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PubMed2001

Simian virus 40 regulatory region structural diversity and the association of viral archetypal regulatory regions with human brain tumors.

The regulatory region (RR) of simian virus 40 (SV40) contains enhancer/promoter elements and an origin of DNA replication. Natural SV40 isolates from simian brain or kidney tissues typically have an archetypal RR arrangement with a single 72-basepair enhancer element. A rare simpler, shorter SV40 RR exists that lacks a duplicated sequence in the G/C-rich region and is termed protoarchetypal. Occasionally, SV40 strain variants arise de novo that have complex RRs, which typically contain sequence reiterations, rearrangements, and/or deletions. These variants replicate faster and to higher titers in tissue culture; we speculate that such faster-growing variants were selected when laboratory strains of SV40 were initially recovered. SV40 strains with archetypal RRs have been found in some human brain tumors. The possible implications of these findings and a brief review of the SV40 RR structure are presented.

Animals↗
PubMed1996

Structure-function analysis of the Autographa californica multinucleocapsid nuclear polyhedrosis virus homologous region palindromes.

Homologous regions (hrs), which are present at eight dispersed locations on the Autographa californica multinucleocapsid nuclear polyhedrosis virus genome, are composed of repeated imperfect palindromes within directly repeated sequences. Hrs act as transcriptional enhancers of RNA polymerase II-mediated transcription and as origins of DNA replication when incorporated into plasmids and tested in transient replication assays. To characterize the physical structure of these elements and to determine the role that mismatched nucleotides may play in hr function, oligonucleotides containing a consensus mismatched "imperfect" palindrome and a closely related "perfect" palindrome were synthesized. These sequences were cloned into individual plasmids and tested for their ability to form cruciform structures using nuclease P1 assays and two-dimensional (2-D) gel analyses of topoisomers. The perfect palindrome formed a cruciform structure and the energy requirement for its formation was predicted to occur under physiological conditions. In contrast, the construct containing an imperfect palindrome did not form a cruciform under these conditions. Both hr constructs were found to bind IE-1 in electrophoresis mobility shift assays and act as enhancers when cis-linked to the baculovirus 39K early gene promoter. However, a single oligonucleotide containing the palindrome sequence did not bind IE-1 when annealed under conditions conducive to hairpin formation.

Animals↗
PubMed1989

Transcriptional activation is not responsible for increased levels of autonomously expressed simian virus 40 T-antigen in herpes simplex virus-infected cells.

Herpes simplex virus type 1 (HSV-1) superinfection of CV-1 cells weakly transactivated a plasmid-borne metallothionein 1 (MT-1) promoter, but activated the expression of a marker gene controlled by an authentic HSV-1 promoter to a high level. In contrast, CMT-3 cells, which are CV-1 cells stably transformed with the simian virus 40 (SV40) large T-antigen (T-Ag) gene controlled by the MT-1 promoter, contained high levels of T-Ag following HSV-1 superinfection, but only if cells were preincubated in the presence of heavy-metal ions. This T-Ag was functional in that it could mediate the increase in copy number of a marker plasmid containing the SV40 origin of DNA replication. Pulse and continuous labeling of preinduced CMT-3 cells showed that T-Ag expression was not induced by HSV-1; but rather, HSV-1 superinfection resulted in the stabilization of pre-existing protein.

Animals↗
PubMed1994

An 'instant gene bank' method for gene cloning by mutant complementation.

We describe a new method of gene cloning by complementation of mutant alleles which obviates the need for construction of a gene library in a plasmid vector in vitro and its amplification in Escherichia coli. The method involves simultaneous transformation of mutant strains of the fungus Aspergillus nidulans with (i) fragmented chromosomal DNA from a donor species and (ii) DNA of a plasmid without a selectable marker gene, but with a fungal origin of DNA replication ('helper plasmid'). Transformant colonies appear as the result of the joining of chromosomal DNA fragments carrying the wild-type copies of the mutant allele with the helper plasmid. Joining may occur either by ligation (if the helper plasmid is in linear form) or recombination (if it is cccDNA). This event occurs with high efficiency in vivo, and generates an autonomously replicating plasmid cointegrate. Transformants containing Penicillium chrysogenum genomic DNA complementing A. nidulans niaD, nirA and argB mutations have been obtained. While some of these cointegrates were evidently rearranged or consisted only of unaltered replicating plasmid, in other cases plasmids could be recovered into E. coli and were subsequently shown to contain the selected gene. The utility of this "instant gene bank" technique is demonstrated here by the molecular cloning of the P. canescens trpC gene.

Aspergillus nidulans↗
PubMed1975

Mutagenic topography of the E. coli chromosome.

The lactose fermenting genes in E. coli have been transposed to various chromosomal locations. The bacterial strains were mutagenized with different chemical mutagens and the frequency of Lac negative mutant colonies was measured as a function of lactose gene location in the chromosome. There appears to be a highly mutable location between 58-60 minutes on the E. coli map. This region does not appear to be correlated with the origin of DNA replication or with the terminus. The possible significance of this mutable region in the evolution of new bacterial genes is discussed.

Biological Evolution↗
PubMed1981

The density distribution of gene loci over the genetic map of Escherichia coli: its structural, functional and evolutionary implications.

A quantitative analysis was carried out on the dispersion of gene loci over the E. coli genetic map. Therefore, the map was divided into regions characterized by an homogeneous gene density. This created a distribution pattern of gene loci that contained a symmetry axis located near to the origin of DNA replication. The pattern could be subdivided into a set of 22 functional domains containing gene loci whose products revealed a biochemical or functional relatedness. A correlation was found between the boundary positions of these domains and the distribution of F plasmid- and DNA insertion sites over the E. coli chromosome. The structural, functional and evolutionary implications of these findings are discussed.

Biological Evolution↗
PubMed1991

Evaluation of the magnetic immuno PCR assay for rapid detection of Salmonella.

A new technique, the Magnetic Immuno PCR Assay (MIPA), has been developed for the detection of Salmonella. The assay utilizes magnetic particles coated with monoclonal antibodies against Salmonella to extract these bacteria from the sample. Trapped bacteria are lysed, and the supernatant, which contains bacterial DNA, is then subjected to the polymerase chain reaction (PCR) using primers from the Salmonella typhimurium origin of DNA replication to amplify a 163 bp region. The specificity of the primer set was tested in the PCR; amplification occurred with all 25 Salmonella strains tested but not with 19 other species of Enterobacteriaceae tested. A sensitivity of 100 cfu Salmonella typhimurium was achieved for the MIPA by visualization of the amplified products by ethidiumbromide stained agarose gel electrophoresis. A ten-fold higher sensitivity was obtained by Southern blotting of the amplified products. The presence of 10(7) cfu Escherichia coli did not interfere with these detection levels. The MIPA thus specifically detected 100 cfu of Salmonella within 5 h and may be potentially useful for rapid detection of Salmonella in clinical specimens and food.

Base Sequence↗
PubMed1990

Regulatory DNA sequence conserved in the course of BK virus evolution.

Within the genome of human polyomavirus BK (BKV), there exists a noncoding regulatory region toward the late region side of the origin of DNA replication. In most BKV strains isolated by viral culture, this regulatory region contains tandem repeats varying in size. Recently, however, several laboratories isolated new BKV strains (designated as archetypal strains) lacking such repeat sequences. To examine the genetic relationship between archetypal strains, a phylogenetic tree was constructed for seven BKV strains, including three archetypal strains, from DNA sequence data on the late genes, those for leader protein (agnoprotein), and those for structural proteins (VP1, VP2, and VP3). For three strains data previously reported were used, whereas for the others sequences were determined in this study. From total numbers of nucleotide substitutions in each pair of strains, a phylogenetic tree was constructed by the unweighted pair-group method. The phylogenetic tree obtained reveals that BKV strains containing the archetypal regulatory region do not constitute a cluster of closely related strains and that these strains, together with those carrying the major part of the archetypal regulatory region, are widespread in the BKV population. This finding suggests that the basic structure of the archetypal regulatory region has been conserved in the course of BKV evolution.

BK Virus↗
PubMed2000

Development of a plasmid vector for easy selection of strong promoters.

A promoter vector pACPR33 for Escherichia coli based on the promotorless ampicillin-resistance gene from pBR322 has been constructed. The promoter of the ampicillin-resistance gene was deleted and replaced by a suitable multiple cloning site. Molecular cloning of promoters into the polylinker resulted in activation of the ampicillin resistance in E. coli. The plasmid contains a functional origin of DNA replication and a tetracycline resistance gene for E. coli, and a chloramphenicol resistance gene for S. aureus. The vector permitted direct detection of promoter activity, especially strong promoters, by easy iodometric determination of beta-lactamase activity in liquid or solid media.

Ampicillin Resistance↗
PubMed1990

The dynamic distribution and quantification of DNA cruciforms in eukaryotic nuclei.

Cruciforms have been suggested as potential recognition structures at or near origins of DNA replication in eukaryotic cells. Monoclonal antibodies specific for cruciforms have been produced. The antibody binds to structural determinants at the base of the cruciform stem, the "elbow." Labeling of nuclei with anti-cruciform antibodies produces a nonuniform pattern of fluorescence in cells arrested at the G1/S boundary. This pattern of fluorescence changes when these cells are released from synchrony. Using fluorescence flow cytometry to quantify the number of DNA cruciform structures in cells throughout the cell cycle, we observed two major populations of nuclei with different numbers of cruciforms; the modal number of cruciforms in these populations was 0.6 x 10(5) and 3 x 10(5) cruciforms per nucleus. Synchronized cells (doubly arrested by serum starvation and aphidicolin) displayed a biphasic distribution of the number of cruciforms over the first 6 h after release from synchrony with maxima at 0 and 4 h after release.

Animals↗
PubMed1984

A rho-dependent transcription termination signal in bacteriophage f1.

The bacteriophage f1 intergenic region distal to gene IV encodes a rho-dependent transcription termination signal. Terminator function in vivo and in vitro is dependent upon active Escherichia coli rho protein, although the RNA 3' ends detected in vivo differ from those seen in vitro. The minimal sequence required for terminator function in a heterologous plasmid system encompasses approximately 100 nucleotides distal to gene IV, which can be drawn as a large hairpin structure. The in vivo rho-dependent 3' end occurs within this sequence, while the in vitro rho-dependent 3' ends occur just distal to it. In vivo in a rho mutant host, f1 transcripts pass through the rho-dependent sites and stop within a sequence of high potential secondary structure near the f1 origin of DNA replication. This sequence alone causes transcription termination in the heterologous plasmid system in vivo. In vitro in the absence of rho protein, transcription does not terminate within this sequence. The RNA 3' ends detected in these studies do not occur within A + T-rich sequences.

Base Sequence↗
PubMed1983

The morphogenetic signal of bacteriophage f1.

The genome of the single-stranded DNA phage f1 contains an intergenic region (IG), 508-nucleotides long, that does not code for any known protein. By use of a system of chimeric plasmids haboring different f1 fragments, we had previously shown that this region contains, in addition to the f1 'functional origin' of DNA replication, a signal of less than 300 nucleotides required for efficient morphogenesis to occur ('morphogenetic signal'). In the present study, we have localized this signal to within a sequence of less that 60 nucleotides of almost perfect palindromic symmetry at the genet IV/IG border. We also present data indicating that the morphogenetic signal is not necessary for the synthesis of single-stranded DNA, but is necessary only at some later step during virion maturation.

Base Sequence↗
PubMed1984

The bacteriophage f1 morphogenetic signal and the gene V protein/phage single-stranded DNA complex.

The region of the bacteriophage f1 genome near the gene IV/intergenic region (IG) junction previously was shown to contain a sequence necessary for efficient packaging of single-stranded (SS) DNA into phage particles (the f1 "morphogenetic signal") [G. P. Dotto, V. Enea, and N. D. Zinder (1981) Virology 114, 463-473]. The DNA content of f1 phage/pBR322 chimeric plasmid-transformed, phage-infected bacteria has been investigated. The chimeric plasmids, constructed by Dotto et al. (1981), contained the f1 origins of DNA replication and, in some cases, also contained the "morphogenetic signal." Chimeric plasmid SS DNA was detected in phage-infected bacteria harboring any one of these chimeric plasmids, and the majority of this SS DNA was found complexed to the phage gene V protein. Therefore, the "morphogenetic signal" is not required for the formation of the gene V protein/f1 SS DNA complex but instead must function at a later stage of filamentous phage morphogenesis.

Base Sequence↗
PubMed1991

Expression of SV40 large T antigen, but not small t antigen, is required for the induction of chromosomal aberrations in transformed human cells.

Expression of the Simian virus 40 (SV40) early region in human cells results in the induction of chromosomal aberrations and polyploidy, and in transformation. To understand how genetic damage occurs and what role it plays in transformation, human diploid fibroblasts and embryonic kidney cells were transfected with plasmids encoding wild type or mutant forms of the viral early region, and the neo gene. Clones selected for G418 resistance and expressing viral genes were initially analyzed within 20 cell divisions. Our results demonstrate that expression of the SV40 large T antigen is sufficient for the induction of chromosomal damage and ploidy changes, and that small t does not contribute to these processes. Mutant plasmids lacking the SV40 origin of DNA replication were as proficient as wild type plasmids, indicating that viral DNA replication is not required for cytogenetic damage. We have also shown that chromosome aberrations, but not necessarily polyploidy, increase in frequency and complexity upon subculturing of the clones regardless of whether such populations arrest at crisis or yield immortal lines. Our results are compatible with the hypothesis that large T antigen destabilizes the cellular genome, and that specific mutations arising from this process may contribute to cell immortalization.

Antibodies, Monoclonal↗
PubMed1991

Two bacteriophage T4 base plate genes (25 and 26) and the DNA repair gene uvsY belong to spatially and temporally overlapping transcription units.

The bacteriophage T4 DNA recombination-repair gene uvsY located at or near an origin of DNA replication and adjacent to the late base plate genes 25 and 26. Our present results reveal a complex transcription pattern in the region encompassing these genes. Most significantly, uvsY and two ORFs, downstream of it, all of which are transcribed from a middle promoter before the onset of DNA replication, are also part of a larger late transcription unit which includes the base plate genes 25 and 26. The late genes 25 and 26 are transcribed not only late, but also early from one or several early promoters further upstream. Translation, however, is inhibited by secondary structures which sequester the ribosome binding site in the early transcript. We discuss possible advantages of these transcriptional patterns for T4 DNA recombination, replication, and repair. The predicted and in vivo-expressed 23.9-kDa product of gene 26 is smaller than the reported size of gene 26 protein isolated from base plates, suggesting that nascent gp26 might be processed to a larger protein during assembly.

Base Sequence↗
PubMed1983

Prediction of melting profiles and local helix stability for sequenced DNA.

The utility and limits of the current theory of helix-coil transition of DNA were intensively studied. Comparison of calculated and observed melting profiles affords solid criteria of a theoretical model and parameter values. Our model differs from others in that heterogeneity in stacking interactions is taken into consideration (Section II). The parameter values were estimated by fitting calculated to observed profiles for several known-sequenced DNA fragments (Section III). Calculated profiles agree quite well with observed, not only for DNAs used for the fitting but also for those not used. Such good agreement was never obtained with traditional two-term (A X T and G X C) stability assignment. It was shown that the all-or-none approximation for local melting transition (Azbel's model) provides informative but somewhat erroneous estimates of local stability. The stabilities of 10 kinds of stacking doublets estimated by the fitting accord well with known properties of DNA double helix (Subsection III-4); three-dimensional structure, calculated stacking energies, and binding specificity of some intercalative drugs are all consistent with the estimated order of stacking stabilities. Relationship between triplet stability and genetic code was discussed. The mechanisms of ionic-strength dependence of melting profiles were discussed in Section IV. Four different mechanisms were proposed and their relative contributions in various salt ranges were considered. It was suggested that at low ionic strengths melting processes deviate significantly from equilibrium, which fixes a limit to the applicability of the present theoretical mode. Locally unstable regions and frequently opening regions were located on several natural DNAs by calculating probabilities of individual base-pair doublets being in an unstacked state. With few exceptions, origins of DNA replication on various DNAs fall on frequently opening regions, indicating their functional importance. Positive correlations were also obtained between frequently opening regions and transcriptional promoters, terminators and gene boundaries (Section V).

Base Composition↗
PubMed1976

Topography of polyoma virus messenger RNA molecules.

The different species of polyoma virus-spedific RNA molecules present in the cytoplasm of 3T6 cells 30 hr after viral infection have been characterized by molecular hybridization between nonradioactive polyadenlated RNA, fractionated by sedimentation through sucrose-formamide density gradients, and the 32P-labeled separated strands of restriction endonuclease fragments of polyoma DNA. Two relatively abundant RNA molecules, sedimenting at 16S and at 19S, transcribed from the L strand of the viral DNA, as well as a minor 20S species transcribed from the E strand of the DNA, were detected. The most abundant viral transcript, the 16S RNA molecule, was estimated to be complementary to the 22% of the L-strand DNA extending from 47 to 25 map units. The less abundant 19S L DNA strand transcript included all the sequences present in the 16S RNA and mapped between 68 and 25 map units. The minor 20S RNA molecule was tentatively identified as a transcript of the E-strand DNA from the entire early region of the polyoma genome. These three viral RNA molecules together exhaust greater than 95% of the coding capacity of the viral DNA. A small region of the DNA (4-5%), including the origin of DNA replication, does not appear to determine sequences present among the major stable species of vital mRNA.

Cell Line↗