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PubMed2001

Role for the adenovirus IVa2 protein in packaging of viral DNA.

Although it has been demonstrated that the adenovirus IVa2 protein binds to the packaging domains on the viral chromosome and interacts with the viral L1 52/55-kDa protein, which is required for viral DNA packaging, there has been no direct evidence demonstrating that the IVa2 protein is involved in DNA packaging. To understand in greater detail the DNA packaging mechanisms of adenovirus, we have asked whether DNA packaging is serotype or subgroup specific. We found that Ad7 (subgroup B), Ad12 (subgroup A), and Ad17 (subgroup D) cannot complement the defect of an Ad5 (subgroup C) mutant, pm8001, which does not package its DNA due to a mutation in the L1 52/55-kDa gene. This indicates that the DNA packaging systems of different serotypes cannot interact productively with Ad5 DNA. Based on this, a chimeric virus containing the Ad7 genome except for the inverted terminal repeats and packaging sequence from Ad5 was constructed. This chimeric virus replicates its DNA and synthesizes Ad7 proteins, but it cannot package its DNA in 293 cells or 293 cells expressing the Ad5 L1 52/55-kDa protein. However, this chimeric virus packages its DNA in 293 cells expressing the Ad5 IVa2 protein. These results indicate that the IVa2 protein plays a role in viral DNA packaging and that its function is serotype specific. Since this chimeric virus cannot package its own DNA, but produces all the components for packaging Ad7 DNA, it may be a more suitable helper virus for the growth of Ad7 gutted vectors for gene transfer.

Adenoviridae↗
PubMed2004

The Sleeping Beauty transposable element: evolution, regulation and genetic applications.

Members of the Tc1/mariner superfamily of transposable elements isolated from vertebrate species are inactive due to the accumulation of mutations. A representative of a subfamily of fish elements estimated to be last active > 10 million years ago has been reconstructed, and named Sleeping Beauty(SB). This element opened up new avenues for studies on DNA transposition in vertebrates, and for the development of transposon tools for genetic manipulation in important model species and in humans. Multiple transposase binding sites within the terminal inverted repeats, a transpositional enhancer sequence, unequal affinity of the transposase to the binding sites and the activity of the cellular HMGB1 protein all contribute to a highly regulated assembly of SB synaptic complexes, which is likely a requirement for the subsequent catalytic steps. Host proteins involved in double-strand DNA break repair are limiting factors of SB transposition in mammalian cells, underscoring evolutionary, structural and functional links between DNA transposition, retroviral integration and V(D)J recombination. SB catalyzes efficient cut-and-paste transposition in a wide range of vertebrate cells in tissue culture, and in somatic tissues as well as the germline of the mouse and zebrafish in vivo, indicating its usefulness as a vector for transgenesis and insertional mutagenesis.

Animals↗
PubMed1985

Amplification and stability of recombinant plasmid molecules with a very large insert of foreign genetic material.

The capacity of bacterial plasmid vector pAT 153 for harbouring and for amplifying in a stable way a very large insert of foreign genetic material was screened. The DNA of a tree shrew herpesvirus (strain 2) with a molecular weight of about 130 megadalton (200 kilobasepairs) consisting of a long unique DNA sequence without direct and inverted repeats (greater than 40 basepairs), was used for molecular cloning. A successful insertion and stable amplification of a DNA fragment with 49 megadalton (74 kilobasepairs) cloned in Eco RI site of pAT 153 was performed, which resulted in the construction of recombinant DNA plasmid pTH2-E-C1. The stability of the recombinant plasmid pTH2-E-C1 with a super insert of 74 kilobasepairs (49 MD) indicates that the bacterial plasmid vector pAT 153 has really a high capacity for harbouring foreign genetic material.

Bacteria↗
PubMed1984

Cruciform transitions in DNA.

The rates of transition between the cruciform and linear conformations of a perfectly inverted repeated lac operator DNA sequence have been measured using a trimethylpsoralen intrastrand cross-linking assay. The rate and extent of the linear to cruciform transition were dependent on temperature and on the superhelical density of the DNA. Apparent half-lives for this transition were between 4-9 min at 37 degrees C for supercoiled DNAs as isolated from cells. The half-life for the cruciform to linear transition in relaxed DNA was about 30 s at 37 degrees C. Mg2+ stabilized both conformations but stabilized the linear form to a greater degree than the cruciform. The rates of transition were temperature dependent suggesting enthalpies of activation of 26.3 kcal mol-1 for the cruciform to linear transition and 33.4 kcal mol-1 for the linear to cruciform transition. The rate of the linear to cruciform transition was slower at 50 than 37 degrees C. Heating above 70 degrees C resulted in the loss of the cruciform structure.

Cloning, Molecular↗
PubMed1987

Observation of a possible pause mutant in the total synthesis of T4 lysozyme.

A DNA of 495 bp coding for T4-lysozyme was chemically synthesized and cloned in Escherichia coli. On DNA sequence analysis, clones pTLY.10 and pTLY.9 were identified to contain identical and complete T4-lysozyme coding sequences except that pTLY.9 had an additional 23 bp inverted repeat DNA at the 3'-end of the coding sequence. On expression and purification under similar conditions, T4-lysozymes from these two clones showed different degrees of retention time on HPLC as well as in the rate of enzymatic reaction. We speculate that this difference could be due to the generation of a pause mutant of T4-lysozyme in pTLY.9 under the influence of 3'-inverted repeat DNA that alters the rate of protein synthesis.

Base Sequence↗
PubMed1985

The internally located telomeric sequences in the germ-line chromosomes of Tetrahymena are at the ends of transposon-like elements.

The germ-line micronuclear genome of the ciliate Tetrahymena thermophila contains approximately 10(2) chromosome-internal blocks of tandemly repeated C4A2 sequences (mic C4A2). This repeated sequence is the telomeric sequence in the somatic macronucleus. Each of six cloned micC4A2 was found to be adjacent to a conserved 30 bp sequence, which we propose is the terminal inverted repeat of a family of DNA elements (the Tel-1 family). This 30 bp sequence contains a site for the infrequently cutting restriction enzyme Bst XI, which allows full-length Tel-1 elements to be cut out of the micronuclear genome. BAL 31 exonuclease digestion of Bst XI-cut micronuclear DNA showed the majority of micC4A2 blocks to be associated with the ends of the Tel-1 family. We propose that Tel-1 elements are transposable and suggest a novel mechanism to account for the origin of micC4A2, in which telomeric repeats are added to the ends of free linear forms of the transposable elements prior to reintegration.

Animals↗
PubMed2003

Insights into the genetic organization of the Corynebacterium diphtheriae erythromycin resistance plasmid pNG2 deduced from its complete nucleotide sequence.

The complete nucleotide sequence of the erythromycin resistance plasmid pNG2 from the human pathogen Corynebacterium diphtheriae S601 was determined. The plasmid has a total size of 15,100 bp and contains at least 17 coding regions. Comparative genomics identified conserved motifs within replication initiator proteins of corynebacterial plasmids and a novel nucleotide sequence feature, termed 22-bp box, located downstream of the repA gene. The erythromycin resistance determinant erm(X) is flanked by inverted repeats of the novel insertion sequence IS3504, which may be responsible for a spontaneous deletion of the antibiotic resistance gene region. Furthermore, pNG2 encodes a putative conjugative relaxase, a membrane protein of the natural resistance-associated macrophage protein (Nramp) family and a protein with Nudix hydrolase signature. Expression of the predicted coding regions of pNG2 in Escherichia coli JM109 was demonstrated by reverse transcription-polymerase chain reaction (RT-PCR) assays. The detailed annotation of the entire pNG2 sequence provided genetic information regarding its molecular evolution and its role in dissemination of antibiotic resistance genes by horizontal gene transfer.

Amino Acid Sequence↗
PubMedDate not supplied

[Repeating sequences in promoter regions of eukaryotic genes].

Promoter regions of eukaryotic genes were analyzed for the presence of repeated fragments. It was found that the promotor sequences are abundant in direct, symmetrical, and inverted repeats. A computer system for searching and visualizing the repeats was developed.

Animals↗
PubMedDate not supplied

Identification of new medium reiteration frequency repeats in the genomes of Primates, Rodentia and Lagomorpha.

We report eleven new families of MEdium Reiteration frequency (MER) interspersed repeats in the genomes of Primates, Rodentia, and Lagomorpha. Two families of the human repeats, MER 46 and MER 47, represent non-autonomous DNA transposons. These sequences are flanked by TA target site duplications and have terminal inverted repeats (TIRs) similar to TIRs of DNA transposons. The sequences of five other families of repeats, MER41, MER48, MER50, MER51, and RMER3, resemble long terminal repeats of retroviruses. A potential involvement of some of the reported MER repeats in the regulation of transcription and genetic rearrangements is suggested. Age estimations place the origin of most MER repeats at the time of decline in MIR (Mammalian-wide Interspersed Repeats) retroposition and before the origin of the Alu family.

Animals↗
PubMed1997

Use of an automatic DNA sequencer for S1 mapping: transcriptional analysis of the Streptomyces coelicolor A3(2) dnaK operon.

The transcription start point of the dnaK operon of Streptomyces coelicolor A3(2) has been determined by S1 mapping, using the EMBL automated fluorescent DNA sequencer. The -35 and -10 hexamers correspond to a sigma 70-type promoter. This promoter responds to heat shock and involves an inverted repeat different from the CIRCE sequence characteristic of the Gram-positive heat-shock promoters.

Base Sequence↗
PubMed1982

Structural features of the herpes simplex virus alpha gene 4, 0, and 27 promoter-regulatory sequences which confer alpha regulation on chimeric thymidine kinase genes.

Previous studies have shown that herpes simplex virus genes form three groups, alpha, beta, and gamma, whose expression is coordinately regulated and sequentially ordered in a cascade fashion. Chimeric genes constructed by fusion of the coding and 5' nontranslated leader sequences of the thymidine kinase (TK) gene to the sequences upstream from the site of initiation of transcription of alpha genes 4 and 27 are regulated as alpha genes and are induced in cells converted to TK+ phenotype by infection with TK- virus. In alpha gene 4 (S. Mackem and B. Roizman, Proc. Natl. Acad. Sci. U.S.A. 79:4917-4921, 1982), both the promoter and the regulatory region are separable and movable. The promoter permits expression but not induction when fused to TK in the noncoding leader region of the gene. The regulator, when fused to the promoter of an expressible but noninducible portion of the natural beta TK, renders the gene inducible as an alpha gene; it consists of multiple regulatory units acting cumulatively. In this paper, we report on the precise site of initiation of transcription of alpha gene 0 within the inverted b sequences of the L component of viral DNA. We also report the following. (i) The chimeric gene consisting of the coding and 5' nontranslated leader regions of the TK gene fused to portions of the domain of alpha gene 0 extending largely upstream from the site of initiation of transcription of alpha gene 0 was regulated in the same fashion as the alpha 4- and alpha 27-TK chimeras. The regulatory region in the alpha gene 0 is largely upstream from nucleotide - 140. (ii) The promoter-regulatory regions of alpha genes 0, 4, and 27 share TATA sequences, A + T-rich (consensus) sequences occurring in regulating regions of alpha genes 0 and 4 in more than one copy, and multiple G + C-rich inverted repeats. The relation of these sequences to the function of the promoter-regulatory regions of the alpha genes is discussed.

Animals↗
PubMed1986

Stable stem-loop and cruciform DNA structures: isolation of mutants with rearrangements of the palindromic sequence at the simian virus 40 replication origin.

With the objective of generating DNA molecules that form stable stem-loop structures or cruciform structures in solution, we have altered the palindromic sequence at the Simian Virus 40 (SV40) replication origin. These alterations include: deletion of 18 of the 27 base pairs (bp) in the 13-bp inverted repeat; deletion of 26 of the 27 bp; substitution of the entire 27 bp with a totally different 26-bp sequence containing a 13-bp inverted repeat; and substitution of the 27 bp with an 8-bp sequence containing a 4-bp inverted repeat. The DNA from these mutants was purified. Mutant DNAs were hybridized to wild-type SV40 DNA or to each other, and the heteroduplexes were purified. The predicted structures were verified by S1 and restriction endonuclease digestion. The mutants - the heteroduplexes have not been tested - are capable of complementing tsA mutants and/or transforming mouse cells.

Autoradiography↗
PubMed2001

The terminal inverted repeats of IS911: requirements for synaptic complex assembly and activity.

The bacterial insertion sequence IS911 transposes via a covalently closed circular intermediate. Circle formation involves transposase-mediated pairing of both insertion sequence ends. While full-length transposase, OrfAB, binds poorly in vitro to IS911 DNA fragments carrying a copy of the IS911 end, truncated protein derivatives carrying the first 135 (OrfAB[1-135]) or 149 (OrfAB[1-149]) amino acid residues bind efficiently. They generate a paired-end complex containing two such fragments which resembles that expected for the first synaptic complex. Shorter protein derivatives lacking a region involved in multimerisation do not form these complexes but modify the binding of OrfAB[1-135] and OrfAB[1-149]. DNaseI footprinting demonstrated that OrfAB[1-149] protects a sub-terminal (internal) region of the inverted repeats which includes two blocks of sequence (beta and gamma) conserved between the left (IRL) and right (IRR) ends. DNA binding assays in vitro and measurement of recombination activity in vivo of sequential deletion derivatives of the two inverted repeats suggested a model in which the N-terminal region of OrfAB binds the conserved boxes beta and gamma in a sequence-specific manner and anchors the two insertion sequence ends into a paired-end complex. The external region of the inverted repeat is proposed to contact the C-terminal transposase domain carrying the catalytic site.

Base Sequence↗
PubMed1988

The site of deletion of the inverted repeat in pea chloroplast DNA contains duplicated gene fragments.

Analysis of a nucleotide sequence from pea chloroplast DNA which spans the site of deletion of one copy of the large inverted repeat (IR) element suggests that the IR unit has been entirely deleted, but that the single-copy coding sequences which flanked it (trnH and ndh5) have been left intact. However, these flanking sequences have not simply been ligated together: between them there is instead a novel approximately 200 bp DNA sequence which includes two regions apparently derived by the duplication of fragments of the psbA and rbcL genes.

Base Sequence↗
PubMed1988

Nucleotide sequence of integrated hepatitis B virus DNA and human flanking regions in the genome of the PLC/PRF/5 cell line.

Three fragments of cellular DNA containing integrated hepatitis B virus (HBV) sequences have been cloned from the genomic library of a PLC/PRF/5 cell line. The complete nucleotide sequences of the HBV DNA inserts have been determined, including the S gene and the cellular flanking DNA. Viral sequences were found to be fragmented and rearranged. The nucleotide sequences of the HBV-HBV and HBV-human DNA junctions in two of the clones were precisely the same as described by others for the analogous HBV-DNA inserts, providing direct evidence for the stability of HBV-DNA integration pattern and sequence in the genome of the PLC/PRF/5 line. Two clones contain the HBV surface antigen gene which is well conserved. According to the amino acid sequence it could be related to the adw subtype. HBV DNA and contiguous human sequences in HC217 clone are flanked by the cellular perfect inverted repeat of at least 3.5 kb. Similar sequences have been found in the genome of the original PLC/PRF/5 cell line and human placental DNA.

Amino Acid Sequence↗
PubMed1981

Structural variation among human beta-tubulin genes.

A chicken beta-tubulin cDNA probe has been used to screen two independently generated human genomic libraries. Of 13 EcoRI fragments detectable in a human genomic Southern blot experiment, 7 correspond in size to EcoRI fragments isolated from recombinant bacteriophage. The location of beta-tubulin-specific regions and the direction of transcription were determined within each cloned fragment. One clone (5 beta) contained a beta-tubulin-specific region of 6.8 kilobase pairs (kbp) that included three intervening sequences as well as a number of inverted repeat structures. The remaining clones contained beta-tubulin-specific sequences that were close to or, in two cases, substantially less than 1.9 kbp long. Because mature human beta-tubulin mRNA is approximately 1.9 kbp long, these short DNA regions cannot on their own encode a functional beta-tubulin mRNA. Analysis using 3'- and 5'-specific probes derived from the chicken cDNA clone showed the presence of both of these end regions within one truncated tubulin-like sequence. A second short tubulin-specific region failed to hybridize with a 3'-specific probe. These short sequences are therefore likely to be examples of pseudogenes that have arisen by loss of a portion of DNA essential to the production of functional human beta-tubulin mRNA.

Animals↗
PubMed2005

Identification of novel non-autonomous CemaT transposable elements and evidence of their mobility within the C. elegans genome.

We describe here two new transposable elements, CemaT4 and CemaT5, that were identified within the sequenced genome of Caenorhabditis elegans using homology based searches. Five variants of CemaT4 were found, all non-autonomous and sharing 26 bp inverted terminal repeats (ITRs) and segments (152-367 bp) of sequence with similarity to the CemaT1 transposon of C. elegans. Sixteen copies of a short, 30 bp repetitive sequence, comprised entirely of an inverted repeat of the first 15 bp of CemaT4's ITR, were also found, each flanked by TA dinucleotide duplications, which are hallmarks of target site duplications of mariner-Tc transposon transpositions. The CemaT5 transposable element had no similarity to maT elements, except for sharing identical ITR sequences with CemaT3. We provide evidence that CemaT5 and CemaT3 are capable of excising from the C. elegans genome, despite neither transposon being capable of encoding a functional transposase enzyme. Presumably, these two transposons are cross-mobilised by an autonomous transposon that recognises their shared ITRs. The excisions of these and other non-autonomous elements may provide opportunities for abortive gap repair to create internal deletions and/or insert novel sequence within these transposons. The influence of non-autonomous element mobility and structural diversity on genome variation is discussed.

Animals↗
PubMed2000

Characterization of a highly conserved satellite DNA from the parasitoid wasp Trichogramma brassicae.

An EcoRI satellite DNA has been isolated, cloned and sequenced from Trichogramma brassicae, a minute parasitic wasp. This repeated family represents 16% of the genome. The monomer is 385 base pairs (bp) long and has an A+T content of 64.5%. The average nucleotide sequence variability among 12 randomly chosen monomers is extremely low (0.5%), suggesting that the amplification of the monomer into a high-copy-number family occurred recently. An EcoRI satellite DNA probe has been developed and used, at high stringency, as an identification tool to unambiguously discriminate T. brassicae from nine other Trichogramma species. However, at a lower stringency, a hybridization signal can be detected in two closely related Trichogramma species, and, using PCR assay, the presence of the T. brassicae EcoRI monomer has been detected in several other species of Trichogramma. These results argue in favor of the 'library' model of satellite DNA evolution that predicts that related species share a number of low-copy satellite sequences, some of which could be amplified into a major satellite family in each of the species. Furthermore, this T. brassicae EcoRI satellite DNA sequence exhibits particular internal features such as a long inverted repeat that can form a dyad structure. Such sequence motifs seem to be a common characteristic of satellite DNAs, suggesting that they could result from selective forces acting on repetitive DNA.

Animals↗