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PubMed1989

Structural analyses of the polymorphic area in type II collagen gene.

The structure of type II collagen gene is extremely well conserved but contains a cluster of high frequency polymorphisms in a 2.2 kb area. Here we report the nucleotide sequence of this DNA area, essential for the PCR-facilitated RFLP-analyses of this gene. In the structural analyses we found four differences in the deduced human amino acid sequence when compared to the published bovine amino acid sequence. The donor and acceptor signals and branch point signals required for the splicing events were in agreement with mammalian consensus sequences. The frequency of inverted repeats which could provoke the DNA strand to loop formation and consequently to deletion mutations did not differ from that found in other sequenced genes coding for fibrillar collagens. mutations did not differ from that found in other sequenced genes coding for fibrillar collagens.

Collagen↗
PubMed1988

Repetitive extragenic palindromic sequences, mRNA stability and gene expression: evolution by gene conversion? A review.

Repetitive extragenic palindromic (REP) sequences are highly conserved inverted repeats present in up to 1000 copies on the Escherichia coli chromosome. We have shown both in vivo and in vitro that REP sequences can stabilize upstream mRNA by blocking the processive action of 3'----5' exonucleases. In a number of operons, mRNA stabilization by REP sequences plays an important role in the control of gene expression. Furthermore, differential mRNA stability mediated by the REP sequences can be responsible for differential gene expression within polycistronic operons. Despite the key role of REP sequences in mRNA stability and gene expression in a number of operons, several lines of evidence suggest that this is unlikely to be the primary reason for the exceptionally high degree of sequence conservation between REP sequences. Other possible functions for REP sequences are discussed. We propose that REP sequences may be a prokaryotic equivalent of 'selfish DNA' and that gene conversion may play a role in the evolution and maintenance of REP sequences.

Biological Evolution↗
PubMed1991

Structural and functional analysis of the origin of conjugal transfer of the broad-host-range IncW plasmid R388 and comparison with the related IncN plasmid R46.

We cloned and sequenced a 402 bp DNA segment containing the origin of conjugal transfer (oriT) of the IncW plasmid R388. Progressive deletions from each end of the sequence were assayed for oriT activity. Stepwise reductions in mobilization frequencies, representing the loss of functional elements, correlated with deletion of structural motifs in the sequence. A sequence of 330 bp of oriT was sufficient for efficient mobilization. The first 86 bp of the sequence contains five tandemly repeated DNA sequences of 11 bp, followed by a 10 bp perfect inverted repeat. Deletion of the first 95 bp reduced the frequency of transfer by a hundred-fold. The sequence between bp 183 and 218 was necessary and sufficient for low frequency mobilization and, thus, it was assumed to contain the nick site. This basis core was cloned as a 60 bp segment (from bp 176-236) that could be mobilized at low frequency. It includes two inverted repeats and a perfect integration host factor (IHF) consensus binding site. A third functionally important segment in oriT was located between bp 260 and 330. The DNA sequence of the oriT of R388 could be aligned with that of the broad-host-range IncN plasmid R46. Moreover, the relative positions of the three inverted repeats are also conserved. Overall sequence similarity was 52%, but was significantly higher in particular regions, which coincided with the functionally important segments mapped by deletion analysis. Conservation of these segments provided independent support for their essential role in oriT function.

Base Sequence↗
PubMed1989

Tissue-specific DNA binding of nuclear proteins that bind to the adenovirus inverted terminal repeat.

Adenovirus (Ad) DNA replication requires nuclear factor I (NFI), a cellular sequence-specific DNA binding protein which binds to the Ad DNA inverted terminal repeat (ITR). The NFI binding consensus sequence, TGGN6-7GCCAA, possibly includes the CCAAT-box which is often observed in regulatory elements of transcription. Adjacent to the NFI binding site on the Ad ITR is the binding site for nuclear factor III (NFIII), another cellular factor that stimulates Ad DNA replication in a cell-free system. Using gel retardation assay, we have examined the tissue specificity of these DNA binding activities in mouse. High NFI activity was detected in nuclear extracts from mouse liver, kidney, and spleen. Competition gel retardation assay with double-stranded oligonucleotides containing herpes simplex virus thymidine kinase (HSV-TK) gene or hsp70 gene CCAAT-box showed no effect on mouse NFI binding to its binding site. In the course of competitive DNA-binding assay, we detected a novel DNA binding protein in mouse kidney nuclear extracts, designated nuclear factor K (NFK). The NFK binding site is included in the NFIII binding site on the Ad ITR. NFK seems to be different from NFIII, in mode of binding to its binding site.

Adenoviridae↗
PubMed1996

IS1394 from Pseudomonas alcaligenes N.C.I.B. 9867: identification and characterization of a member of the IS30 family of insertion elements.

A new insertion sequence designated IS1394 was isolated from Pseudomonas alcaligenes NCIB 9867 (P25X) by entrapment in plasmid pUCD800 which carries the Bacillus subtilis sacB and sacR genes. The 1100-bp sequence contains 27-bp inverted repeats with 4 bp mismatch and has one long open reading frame, spanning 92.1% of the entire IS. The deduced 338 amino-acid sequence demonstrated homology (varying from 65% to 78% similarity and 36-67% identity) to transposases encoded by the IS30 family of IS elements. Comparison of four different IS-sacB junction sequences showed that IS1394 generated 3-bp direct repeats of target DNA upon insertion. IS1394 is present in at least 10 copies in the P25X genome but none was detected in its endogenous plasmid pRA2. Hybridization experiments revealed that the distribution of IS1394 is limited to closely related strains, being present in three copies in Pseudomonas putida NCIB 9869 (P35X) and two copies in Pseudomonas alcaligenes ATCC type strain (ATCC 14904).

Base Sequence↗
PubMed2004

Common structure of rare replication-deficient E1-positive particles in adenoviral vector batches.

The use of the PER.C6 adenovirus packaging cell line in combination with a designated vector plasmid system, whereby the cell line and vector with E1 deleted have no sequence overlap, eliminates the generation of replication-competent adenovirus during vector production. However, we have found cytopathic effect (CPE)-inducing particles in 2 out of more than 40 large-scale manufacturing lots produced in PER.C6 cells. The CPE inducer was detected at a frequency of 1 event in 7.5 x 10(12) vector particles. Despite amplification, it was not readily purified, indicating that the agent itself is replication deficient and requires the parental recombinant adenovirus serotype 5 (rAd5) vector for replication and packaging. Therefore, we designated the agent as a helper-dependent E1-positive region containing viral particle (HDEP). Here, we report the molecular structure of the HDEP genome, revealing an Ad comprised of E1 sequences derived from PER.C6 cells flanked by inverted terminal repeat, packaging signal, and transgene sequences. These sequences form a palindromic structure devoid of E2, E3, E4, and late genes. Since only 5 bp were shared between E1 sequences in the PER.C6 genome and viral vector sequences, the data strongly suggested that insertion of genomic DNA into an adenoviral genome had occurred essentially via nonhomologous recombination. HDEPs have been found in unrelated virus batches and appear to share a common structure that may explain their mechanism of generation. This finding allowed development of an HDEP assay to screen batches of rAd5 produced on the PER.C6 cell line and resulted in detection of seven HDEP agents from four different transgene-virus vector constructs in separate batches of Ad.

Adenovirus E1 Proteins↗
PubMed1993

Murine radiation myeloid leukaemogenesis: relationship between interstitial telomere-like sequences and chromosome 2 fragile sites.

While the specific nature of chromosomal fragile sites and their relationship to human leukaemogenesis remain obscure, there is evidence that chromosomal fragility may, in some circumstances, be associated with telomere-like repeat sequences and that chromosome 2 fragility in the mouse is involved in the initiation of myeloid leukaemia by ionising radiation. Here we describe the molecular cloning and characterisation of two murine telomere-like sequences, one having an inverted repeat structure and the other a simple tandem repeat organisation. The inverted telomere repeat clone generates an in situ chromosome 2 hybridisation pattern very similar to the distribution of the radiation-sensitive fragile sites previously found to be associated with leukaemogenic initiation. Furthermore, statistical comparison of the distributions of radiation induced breakpoints and sites of inverted telomere repeat hybridization indicates concordance at all chromosome 2 sites excluding the terminal regions. These data are discussed with respect to mechanisms of radiation-induced, site-specific chromosome 2 rearrangement and their implications for leukaemogenic initiation.

Acute Disease↗
PubMed1988

Characteristics of the X gene of hepatitis B virus.

The 465-nucleotide sequence of the X gene from our cloned hepatitis B virus (HBV) DNA (subtype adw) was determined and compared to the same gene of 10 other published HBV sequences (3 of adw, 4 of adr, 2 of ayw, and 1 of ayr). We found (i) a total of 56 base differences among the 11 sequences (without counting the 27-base deletion in one adr) which resulted in 88% nucleotide homology, and (ii) 5 pairs of repeated sequence (3 direct repeats and 2 inverted repeats) that were highly conserved. Comparison of the protein amino acid sequences indicated that (i) there is 80% amino acid homology in total, and (ii) there are four highly conserved cysteine residues. In addition, the X gene of the adw subtype is more conserved than that of adr.

Amino Acid Sequence↗
PubMed1995

An improved TnMax mini-transposon system suitable for sequencing, shuttle mutagenesis and gene fusions.

A new collection of mini-transposons (mini-Tn) of the previously described TnMax series [Haas et al, Gene 130 (1993a) 23-31] has been constructed. The transposons (Tn) bear genes conferring resistance to either chloramphenicol (Cm) or kanamycin (Km). Each member of the new series (TnMax5-TnMax11) contains the general M13 forward (M13-FP) and reverse (M13-RP1) sequencing primers close to the inverted repeats (IR), facilitating the rapid and convenient determination of the DNA sequences flanking the transposon insertion site. Furthermore, the mini-Tn possess the infrequently occurring NotI sites, allowing the localization of genes on macro-restriction maps of bacterial species. Some derivatives contain promoterless trp-lacZ (TnMax11), xylE (TnMax10), phoA (TnMax6) or blaM (TnMax7, TnMax9) genes next to the IR, suitable for the generation of in vivo gene- and operon fusions to study gene regulation, protein export, or to determine the topology of proteins in bacterial membranes. A set of conjugative minimal plasmid vectors (pMin1, pMin2) are used to select for TnMax insertions into the cloned insert, rather than the vector sequences. Due to the small size of the mini-Tn, and a simple and efficient mutagenesis procedure, the TnMax system is a useful tool for targeting and sequencing of cloned genes in Escherichia coli, and especially for shuttle mutagenesis of bacterial species which cannot be targeted by direct transposition.

Base Sequence↗
PubMed1995

Amplification of a circular episome carrying an inverted repeat of the DFR1 locus and adjacent autonomously replicating sequence element of Saccharomyces cerevisiae.

Lack of suitable amplification markers has hindered the use of the yeast system for investigating the mechanism of gene amplification in a eukaryote with a simple genome and well defined genetic system. Recently, methotrexate has been used to select for Saccharomyces cerevisiae mutants with de novo amplification of the dihydrofolate reductase gene (DFR1) (Huang, T. (1993) In Vivo Disruption and de Novo Amplification of the DFR1 Gene Encoding Dihydrofolate Reductase in Saccharomyces cerevisiae. Ph. D. thesis, University of Alberta, Edmonton, Canada). We report here the detailed structure of a DFR1 episome amplified in methotrexate-resistant strain 25-1. The extrachromosomal DNA is found predominantly as a single 11-kilobase circular molecule. It consists of a 5.5-kilobase inverted duplication that contains the DFR1 locus and adjacent ARS (autonomously replicating sequence) element. This molecular configuration mimics the inferred structure of double minute chromosomes observed in a number of mammalian amplification systems and suggests that mechanisms that generate amplified DNAs are conserved from yeast to mammals.

Antimycin A↗
PubMed1996

Characterisation of the novel junctions of two minichromosomes of Leishmania major.

The minichromosomes M210 and M230 of Leishmania major are linear amplicons which have an inverted repeat structure. We report here the molecular cloning of the megabasic chromosome region where M210 and M230 recombination points are located. The analysis of this chromosomal region revealed a 374 bp fragment directly repeated 3 times, which was also repeated elsewhere in the genome. DNA fragments containing the novel junctions of the minichromosomes M210 and M230 have been likewise cloned. The nucleotide sequence of these fragments was determined and compared with their corresponding DNAs in the source chromosome. It revealed that M210 contains a central 226 bp non-repeated fragment, whereas the central fragment of M230 was 5 kb long. A 9 bp sequence which was repeated in inverted orientation around the recombination points of M230 was found. M210 showed instead an A + T rich DNA which could be implicated in its generation.

Animals↗
PubMed1987

Isomerization of the UL region of varicella-zoster virus DNA.

Varicella-zoster virus (VZV) DNA exists principally as two isomers. Despite the presence of inverted repeats bounding the long sequence region, the unique long sequence, UL, is found in one (prototype) orientation in 95-98% of VZV DNA molecules and in the inverted orientation in only 2-5% of the molecules. In searching for an explanation for this disparity, we superinfected VZV-infected cells with herpes simplex virus type 1 or pseudorabies virus. Neither superinfecting virus produced a measurable change in the frequency of isomerization of the VZV DNA long sequence region.

Base Sequence↗
PubMed1981

Evolutionary variants of simian virus 40 which are impaired in early lytic functions but transform nonpermissive cells.

From an undiluted passaged virus stock, two size classes of defective simian virus 40 (SV40) DNA were isolated from which two evolutionary variants were cloned. By means of restriction enzyme and heteroduplex analysis, physical maps of the mutants have been constructed. Both mutants contained the region of SV40 DNA coding for the early proteins plus some adjacent sequences (the region from 0.120 to 0.685 map unit, clockwise, on the standard SV40 DNA map). Furthermore, each mutant contained, in the form of two inverted repeats, four times the sequences from the region 0.625 to 0.685 map unit, clockwise. Some biological properties of the mutant DNA were examined, and we found that the mutant DNA (i) has, as compared with SV40 DNA, an impaired ability to induce T antigen in permissive and nonpermissive cells; (ii) does not complement a thermosensitive A mutant of SV40; (iii) replicates very inefficiently without a helper; and (iv), as an apparent contradiction, transforms nonpermissive baby rat kidney cells as well as SV40 DNA. A hypothetical mechanism for the expression of the mutant DNA that might explain the observed biological properties is presented.

Animals↗
PubMed1995

Intron length variation of the Adh gene in Brachyscome (Asteraceae).

Eighteen polymerase chain reaction (PCR) products of the partial sequence of the Adh (alcohol dehydrogenase) gene from 10 Brachyscome species were sequenced and compared. These products contained the 5' three fourths of exon 4 and whole sequences of intron 3. They varied extensively in length due to the differences in length of intron 3. A total of 10 long insertions were flanked by direct repeats of 5 to 12 bp sequences, indicating inserted elements. These inserted elements were classified into the following five categories based on nucleotide sequence characteristics and length; (1) a region homologous to that of 5S RNA genes (5S DNA), (2) A-rich structure at the 3' end-like short interspersed elements (SINEs) in animals, (3) a sequence of 280 bp with no characteristic features, (4) a sequence of 125 bp with no characteristic features, (5) termini of 11 bp inverted repeats flanked by 5 bp sequence of direct repeats characteristics of a transposon.

Alcohol Dehydrogenase↗
PubMed1989

Characterization of the termini of linear plasmids from Nectria haematococca and their use in construction of an autonomously replicating transformation vector.

The mitochondria of isolate FS37 from Nectria haematococca mating population I (Fusarium solani f. sp. cucurbitae) contain two linear plasmids, pFSC1 and pFSC2, of 9.2 and 8.3 kbp, respectively. Evidence for a protein blocking the 5' termini of these plasmids was obtained from exonuclease digestion experiments. A single protein band with an apparent Mr of 80 K was labeled when the DNA-protein complex of either plasmid was reacted with [125I] Bolton-Hunter reagent and then digested with DNase I. DNA sequence analysis of the termini of both plasmids revealed long inverted repeats of 1,211 bp (pFSC1) and 1,027 bp (pFSC2). No sequence similarity was found between the terminal inverted repeats (TIRs) of pFSC1 and pFSC2, nor was any similarity identified between the TIRs of these plasmids and sequences of TIRs from other linear DNAs. A restriction fragment containing the TIR of pFSC1 conferred autonomous replication when incorporated into an integrative transformation vector of Ustilago maydis.

Base Sequence↗
PubMed1992

Perturbing highly conserved spatial relationships in the regulatory domain that controls inducible cat translation.

Chloramphenicol activates translation of cat-86 mRNA by stalling a ribosome in the leader of individual transcripts. Stalling triggers two sequential events: the destabilization of a region of secondary structure that sequesters the cat ribosome-binding site (RBS-C), and the initiation of cat translation. The site of drug-dependent ribosome stalling is dictated by the leader sequence, crb; crb causes a ribosome to stall with its aminoacyl site at leader codon 6. We demonstrate that induction requires the maintenance of a precise spatial relationship between crb and sequences within the left inverted repeat of the secondary structure. Therefore, destabilization of the secondary structure during chloramphenicol induction may result from the interaction of a stalled ribosome with a specific sequence in the secondary structure rather than from non-specific masking of RNA sequences. cat-86 regulation also depends on the distance that separates crb from RBS-C. This interval of 33 nucleotides was incrementally increased and decreased by mutations within a loop in the secondary structure. Shortening the distance between crb and RBS-C by three nucleotides reduced induction by half and a deletion of nine nucleotides abolished induction. Insertion mutations were without effect on induced expression but elevated basal expression. The results indicate that when the A site of a ribosome occupies leader codon 6 the secondary structure is destabilized and there is no interference with entry of a second ribosome at RBS-C. The data further demonstrate that when the A site of a ribosome in the leader is within 30 nucleotides of RBS-C, cat expression decreases. This decrease probably results from competition of the leader ribosome with the ribosome initiating cat translation. Our observations demonstrate that in wild-type cat-86 the distances between crb and the secondary structure, and between crb and RBS-C provide the precise spacing necessary to achieve three interdependent effects: the destabilization of the RNA secondary structure by a ribosome stalled at crb; a lack of competition between a ribosome stalled at crb and the initiating ribosome; and maintenance of a low, but measurable, basal level of cat expression. The spatial relationships identified as necessary for the regulation of cat-86 are conserved in the regulatory regions for five other inducible cat genes.

Base Sequence↗
PubMed1995

Long stretches of short tandem repeats are present in the largest replicons of the Archaea Haloferax mediterranei and Haloferax volcanii and could be involved in replicon partitioning.

We report the presence of long stretches of tandem repeats in the genome of the halophilic Archaea Haloferax mediterranei and Haloferax volcanii. A 30 bp sequence with dyad symmetry (including 5 bp inverted repeats) was repeated in tandem, interspersed with 33-39 bp unique sequences. This structure extends for long stretches--1.4 kb at one location in H. mediterranei chromosome and about 3 kb in the H. volcanii chromosome. The tandem repeats (designated TREPs) show a similar distribution in both organisms, appearing once or twice in the H. volcanii and H. mediterranei chromosomes, and once in the largest, probably essential megaplasmid of each organism but not in the smaller replicons. Sequencing of the structures in both H. volcanii replicons revealed an extremely high sequence conservation in both replicons within the species, as well as in the different organisms. Homologous sequences have also been found in other more distantly related halophilic members of the Archaea. Transformation of H. volcanii with a recombinant plasmid containing a 1.1 kb fragment of the TREPs produced significant alterations in the host cells, particularly in terms of cell viability. The introduction of extra copies of TREPs within the vector significantly alters the distribution of the genome among the daughter cells, as observed by DAPI staining. Although the precise biological role cannot be completely ascertained, all the data conform with the tandem repeats being involved in replicon partitioning in halobacteria.

Base Sequence↗
PubMed2002

A putative iron-regulated TonB-dependent receptor of Mannheimia (Pasteurella) haemolytica A1: possible mechanism for phase variation.

A recombinant plasmid that codes for a novel iron receptor protein (Irp) of Mannheimia (Pasteurella) haemolytica A1 was isolated by the partial complementation of an Escherichia coli fur mutant. The deduced amino acid sequence of Irp exhibited characteristics typical of TonB-dependent receptors. These include: a TonB-box at the N-terminal; a 50 amino acid region homologous to the "plug" domain of the E. coli FhuA and FepA receptors; and a C-terminal TonB-dependent signature which likely functions as an outer membrane anchoring domain. Previously uncharacterized Irp homologues were detected by BLAST analysis of available databases and incomplete microbial genomes. When the irp homologues from Neisseria gonorrhoeae and N. meningitidis were cloned by PCR and expressed in E. coli, novel proteins of the predicted size (84kDa) were detected in cell lysates, demonstrating that these are functional genes. The M. haemolytica A1 irp gene undergoes phase variation at a nucleotide region which contain the sequence AAAAAAATTAAAA (7A-2T-4A) flanked by a short inverted repeat. Site-specific mutagenesis of the 7A-2T-4A sequence as well as replacement of the inverted repeats resulted in a stable construct that expressed the Irp protein without phase variation. The expression of irp in M. haemolytica A1 was regulated by iron concentrations and most likely a Fur homologue, consistent with the proposed function of Irp in iron metabolism. The irp genes may represent contingency loci that play a role in iron acquisition during infection.

Amino Acid Sequence↗