Right-hand End Research Articles

Minute virus of mice (MVM) mRNAs predominantly polyadenylate at a single site

The polyadenylation sites for MVM (p) and MVM (1) mRNAs were determined by a quantitative hybridization-S1 protection assay. mRNAs produced by MVM (p) both early and late in infection of mouse A9 fibroblasts, and by MVM (1) and MVM (1) late in infection of human NB324K cells, polyadenylate predominantly at a single site, at nucleotide 4908 ± 2 for MVM (p) and 4843 ± 2 for MVM (1) shortly downstream of the final AATAAA in each viral genome. These results demonstrate that although the right-hand end of MVM has multiple AATAAA signals, and MVM (p) and MVM);) vary significantly within this region, 3′ end processing of viral mRNAs is not a prevalent mechanism for the regulation of MVM gene expression.

Processing of concatemers of bacteriophage T7 DNA in vitro.

The T7 chromosome is a double-stranded linear DNA molecule flanked by direct terminal repeats or so-called terminal redundancies. Late in infection bacteriophage T7 DNA accumulates in the form of concatemers, molecules that are comprised of T7 chromosomes joined in a head to tail arrangement through shared terminal redundancies. To elucidate the molecular mechanisms of concatemer processing, we have developed extracts that process concatemeric DNA. The in vitro system consists of an extract of phage T7-infected cells that provides all T7 gene products and minimal levels of endogenous concatemeric DNA. Processing is analyzed using a linear 32P-labeled substrate containing the concatemeric joint. T7 gene products required for in vitro processing can be divided into two groups; one group is essential for concatemer processing, and the other is required for the production of full length left-hand ends. The products of genes 8 (prohead protein), 9 (scaffolding protein), and 19 (DNA maturation) along with gene 18 protein are essential, indicating that capsids are required for processing. In extracts lacking one or more of the products of genes 2 (Escherichia coli RNA polymerase inhibitor), 5 (DNA polymerase), and 6 (exonuclease), full length right-hand ends are produced. However, the left-hand ends produced are truncated, lacking at least 160 base pairs, the length of the terminal redundancy. Gene 3 endonuclease, required for concatemer processing in vivo, is not required in this system. Both the full length left- and right-hand ends produced by the processing reaction are protected from DNase I digestion, suggesting that processing of the concatemeric joint substrate is accompanied by packaging.

The structure of barley stripe mosaic virus double-stranded RNAs

The terminal structures of the double-stranded replicative forms (RFs) of barley stripe mosaic virus (BSMV) RNAs 1–3 have been investigated. All three BSMV RFs have identical right-hand ends but unique left-hand ends. The plus (+) strands of RFs lack the 3′-ultimate A typical for the encapsidated BSMV RNAS. The 3′-termini of the minus (−) strands contain an unpaired G. It was demonstrated that the internal poly(A) tract of BSMV genome has an equivalent poly(U)-counterpart in the RF (−) strands. The possible role of these peculiarities of BSMV RF structure in RNA replication is discussed.

The nature and biological significance of linear potato spindle tuber viroid molecules

“Naturally occurring” linear potato spindle tuber viroid (PSTV L) was shown to be as infectious as circular PSTV (PSTV c). The occurrence of PSTV L was shown not to be (a) an artifact of the extraction procedure per se; (b) due to the presence of metal ions in extraction buffers; or (c) related to the host species used for propagation, to a particular PSTV strain, or to the duration of infection. From labeling and blot-hybridization experiments, it was concluded that in infected tissue PSTV c appears first followed by PSTV L, which is produced as the result of cleavage of PSTV c at specific sites. One of the sites of cleavage of PSTV c was delineated by 5′-end labeling PSTV L, digesting it with RNase T 1, separating the fragments by two-dimensional gel electrophoresis, and sequencing the major RNase T 1-resistant fragments. The major site of in vitro labeling was shown to be variable, but was confined to the right-hand loop of PSTV c, i.e., between nucleotides 177 and 182. Furthermore, the right-hand end stem and loop of PSTV c contains sequences which are similar to a rDNA gene promoter sequence, and thus we suggest that DNA-dependent RNA polymerase I may be involved in viroid biosynthesis. Other cleavage sites in PSTV were determined by primer extension cDNA synthesis and by dideoxynucleotide chain termination, and were shown to correspond to nucleotides 113–114 and 80–81 and to sequences in the region of 334–340, 300–312, and 271–275. The significance of these cleavage sites is discussed.

Induction of cellular functions in spontaneously immortalized Rat-2 cells transfected with cloned herpes simplex virus type 2 (HSV-2) DNA

Experiments were done to determine if cloned transforming sequences from herpes simplex virus type 2 (HSV-2) DNA confer upon transfected Rat-2 cells the capacity to be stimulated in phospholipase and cyclooxygenase activities following 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment. Tumor-derived Rat-2 cells transformed with sub-fragments (BamHI-E, HindIII/HpaI-ED, or PstI-C) overlapping the right-hand end of the BglII-C transforming region of HSV-2 DNA were stimulated by TPA in both phospholipase activity, measured by deacylation of arachidonic acid, and cyclooxygenase activity, measured by prostaglandin synthesis. Non-transfected Rat-2 control cells showed no increase in these enzyme activities following TPA treatment. To our knowledge, this is the first evidence that cloned HSV-2 DNA has the capacity to induce cellular functions (phospholipase and cyclooxygenase) in transformed mammalian cells.

Nucleotide sequence of IS110, an insertion sequence of Streptomyces coelicolor A3(2).

The nucleotide sequences of the Streptomyces transposable element IS110 and its insertion site in the DNA of a derivative of the temperate phage luminal diameter C31 were determined. The element is inserted about 460 bp from the right-hand end of luminal diameter C31 DNA, in a region of apparently non-coding DNA. The target site (in a run of seven C residues) is within an 11 bp sequence homologous with one end of IS110. The inserted element is flanked by runs of 11 and 15 C residues which form part of more extensive regions of homology between the left and right junction regions. Imperfect inverted repeats (10 matches out of 15 bp) are present near (but not at) the ends of IS110. The whole IS110 element contains about 1550 bp of which 71% are G-C bp. One major potentially protein-coding region (ORF 1215) was detected, of 1215 bp, the product of which, a presumptively soluble protein of MR 43,563, was not overtly related to any entry in a protein sequence database. A smaller open reading frame (ORF 330) was tentatively identified in the opposite strand of the ORF 1215 region.

Analysis of Tn7 transposition

Five gene products required for Tn7 transposition were identified using genetic complementation tests. Four of these (tnsA, tnsB, tnsC and tnsD) are essential for insertion into the attachment site, whereas the fifth (tnsE) is required for transposition to plasmids which lack this site. tnsD is not required for transposition to plasmids lacking the attachment site. This analysis used a chloramphenicol-resistant 'mini' transposon containing Tn7 termini but no complete Tn7 gene product and several compatible expression vectors containing cloned Tn7 fragments. The use of transcriptional and translational fusions allowed the identification of two promoters (P1 and P2) at the righthand end of the transposon and indicated that at least tnsA, tnsB and tnsC are translated in the same direction. Expression from P1 appears to be repressed by tnsB.

Genomic organization of the simian virus 40-adenovirus 7 hybrid virus, PARA(cT), that encodes a nuclear transport defective simian virus 40 T antigen

The genomic organization of the simian virus 40 (SV40)-adenovirus (Ad)7 hybrid virus, PARA(cT), was examined. A deletion of approximately 5529 by of Ad7 DNA extends from 78.8 map units to 94.0 map units and is replaced by an SV40 DNA insert of 3809 bp. The left-hand end of the insertion begins at SV40 nucleotide 5168, 5 by upstream of the ATG initiation codon for T-ag synthesis. The sequence extends counterclockwise through the T-ag encoding sequences and into SV40 late region DNA. Most of the late region DNA has been removed in a deletion between nucleotides 2464 and 301. One of the 72-bp repeats has also been deleted. The right-hand end of the SV40 DNA insert is at nucleotide 4366. Thus, a portion of the SV40 DNA early region is repeated at both ends of the insert (nucleotides 5168-4366).

Relaxation motions induced in Bacillus subtilis macrofibres by cleavage of peptidoglycan.

Bacillus subtilis macrofibres exposed to lysozyme underwent characteristic rotations, termed relaxation motions, in which their twist changed. Intact macrofibres and macrofibre fragments devoid of loop ends responded in the same way. Macrofibre strains for which the helix hand is temperature-dependent and also those of fixed-hand (both left and right) underwent initial relaxation motions towards the right-hand end of the twist spectrum, the only exception being those in which the initial twist state was at or near the right-hand maximum. Often when the initial relaxation motions were completed immediately before structure breakdown the macrofibres underwent one or a few rotations in the opposite direction (towards the left-hand end of the twist spectrum). Crude autolysin extract obtained from wild-type B. subtilis also caused macrofibre relaxation motions at pH 5.6 but at pH 8.0 macrofibre breakdown occurred as a result of septal cleavage. This resulted in the release of helically shaped individual cellular filaments. These findings suggest that strain in the cell wall associated with helical shape was dependent on the integrity of the glycan backbone rather than peptide cross-bridges. In contrast, cleavage of peptide cross-bridges apparently was instrumental in the cell separation process. Left- and right-hand macrofibres, when exposed to lysozyme, exhibited different rates of relaxation, breakdown of fibre structure and protoplast formation. Similarly, the rate of macrofibre breakdown during the lag between temperature shift and inversion reflected the replacement of septal wall material by that of a new conformation corresponding to the new helix hand.(ABSTRACT TRUNCATED AT 250 WORDS)

Localization and fine structure of a vaccinia virus gene encoding an envelope antigen

The major antigen on the envelope of extracellular vaccinia virus particles is a polypeptide with an apparent molecular weight of 37,000 (p37K; G. Hiller and K. Weber, J. Virol. 55:651-659, 1985). The gene encoding p37K was mapped in the vaccinia virus genome by hybrid selection of RNA followed by in vitro translation. p37K was then identified among the in vitro translation products by immunoprecipitation with a monoclonal antibody. The gene is located close to the right-hand end of the HindIII F fragment. The corresponding region of the DNA was sequenced, and an open reading frame encoding a polypeptide of 41,748 daltons was observed. The 5' end of the mRNA, as defined by nuclease S1 analysis, maps within only a few nucleotides of the translation initiation codon. Examination of the DNA sequence around the putative initiation site of transcription revealed a characteristic sequence, TAAATG, which includes the ATG translation initiation codon and which is conserved in all but one late gene so far analyzed. It is therefore likely that this sequence is an important regulatory signal for late gene expression in vaccinia virus.

The role of RNA molecules in transduction of the proto-oncogene c- fps

Transduction of cellular genes by retroviruses requires two recombinations: one to form the left-hand junction between cellular gene and viral genome, the other to form the right-hand junction. Previous findings raised the possibility that the right-hand recombination might use RNA molecules as intermediates. We now provide direct experimental support for this view by showing that the right-hand end of v- fps in the avian sarcoma virus PRCII was formed by recombination within the poly(A) tract at the 3′ end of the mRNA for the proto-oncogene c- fps. Recombination of this sort may be mediated by “copy-choice” during reverse transcription, acting on either homologous or non-homologous nucleotide sequences.

Temperature-pulse-induced "memory" in Bacillus subtilis macrofibers and a role for protein(s) in the left-handed-twist state

Macrofibers in steady-state growth at one temperature were subjected to pulses of various durations at a temperature at which the opposite helix hand would form and then returned to the initial temperature. In an upshift pulse (20 to 48 degrees C), at least 3 min of incubation was required to induce a transient inversion that occurred later after return to 20 degrees C. Longer pulses resulted in shorter delays in onset of the transient inversion. This "memory" of a brief high-temperature pulse suggests that even a small amount of material can influence the twist of the entire macrofiber. Similar results were found for temperature downshift pulses corresponding to the opposite inversion. Adding chloramphenicol during the temperature pulse blocked the establishment of memory associated with the right-to-left inversion but not that associated with left-to-right inversion. In contrast, inhibiting peptidoglycan synthesis with D-cycloserine during the temperature pulse did not prevent establishment of memory. Inhibiting protein synthesis in mutants fixed as left-handed structures over the entire temperature range induced conversion to right-handedness but did not affect mutants fixed as right-handed structures. Adding protease to either live or formaldehyde-killed macrofibers always induced rotations of right-handed orientation. Steady-state growth in the presence of protease was found to shift the initial macrofiber twist towards the right-hand end of the twist spectrum. The phenomenon was observed in several mutants with different initial twists.

Tn7-encoded proteins.

Proteins encoded by Tn7 have been studied in Escherichia coli maxicells harbouring either various deleted ColE1::Tn7 plasmids or Tn7 fragments cloned in pBR322. Six Tn7-encoded proteins were detected and named p18, p32, p40, p54, p85-a and p85-b according to their apparent molecular weight. Protein p18 is dihydrofolate reductase type I and p32 is probably the protein conferring resistance to streptomycin/spectinomycin. Both genes map on the left-hand part of Tn7. The genes for the four other proteins are located on the right-hand part of Tn7. We propose that they fully cover a 6.9 kb DNA fragment without any overlapping. Starting from the right-hand end towards the middle of the transposon, these four genes are in the following order: p85-a, p54, p40 and p85-b. Transposition of Tn7 onto E. coli plasmids requires the proteins p85-a, p85-b, p54 and p40. However, transposition onto the chromosome does not require the p85-b and p40 products.

Transcription mapping of the varicella-zoster virus genome.

RNA was isolated from varicella-zoster virus-infected Flow 5000 cells (diploid fibroblasts) at late times after infection. With the use of overlapping DNA probes representing all regions of the varicella-zoster genome, an extensive Northern blot analysis of the RNA was carried out. The analysis revealed at least 58 discrete transcripts ranging in size from approximately 0.8 to 6.5 kilobases. RNAs were found to be homologous to all probes used except for those mapping at approximately map unit 0.3, where no RNA transcripts could be detected. Comparison of the sizes and locations of RNA transcripts mapping in the right-hand ends of the varicella-zoster virus and the herpes simplex virus DNAs shows a number of striking analogies, suggesting their similar genomic organization.

Promotion of RNA transcription on the insertion element IS30 of E. coli K12.

Two promoters of RNA transcription have been identified on IS30 by an in vivo assay, in which various DNA fragments with IS30 sequences were inserted in front of the promoterless galK gene of plasmid pFD51. Both promoters have a similar activity of approximately 10% of the activity of the lacUV5 promoter. Promoter P30A precedes the long open reading frame (ORFA), and its proposed -35 region lies within the left-hand terminal inverted repeat of IS30. However, the apparent activity of promoter P30A is significantly reduced when measured in the 3' region of ORFA. Thus, either the activity of promoter P30A is controlled by an IS30-encoded product from the same element, or some termination of transcription from P30A occurs within the coding region of ORFA. Promoter P30C precedes a short open reading frame (ORFC) in-frame with ORFA, but in the opposite strand. Reading frame ORFC is closely followed by a terminator of RNA transcription, T30C. None of the other potential open reading frames predicted from the DNA sequence, with one possible exception, are preceded by a promoter of RNA transcription active in the assay. No significant transcription was detected out of the left-hand end of the complete element. However, a small amount, probably due to read-through from promoter P30A, was detected out of the right-hand end of a complete copy of IS30. In addition the right-hand end of IS30 has been shown to have the potential to create promoters by insertion.

Analysis of the flanking regions from different haemolysin determinants of Escherichia coli.

The haemolysin (hly) determinant of the plasmid pHly152 contains an IS2 element at 469 bp upstream of the hlyC gene. The sequence at the other (right-hand) end (RS) also shows multiple hybridization with the plasmid pHly152 and the chromosome of some Escherichia coli strains but the nucleotide sequence of this region does not reveal the typical properties of an IS element. Similar arrangements in the regions flanking the hly determinant are also found on various Hly plasmids from uropathogenic E. coli strains. Chromosomal hly determinants lack both flanking sequences (IS2 and RS) in the immediate vicinity of the hly genes. The sequences immediately upstream of the hlyC gene have been determined from several chromosomal hly determinants and compared with the corresponding sequence of the hly determinant of the plasmid pHly152. We show that these sequences, which contain one promoter (left promoter, phlyL) in all hly determinants tested, vary considerably although common sequence elements can still be identified. In contrast, only relatively few nucleotide exchanges have been detected in the adjacent structural hlyC genes. The A + T content of the 200 bp sequence upstream of hlyC is very high (72 mol% A + T) but even the structural hly genes show a considerably higher A + T content (about 60 mol%) than the E. coli chromosome on average (50 mol% A + T) suggesting that the hly determinant may not have originated in E. coli.

Negative control of oriC plasmid replication by transcription of the oriC region.

We have demonstrated that the replication of the oriC plasmid, carrying the replication origin of the Escherichia coli chromosome, is inhibited by transcriptional readthrough from an oriC flanking region of the plasmid. This was drawn from an examination of the replication of an oriC plasmid, pKZ4, which bears the lacOP segment at the right-hand side of oriC (the asnA side) in such an orientation that transcription from the lac promoter proceeds towards oriC. Replication of pKZ4 was found to be drastically inhibited by inducing transcription from the lac promoter with IPTG, an inducer of the lactose operon. When trp transcription attenuator termination sequences were inserted near the right-hand end of the oriC region of pKZ4, the replication of the plasmid became considerably insensitive to the inhibitory effect of IPTG. This indicates that the inhibition is due to the frequent leftward transcription, which initiates at the lac promoter and proceeds into the oriC region. Since IPTG inhibits the replication of pKZ4, but not that of another coexisting oriC plasmid which is devoid of the lacOP segment, the replication inhibition is judged to act only in cis. Transcription from the promoter of the chloramphenicol resistance gene also caused the inhibition of oriC plasmid replication.

Initiation of DNA replication at the primary origin of bacteriophage T7 by purified proteins. Initiation of bidirectional synthesis.

Replication of bacteriophage T7 DNA initiates in vivo at an origin located 15% of the distance from the genetic left end of the chromosome. Bidirectional DNA synthesis from this site results in complete replication of the chromosome. The combination of T7 RNA polymerase, T7 DNA polymerase, and T7 gene 4 protein initiates DNA synthesis in vitro within the cloned origin sequence (Fuller, C. W., and Richardson, C. C. (1985) J. Biol. Chem. 260: 3185-3196). DNA synthesis is primed by T7 RNA polymerase transcripts, and proceeds in the same direction (rightward) as transcription to yield partially replicated Y-form DNA molecules. The DNA product of in vitro synthesis (Y-form DNA) has been characterized by electron microscopic, sedimentation, and gel electrophoretic analyses. These studies show that Y-form DNA is the product of unidirectional replication of both leading and lagging strands from the origin to the right-hand end of the template. The inclusion of either Escherichia coli single-stranded DNA-binding protein or the functionally similar T7 gene 2.5 protein results in marked stimulation of bidirectional synthesis. Studies using purified Y-form DNA provide direct evidence that this species is an intermediate in the complete replication of the linear template. Purified Y-form DNA is converted to linear DNA in a reaction catalyzed by T7 DNA polymerase, T7 gene 4 protein, and single-stranded DNA-binding protein. Y-form DNA is a competent, transient intermediate during the bidirectional replication of linear DNA molecules and DNA-binding protein is essential to initiate leftward synthesis.

Genetic analysis of Tn7 transposition.

The purpose of this work was to localize the DNA regions necessary for the transposition of Tn7. Several deletions of Tn7 were constructed by the excision of DNA fragments between restriction sites. The ability of these deleted Tn7s to transpose onto the recipient plasmid RP4 was examined. All the deleted Tn7s isolated in this work had lost their transposing capability. The possibility of complementing them was studied using plasmids containing all or part of Tn7. Two deleted Tn7s could not be complemented by an entire Tn7 indicating that a DNA sequence greater than the 42 bp terminal sequence is needed for recognition of the transposon by a transposition function. Four other deleted Tn7s could be complemented by Tn7. One of these was studied intensively in complementation experiments using different parts of Tn7 to obtain transposition. The results obtained allow us to propose that all genes needed for transposition of Tn7 onto plasmids are contained in a DNA segment of between 6.0 and 7.4 kb. Furthermore, one essential function must be contained in a DNA fragment longer than 2.5 kb on the right-hand end of Tn7. The classification of Tn7 with regard to the other transposable elements is discussed.

In phage λ, cos is a recombinator in the red pathway

Among λ particles carrying chromosomes that have failed to replicate during a lytic cycle cross there is a high frequency of Red-mediated recombination near the right-hand end. Earlier work has shown that this recombination is dependent on cos ( cohesive end site), the packaging origin of λ. In contrast to the prediction of the break-copy model proposed earlier, we find a high recombination rate near cos even when only one of the two participating parents has a functional cos at that locus. The exchange is accompanied by loss of the stimulating cos in the recombination product, irrespective of the marker configurations: a + b + cos − rather than a + b + cos + is produced in the cross a + b − cos − × a − b + cos + as well as in the cross a + b − cos + × a − b + cos −. Further analyses of these and earlier data allow the formulation of a detailed model for cos-stimulated, Red-mediated genetic exchange. In this model, cos stimulates exchange by virtue of being a double-strand cut site. The model has several features like that proposed for yeast. This role of cos in the Red pathway contrasts with the role of cos in the RecBC pathway, in which cos serves as an entry site for a recombinase that stimulates exchanges far from cos.