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PubMed1985

Mutants and pseudorevertants of Moloney murine leukemia virus with alterations at the integration site.

Soon after infection, retroviruses synthesize a DNA copy of the genomic RNA and insert that DNA into the cellular genome by recombination at inverted repeat sequences at the termini of the viral genome. We have generated mutations that alter one terminus of the genome of Moloney murine leukemia virus (M-MuLV). Some mutations did not prevent integration of the viral DNA even though the very terminal bases were disrupted. Other mutations had dramatic effects on the efficiency of infection; in these cases the formation of preintegrative DNA was normal but the establishment of the productive provirus was prevented. One of these defective mutants gave rise to a pseudorevertant which differed from the wild type but displayed normal infectivity. An unusual number of bases of viral DNA were removed during the integration reaction carried out by this virus.

Animals↗
PubMed1994

Isolation and characterization of cDNA and genomic promoter region for a heat shock protein 30 from Aspergillus nidulans.

A cDNA encoding for a heat shock protein 30 (HSP30) of Aspergillus nidulans and the promoter region of its gene were analyzed for their primary structures. The promoter region had no heat shock element but possessed three inverted repeat sequences. Northern blot hybridization indicated that the expression of the HSP30 gene was high at a normal temperature and was slightly accelerated at elevated temperatures in A. nidulans cells. Although the deduced amino acid sequence of the A. nidulans HSP30 had a domain highly conserved among other small HSPs from different species, it showed a sequence homology of only 42% even in comparison with the most closely related molecular species, Neurospora crassa HSP30. These findings suggest that the present HSP30 belongs to a novel subfamily of low-molecular-weight HSPs.

Amino Acid Sequence↗
PubMed1987

Characterization and mapping of equine herpesvirus type 1 immediate early, early, and late transcripts.

Northern blot analysis was used to characterize and map equine herpesvirus type 1 (EHV-1) immediate early (IE), early, and late transcripts. Genomic EHV-1 DNA and cloned EHV-1 restriction endonuclease fragments, representing the entire genome, were 32P-labeled and hybridized to immobilized total cell RNA isolated from EHV-1 infected rabbit kidney cells incubated in the presence or absence of metabolic inhibitors. A single 6.0 kilobase (kb) IE transcript mapped to viral inverted repeat sequences. Approximately 41-45 early transcripts ranging in size from 0.8 to 6.4 kb and 18-20 late transcripts ranging in size from 0.8 to 10.0 kb were identified. These findings demonstrate that EHV-1 gene expression is regulated at the level of transcription, although regulation at the level of translation is also possible. The results provide a basis for examining alterations in viral gene expression in EHV-1 oncogenically transformed and persistently infected cells.

Animals↗
PubMedDate not supplied

Precise and nearly-precise excision of the symmetrical inverted repeats of Tn5; common features of recA-independent deletion events in Escherichia coli.

The transposon Tn5 contains a unique central region bordered by 1.5-kb inverted repeats. The in vitro deletion of the centre of Tn5, with a restriction endonuclease (XhoI) which cuts within the inverted repeats leads to the production of a palindrome on subsequent ligation. This palindromic region is unstable on subsequent transformation into Escherichia coli (Collins, 1981). Precise excision of the Tn5 region plus one copy of the bracketing 9-bp direct repeat occurred in about one-third of the transformants. The rest of the transformants contain only remnants of the inverted repeat. Sequence analysis indicated that deletion had occurred between short direct repeats. The precise excision of these "nearly precise" excision products continued with high frequency and was found to be affected by mutations that interfere with the normal precise excision of transposons. In a recB, sbcB host precise excision was markedly reduced. A common mechanism is proposed for all recA-independent deletions occurring in E. coli.

Bacterial Proteins↗
PubMed1983

Copy number control by a yeast centromere.

Plasmids containing a cloned yeast (Saccharomyces cerevisiae) centromere (CEN3) in combination with a suitable DNA replication system are maintained in yeast at the low copy number typical of a chromosome. In composite plasmids containing CEN3 plus the yeast 2 mu plasmid, the CEN3 copy number control is dominant over the amplification system that normally drives the 2 mu plasmids to high copy number. The CEN3-2 mu composite plasmids are relatively stably maintained in yeast at a copy number of about one per haploid genome, and segregate through meiosis in a typical Mendelian pattern. Some of the CEN3-2 mu composite plasmids isolated from yeast contain deletions of variable size that remove the functional centromere, resulting in loss of the CEN3 control and reversion to high copy number. Formation of the CEN3 deletions requires the specialized recombination system (inverted repeat sequences and FLP gene) of the yeast 2 mu plasmid.

Centromere↗
PubMed1989

Transcriptional analysis of the repressor gene of the temperate Streptomyces phage phi C31.

A 397-bp fragment that contained the 5' end of the coding region of the repressor gene of the temperature Streptomyces phage phi C31 was shown by in vivo promoter-probing to possess bidirectional promoter activity. In vitro runoff transcription experiments, and high resolution transcript mapping of mRNA species produced in vivo using both nuclease S1 and mung bean nuclease, indicated the probable presence of two promoters for the repressor gene with two further promoters oriented in the opposite direction. An inverted repeat sequence is situated 20 bp downstream from the translational stop codon of the repressor gene; high resolution transcript mapping revealed an mRNA endpoint close to this sequence, indicating its likely role as a transcriptional terminator.

Bacteriophages↗
PubMed1992

RAmy2A; a novel alpha-amylase-encoding gene in rice.

The structure and expression of the alpha-amylase-encoding gene, RAmy2A, are described. This only representative of the Amy2 subfamily in rice differs from other cereal alpha-amylase-encoding genes in several respects. It contains the largest introns of all the cereal alpha-amylase-encoding genes examined to date. Moreover, the second of three introns in this gene contains a long inverted repeat sequence that can potentially form a large and stable stem-loop structure in the unspliced RNA transcript. Finally, RAmy2A is constitutively expressed at very low levels in germinated seeds, root, etiolated leaves, immature seeds and callus. This is in marked contrast to the Amy2 genes of wheat and barley which are highly expressed in the aleurone layer of the germinated seeds.

Amino Acid Sequence↗
PubMed2005

Silencing the major apple allergen Mal d 1 by using the RNA interference approach.

BACKGROUND: Apple allergy is dominated by IgE antibodies against Mal d 1 in areas where birch pollen is endemic. Apples with significantly decreased levels of Mal d 1 would allow most patients in these areas to eat apples without allergic reactions. OBJECTIVE: The aim of this study was to inhibit the expression of Mal d 1 in apple plants by RNA interference. METHODS: In vitro -grown apple plantlets were transformed with a construct coding for an intron-spliced hairpin RNA containing a Mal d 1-specific inverted repeat sequence separated by a Mal d 1-specific intron sequence. The presence of the construct in transformants was checked by PCR. Expression of Mal d 1 in leaves was monitored by prick-to-prick skin testing in 3 patients allergic to apples and by immunoblotting with a Mal d 1-reactive mAb and with IgE antibodies against Mal d 1. RESULTS: After transformation, plantlets were selected on the basis of having a normal phenotype and growth rate. With PCR, in 6 of 9 selected plantlets, the presence of the gene-silencing construct was demonstrated. By skin prick test it was shown that a wild-type plantlet had significantly ( P < .05) higher allergenicity than 5 of the transformants. Reduction of expression of Mal d 1 was confirmed by immunoblotting. In wild-type and unsuccessful transformants, a strong band was detected with Mal d 1-reactive mAb 5H8 at the expected apparent M r of 17 kDa. This band was virtually absent in the transformants that carried the gene-silencing construct. With human IgE antibodies, the same observations were made. CONCLUSIONS: Mal d 1 expression was successfully reduced by RNA interference. This translated into significantly reduced in vivo allergenicity. These observations support the feasibility of the production by gene silencing of apples hypoallergenic for Mal d 1.

Adult↗
PubMed2004

Most chloroplast DNA of maize seedlings in linear molecules with defined ends and branched forms.

We used pulsed-field gel electrophoresis, restriction fragment mapping, and fluorescence microscopy of individual DNA molecules to analyze the structure of chloroplast DNA (cpDNA) from shoots of ten to 14 day old maize seedlings. We find that most of the cpDNA is in linear and complex branched forms, with only 3-4% as circles. We find the ends of linear genomic monomers and head-to-tail (h-t) concatemers within inverted repeat sequences (IRs) near probable origins of replication, not at random sites as expected from broken circles. Our results predict two major and three minor populations of linear molecules, each with different ends and putative origins of replication. Our mapping data predict equimolar populations of h-t linear concatemeric molecules differing only in the relative orientation (inversion) of the single copy regions. We show how recombination during replication can produce h-t linear concatemers containing an inversion of single copy sequences that has for 20 years been attributed to recombinational flipping between IRs in a circular chromosome. We propose that replication is initiated predominantly on linear, not circular, DNA, producing multi-genomic branched chromosomes and that most replication involves strand invasion of internal regions by the ends of linear molecules, rather than the generally accepted D-loop-to-theta mechanism. We speculate that if the minor amount of cpDNA in circular form is useful to the plant, its contribution to chloroplast function does not depend on the circularity of these cpDNA molecules.

Chloroplasts↗
PubMed2006

DNA cleavage by the A22R resolvase of vaccinia virus.

Vaccinia virus encodes an enzyme, A22R, required during DNA replication for cleaving viral DNA concatamers to yield unit-length viral genomes. The concatamer junctions contain inverted repeat sequences that can be extruded as cruciforms, yielding Holliday junctions. Previous work indicated that A22R can cleave Holliday junctions in vitro. To investigate the mechanism of action of A22R, we have optimized reaction conditions and characterized the sequence specificity of cleavage. We found that addition of 20% dimethylsulfoxide boosted product formation six-fold, resulting in improved sensitivity of cleavage assays. To analyze cleavage specificity, we took advantage of mobile Holliday junctions, in which branch migration allowed sampling of many DNA sequences. We found that A22R weakly favors cleavage at the sequence 5'-(G/C) downward arrow(A/T)-3', and so is much less sequence specific than its Escherichia coli relative, RuvC. Analysis of the reaction products revealed that A22R cleaves to leave a 3' hydroxyl at the cleaved phosphodiester bond.

Base Sequence↗
PubMed1986

Copy number amplification of the 2 micron circle plasmid of Saccharomyces cerevisiae.

The 2 micron circle is a small double stranded DNA plasmid that occurs at about 60 copies per cell in the nuclei of virtually all strains of Saccharomyces cerevisiae. The plasmid has no apparent phenotypic effect on host cells, and is the basis of many useful vectors for the transformation of yeast. Under certain circumstances, the plasmid is apparently able to replicate more than once per cell cycle; this over-replication allows the maintenance of the plasmid at high copy number. The plasmid has two inverted repeat sequences, and encodes a product that catalyses intra-molecular recombination between these two repeats. Models are proposed whereby recombination leads to copy number amplification. In particular, it is proposed that intra-molecular recombination during replication flips the orientation of one replication fork with respect to the other, so that both forks travel in the same direction around a circular monomer template, generating a large multimer from a monomer and a single initiation of replication.

DNA Replication↗
PubMed1985

G inversion in bacteriophage Mu DNA is stimulated by a site within the invertase gene and a host factor.

The Gin function of bacteriophage Mu catalyzes inversion of the G DNA segment, thus switching the host range of Mu phage particles. This site-specific recombination event takes place between inverted repeat sequences (IR) that border the G segment. Sequences in the Mu beta region extending approximately from position 118 to 178 are essential for efficient inversion. In cis this region, termed sis, stimulates inversion about 15-fold. Neither the relative orientation of sis with respect to the IR sequences nor the distance to IR substantially influences the stimulatory effect. For full activity purified Gin protein must be supplemented with crude host factor from E. coli K12. We suggest that, in addition to Gin, a DNA-binding host protein is required for efficient G inversion.

Bacterial Proteins↗
PubMed1998

Cosolvents facilitate DNA synthesis in the herpes simplex virus 1 unique short (Us) inverted repeat.

DNA synthesis under standard conditions is not successful within a portion of the Us1 gene of HSV-1 which is juxtaposed to an 86% G + C-containing tract in the Us inverted repeat sequence. We report that the independent addition of specific amounts of at least three different types of cosolvents is capable of facilitating DNA synthesis within this G + C-rich region. In addition, this strategy was used to successfully place a specific site-directed mutation in the Us1 gene. Consideration of these observations should enable future site-specific mutational analyses of portions of the HSV-1 genome which have traditionally been unamenable to genetic manipulations.

DNA Restriction Enzymes↗
PubMed1997

Organization, generation and replication of amphimeric genomes: a review.

Genomes comprising a pair of separated inverted repeats and called 'amphimers' are reviewed. Amphimeric genomes are observed in a large variety of different organisms, ranging from archaebacteria to mammals. The widespread existence of amphimeric genomes in nature could be due to their particular dynamic structure. Amphimeric genomes containing long inverted segments may provide the only form in which a duplicated segment is stably retained in genomes. Amphimers are often found in amplified subgenomes, indicating that they could promote a special mechanism of DNA replication and amplification. The possible mechanisms of generation, isomerization and replication/amplification of different types of amphimeric genomes are discussed. The study of amphimeric mitochondrial petite genomes of yeast could be a good model system for the study of the role of inverted repeat sequences in genome dynamics.

Animals↗
PubMed2000

Tn5044, a novel Tn3 family transposon coding for temperature-sensitive mercury resistance.

We report the discovery and characterization of the mercury resistance transposon, Tn5044, from a Xanthomonas strain from the Kamchatka peninsula. In addition to the standard set of merRTPCAD genes, the mer operon of Tn5044 contains a gene named sigY that encodes the RNA polymerase sigma factor-like protein. Mercury resistance determined by Tn5044 is expressed at low (30 degrees C) but not at elevated temperatures (37 degrees C). None of the mer operon genes downstream of merA is responsible for the temperature-sensitive mercury resistance. The transposition module of Tn5044 is closely related to those of Tn1412 isolated from medical sources and to Tn5563 and ISXc5 from environmental sources. However, Tn5044 differs from these transposons in that it has unusually long terminal inverted repeats. Sequence analysis of the transposase (tnpA) genes places Tn5044 and its close relatives into the Tn3 subgroup of the Tn3 family. However, the orientation of their resolvase and transposase genes is unusual for the Tn3 family: tnpR is proximal to the end of the transposon, while divergently transcribed tnpA is oriented inwardly. The region between tnpA and tnpR genes is unusually large and contains two short conserved open reading frames. In addition to the complete set of sequence motifs common to true resolvases, the resolvase of Tn5044 and its close relatives possesses a C-terminal extension showing no homology to known proteins. Despite this peculiarity, Tn5044 resolvase can resolve cointegrates formed during Tn5044 transposition controlled by tnpA. Genetic data suggest that the extension is essential for TnpR functioning.

Amino Acid Sequence↗
PubMed1994

Refinement of gamma delta resolvase reveals a strikingly flexible molecule.

BACKGROUND: gamma delta resolvase is a 20.5 kDa enzyme that catalyzes a site-specific recombination in the second step of the transposition of the gamma delta transposon and requires no cofactors other than Mg2+ for activity. Dimers of resolvase bind cooperatively to DNA at three inverted repeat sequences of differing geometry but catalyze recombination at only one site. RESULTS: The structure of the catalytic domain of gamma delta resolvase, which provides the protein-protein interactions in the synaptic complex, has been refined to an R-factor of 20% at 2.3 A resolution. The structures of the three independent monomers in the asymmetric unit are similar but not identical. Differences occur in the positions of surface loops and in the overall twist of the central beta-sheet of the molecule. The crystal also gives two independent structures for the dimeric form of the molecule, which also show significant differences in the relative orientations of their subunits. CONCLUSION: Resolvase is an unusually flexible protein. This conformational adaptability may be necessary to allow each of the 12 resolvase subunits in the synaptic complex to play a different but specific role in wrapping DNA, binding sites of differing geometry and catalyzing recombination.

Binding Sites↗
PubMed1995

Gin invertase of bacteriophage Mu is a dimer in solution, with the domain for dimerization in the N-terminal part of the protein.

The Gin protein of bacteriophage Mu mediates recombination between two inverted repeat sequences. Gin binds as a dimer to each of these recombination sites. We show that Gin is a dimer in solution also, and that the dimerization is probably stabilized by hydrophobic interactions between the subunits. The subunits of the dimer could efficiently be cross-linked with the 4-A cross-linker diepoxybutane. Spontaneous oxidation of Cys(24) and/or Cys(27) also resulted in intersubunit cross-linking. One or both cysteine residues are located at the interface of the Gin dimer, which maps the dimerization domain in the N-terminal part of the protein. Binding of the disulfide-bonded dimers of Gin to a recombination site was strongly reduced, suggesting that the subunits need to reorient in order to form a stable protein-DNA complex. In the protein-DNA complex, however, oxidation of cysteine residues still seems to be possible, indicating that the N-terminal parts of two Gin subunits are also in close proximity when bound to DNA.

Amino Acid Sequence↗
PubMed1985

Characterization of the bovine prothrombin gene.

The bovine prothrombin gene was characterized by Southern blot analysis of bovine genomic DNA using bovine prothrombin cDNA fragments as hybridization probes. These analyses suggested that the bovine genome contains a single prothrombin gene that is at least 10 kilobase pairs (kbp) in size. To characterize the gene more thoroughly, two bovine genomic phage libraries were screened by using prothrombin cDNAs as hybridization probes. Heteroduplex analysis of the cloned genomic DNA and cDNA showed that the prothrombin gene is 14.9 kbp in size and contains at least 14 exons interrupted by 13 introns. The exons vary in size from 28 to 317 base pairs (bp), while the introns vary in size from less than 100 to 6940 bp. Regions of self-complementarity were observed within some of the introns, suggesting the presence of inverted repeat sequences. The bovine prothrombin gene shows similarities in structure to both the human prothrombin gene and the human factor IX gene.

Amino Acid Sequence↗