 (2.5 kbp, HindIII/EcoRI subfragment of the EcoRI CIV DNA fragment H). This indicates that an inverted repeat sequence is located at this region of the viral genome. The DNA nucleotide sequence of this subfragment was determined (2555 bp) which confirmed the data obtained from electron microscopy. An inverted repeat DNA sequence located at nucleotide positions 304 and 1011 is able to form this type of stem-loop structure.

Base Sequence↗

Tau, sigma, and delta. A family of repeated elements in yeast.

We report here the isolation and structure of a new repeated DNA element, tau. This element, from Saccharomyces cerevisiae, is 371 base pairs long and is flanked on either end by the same invertedly repeated sequence found at the ends of some Ty and sigma elements in yeast, copia elements in Drosophila and spleen necrosis virus. The tau inverted repeats are themselves flanked by a 5-base pair directly repeated genomic sequence that is present only once in a cognate tau-allele. These structural characteristics, the presence of multiple copies of tau in the genome, and the isolation of tau+ and tau- allelic pairs suggest that tau may be capable of transposition either alone or in association with some larger element. Detailed sequence analysis of the tau, sigma, and delta elements revealed that all three contain significant regions of homology, suggesting that they are probably members of a single family derived from a common progenitor.

Base Composition↗

Genomic organization and developmental fate of adjacent repeated sequences in a foldback DNA clone of Tetrahymena thermophila.

DNA sequence elimination and rearrangement occurs during the development of somatic cell lineages of eukaryotes and was first discovered over a century ago. However, the significance and mechanism of chromatin elimination are not understood. DNA elimination also occurs during the development of the somatic macronucleus from the germinal micronucleus in unicellular ciliated protozoa such as Tetrahymena thermophila. In this study foldback DNA from the micronucleus was used as a probe to isolate ten clones. All of those tested (4/4) contained sequences that were repetitive in the micronucleus and rearranged in the macronucleus. The presence of inverted repeated sequences was clearly demonstrated in one of them by electron microscopy. DNA sequence analysis showed that the left portion of this clone contains three tandem, directly repeated copies of a 340-bp sequence, a 120-bp portion of which appears in inverted orientation at a 1.6-kb distance. This clone, pTtFB1, was subjected to a detailed analysis of its developmental fate. Subregions were subcloned and used as probes against Southern blots of micronuclear and macronuclear DNA. We found that all subregions defined repeated sequence families in the micronuclear genome. A minimum of four different families was defined, two of which are retained in the macronucleus and two of which are completely eliminated. The inverted repeat family is retained with little rearrangement. Two of the families, defined by subregions that do not contain parts of the inverted repeat, one in the "loop" and one in the "right flanking region," are totally eliminated during macronuclear development--and contain open reading frames. A fourth family occurs in the "loop" region and is rearranged extensively during development. The two gene families that are eliminated are stable in the micronuclear genome but are not clustered together as evidenced by experiments in which DNAs from nullisomic strains are used to map family members to specific micronuclear chromosomes. The inverted repeat family is also stable in the micronuclear genome and is dispersed among several chromosomes. The significance of retained inverted repeats to the process of elimination is discussed.

Animals↗

Nonreciprocal recombination between alleles of the chloroplast 23S rRNA gene in interspecific Chlamydomonas crosses.

The inheritance of six polymorphic loci mapping in the rRNA-encoding (rDNA) region of the inverted repeat sequence of chloroplast DNA (cpDNA) was scored in hybrid subclones derived from reciprocal interspecific crosses between the green algae Chlamydomonas eugametos and Chlamydomonas moewusii. In order to enhance the detection of cells that had undergone recombination between parental cpDNAs, hybrids were selected that inherited a chloroplast antibiotic-resistance marker contributed by the mating-type-minus(mt(-)) parent, the parent that normally contributes fewer cpDNA molecules. The major findings of this study can be summarized as follows. (i) The majority of the hybrids (14/17) were recombinant for cpDNA markers in the 10-kilobase-pair rDNA region under study. (ii) Only one allele of each polymorphic cpDNA locus was ever detected in the hybrids, thus suggesting that newly recombined rDNA sequences in one copy of the inverted repeat are rapidly spread to the other by a copy-correction mechanism. (iii) Chloroplast streptomycin-resistance (sr-2) and erythromycin-resistance (er-nM1) loci, although showing little or no genetic linkage, were mapped to the 16S and 23S rRNA gene regions of the cpDNA, respectively, by virtue of their perfect coinheritance with polymorphic markers within these genes. (iv) cpDNA markers associated with a putative intron of the C. eugametos 23S rRNA gene were inherited by all 17 hybrids. Such a result is similar to that observed for certain alleles of the large rRNA gene of yeast mitochondria in crosses between omega(+) and omega(-) strains.

Journal Article↗

The mobilization and origin of transfer regions of a Thiobacillus ferrooxidans plasmid: relatedness to plasmids RSF1010 and pSC101.

The components for the mobilization function of a plasmid DNA during conjugation include a cis-acting sequence (the origin of transfer, oriT) and a transacting sequence coding for mobilization (Mob) proteins. By genetic and deletion analysis, we have located the mobilization region of pTF1, a cryptic plasmid previously isolated from a Thiobacillus ferrooxidans strain. Within a 2797 bse-pair sequenced region, several open reading frames (ORFs) were predicted; two of the ORFs are divergently transcribed and they encode proteins of calculated molecular masses, 42.6kD (ORF2) and 11.4kD (ORF6). Surprisingly, these protein sequences are substantially similar to two of the previously characterized mobilization proteins of the Escherichia coli IncQ plasmid, RSF1010. Moreover, the pTF1 ORF2 (now designated MobL) sequence is also found to be similar to a presumed mobilization protein of plasmid pSC101. Regions of sequence identity of plasmids pTF1, RSF1010 and pSC101 include their oriT sites. By alkaline agarose gel electrophoresis and DNA sequencing, we have established the location of the relaxation complex nick site within the oriT of pTF1. An identical nick site, which is adjacent to a characteristic 10 base-pair inverted repeat sequence, is also found for plasmid RSF1010. A recombinant plasmid containing a 42 base-pair synthetic piece of DNA encompassing the pTF1 inverted repeat and nick sequence was shown to be oriT-active.

Amino Acid Sequence↗

A deletion in the endothelin-B receptor gene is responsible for the Waardenburg syndrome-like phenotypes of WS4 mice.

The WS4 mouse is an animal model for human Waardenburg syndrome type 4 (WS4), showing pigmentation anomalies, deafness and megacolon, which are caused by defects of neural crest-derived cells. We have previously reported that the gene responsible for the WS4 mouse is an allele of the piebald mutations of the endothelin B receptor gene (Ednrb). In this study, we examined the genomic sequence of the Ednrb gene in WS4 mice and found a 598-bp deletion in the gene. The deleted region contains the entire region of exon 2 and the 5' part of exon 3 and is flanked by inverted repeat sequences which are suggested to trigger the deletion. We concluded that the deletion in the Ednrb gene is the causative mutation for the phenotype of WS4 mice.

Animals↗

IS1631 occurrence in Bradyrhizobium japonicum highly reiterated sequence-possessing strains with high copy numbers of repeated sequences RSalpha and RSbeta.

From Bradyrhizobium japonicum highly reiterated sequence-possessing (HRS) strains indigenous to Niigata and Tokachi in Japan with high copy numbers of the repeated sequences RSalpha and RSbeta (K. Minamisawa, T. Isawa, Y. Nakatsuka, and N. Ichikawa, Appl. Environ. Microbiol. 64:1845-1851, 1998), several insertion sequence (IS)-like elements were isolated by using the formation of DNA duplexes by denaturation and renaturation of total DNA, followed by treatment with S1 nuclease. Most of these sequences showed structural features of bacterial IS elements, terminal inverted repeats, and homology with known IS elements and transposase genes. HRS and non-HRS strains of B. japonicum differed markedly in the profiles obtained after hybridization with all the elements tested. In particular, HRS strains of B. japonicum contained many copies of IS1631, whereas non-HRS strains completely lacked this element. This association remained true even when many field isolates of B. japonicum were examined. Consequently, IS1631 occurrence was well correlated with B. japonicum HRS strains possessing high copy numbers of the repeated sequence RSalpha or RSbeta. DNA sequence analysis indicated that IS1631 is 2,712 bp long. In addition, IS1631 belongs to the IS21 family, as evidenced by its two open reading frames, which encode putative proteins homologous to IstA and IstB of IS21, and its terminal inverted repeat sequences with multiple short repeats.

Amino Acid Sequence↗

Long tandemly repeated repetitive (LTRR) sequences in the filamentous cyanobacterium Anabaena sp. PCC 7120.

Nucleotide sequence analysis of the DNA region carrying transposon Tn5-1087b from the Anabaena 7120 nitrogen fixation-deficient mutant YC16 revealed the presence of a novel repeated DNA element in cyanobacteria designated long tandemly repeated repetitive (LTRR) sequence. The LTRR element is 37 bp long and contains an inverted repeat sequence. 17 copies of the LTRR element, 13 of which were completely identical, were identified within a 1.3 kb DNA fragment, which was flanked by two divergently transcribed genes homologous to bacteriophage T4 'gene 15' and Rhizobium meliloti exoD, respectively. LTRR-like sequences occur in several DNA regions in Anabaena 7120 and in other cyanobacteria. Furthermore, the presence of an LTRR-like DNA region in mitochondrial plasmids of Vicia faba indicates strong conservation of such structures during evolution.

Amino Acid Sequence↗

Characterization of a Staphylococcus aureus transposon, Tn5405, located within Tn5404 and carrying the aminoglycoside resistance genes, aphA-3 and aadE.

A new staphylococcal composite transposon, designated Tn5405, carrying the genes aphA-3 and aadE, which encode resistance to aminoglycosides, was partially characterized. The transposon is 12 kb long and is flanked by inverted repeated sequences displaying the characteristic features of an insertion sequence, named IS1182. This insertion sequence is 1864 bp long and has 23/33-bp imperfect inverted repeats at its ends. One of the IS1182 copies delimiting Tn5405 contains a copy of IS1181 flanked by 8-bp direct repeats. Tn5405 was found in the chromosome of MRSA clinical isolate BM3121, within a Tn552-related transposon, Tn5404. Tn5404 was previously characterized following its transposition onto a beta-lactamase plasmid harbored by BM3121. Two forms of the recombinant beta-lactamase-encoding plasmid generated by the inversion of Tn5405 within Tn5404 were detected. IS1182 was not detected in the DNA of 4 of the 17 tested MRSA isolates containing aphA-3 and resistant to streptomycin. Thus, aphA-3 and aadE genes are not disseminated only by Tn5405 or related transposons delimited by IS1182.

Amino Acid Sequence↗

Mechanism and application of genetic recombination in herpesviruses.

Herpes simplex virus type 1 (HSV-1) is a ubiquitous human pathogen that latently infects sensory ganglia and encodes over 80 genes in a 152 kbp DNA genome. This well characterised virus provides a model for analysing genetic recombination in herpesviruses, a fundamental biological process by which new combinations of genetic materials are generated. The frequency of homologous recombination was estimated to be 0.0048-0.007 (0.48%-0.7%)/kb of the HSV-1 genome, determined using physical markers. The double-strand break repair model, the current model of homologous recombination, adequately explains L-S inversion of herpesvirus genomes and the recombinogenicity of the a sequence. Several herpesvirus genomes, including HSV-1 consist of a unique sequence bracketed by a pair of inverted repeat sequences. This arrangement is attributed to illegitimate recombination between molecules arranged in an inverse orientation. Junctions of unique and repeated sequences that correspond to the crossover site of illegitimate recombination are recombinogenic. Recombination is important for virus evolution, construction of mutated virus, gene therapy and vaccination in which the potential for recombination between engineered input virus and wild type virus has to be considered.

Animals↗