Simple, inexpensive preparation of T1/T2 ribonuclease suitable for use in RNase protection experiments.
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Aspergillus↗
Biomedical subjects
G G Carmichael
Publications and source records attributed to G G Carmichael.
Splice site requirement for the efficient accumulation of polyoma virus late mRNAs.
Polyoma virus late nuclear primary transcripts are giant and heterogeneous, containing tandem repeats of the late strand of the circular viral genome. Late pre-mRNA processing involves the splicing of noncoding 'leader' exons to each other (removing genome-length introns), with the joining of the last leader to a coding 'body' exon. We have constructed a number of mutants blocked only in leader-leader splicing, or blocked in both leader-leader and leader-body splicing. We examined the accumulation of both nuclear and cytoplasmic late-strand RNAs in NIH3T3 cells. Consistent with our previous results, mutants lacking the 3' splice site of the late leader (leader-leader splicing blocked) showed a 10-20 fold defect in late RNA accumulation. Mutants which lacked the leader 5' splice site (leader-body splicing blocked) had a more profound defect, exhibiting virtually no late-strand cytoplasmic or nuclear RNA. This result was unexpected as a substantial proportion of wild type late cytoplasmic messages are unspliced. A mutant with no intron, but having functional 3' and 5' splice sites bordering the leader exon, is capable of producing large amounts of unspliced late mRNA. This demonstrates that an excisable intron is not a requirement for late mRNA accumulation. The accumulation of polyoma late mRNAs requires the presence of leader exons bordered by functional 3' and 5' splice sites, whether or not these sites are used during pre-mRNA processing.
Base Sequence↗
Splice site skipping in polyomavirus late pre-mRNA processing.
Polyomavirus late nuclear primary transcripts contain tandem repeats of the late strand of the viral genome, as a result of inefficient transcription termination and polyadenylation. Pre-mRNA processing involves the splicing of short noncoding late leader exons to each other (removing genome-length introns) and the splicing of the last leader to a coding body exon (such as for the major virion structural protein, VP1). As a result, cytoplasmic mRNAs contain 1 to 12 tandem leader exons at their 5' ends that are followed by a single coding exon. To understand more about how polyomavirus exons are spliced together, we studied a double-genome construct consisting of two tandem but nonidentical polyomavirus late transcription units. The alternating leader exons are distinguishable from one another but retain identical flanking RNA-processing signals, as for the alternating VP1 exons. We transfected this construct and derivatives of it into mouse cells and determined which leader exons are spliced to which others and which VP1 exons are utilized. Results showed that leader exons are almost never skipped during splicing and are spliced sequentially to one another. On the other hand, VP1 exons were often skipped, with the VP1 exon closest to the polyadenylation site splicing to the nearest upstream leader exon. Splice site replacement experiments showed that VP1 exon skipping is not due to a relative weakness of its 3' splice site or to any sequence upstream of the VP1 3' splice site. Exon skipping is also not the result of sequences within the VP1 exon. Rather, VP1 3' splice site skipping can be eliminated by replacing the inefficient late polyadenylation signal with an efficient one, or by inserting a 5' splice site between the VP1 3' splice site and the late polyadenylation site. Thus, sequences that compose the distal border of the VP1 exon can influence usage of the upstream 3' splice site.
3T3 Cells↗
Splice site choice in a complex transcription unit containing multiple inefficient polyadenylation signals.
The relationship between polyadenylation and splicing was investigated in a model system consisting of two tandem but nonidentical polyomavirus late transcription units. This model system exploits the polyomavirus late transcription termination and polyadenylation signals, which are sufficiently weak to allow the production of many multigenome-length primary transcripts with repeating introns, exons, and poly(A) sites. This double-genome construct contains exons of two types, those bordered by 3' and 5' splice sites (L1 and L2) and those bordered by a 3' splice site and a poly(A) site (V1 and V2). The L1 and L2 exons are distinguishable from one another but retain identical flanking RNA processing signals, as is the case for the V1 and V2 exons. Analysis of cytoplasmic RNAs obtained from mouse cells transfected with this construct and its derivatives revealed the following. (i) V1 and V2 exons are often skipped during pre-mRNA processing, while L1 and L2 exons are not skipped. (ii) No messages contain internal, unused polyadenylation signals. (iii) Poly(A) site choice is not required for the selection of an upstream 3' splice site. (iv) When two tandem poly(A) sites are placed downstream of a 3' splice site, the first poly(A) site is chosen almost exclusively, even though transcription can proceed past both sites. (v) Placing a 3' splice site between these two tandem poly(A) sites allows the more distal site to be chosen. These and other available data are most consistent with a model in which terminal exons are produced by the coordinate selection and use of a 3' splice site with the nearest available downstream poly(A) site.
Animals↗
Polyomavirus late pre-mRNA processing: DNA replication-associated changes in leader exon multiplicity suggest a role for leader-to-leader splicing in the early-late switch.
Polyomavirus late mRNAs contain at their 5' ends multiple, tandem repeats of a 57-base noncoding sequence, the late leader, whose sequence appears only once in the viral genome. Pre-mRNA molecules are processed by a pathway that includes the splicing of late leader exons to each other in giant, multigenome-length precursors which are the result of inefficient transcription termination. We have devised a method involving reverse transcription and the polymerase chain reaction to determine the number of tandem late leader units on polyomavirus late RNA molecules. Using this technique, we have shown that each class of late viral mRNA (mVP1, mVP2, and mVP3) consists of molecules with between 1 and 12 tandem leader units at their 5' ends. Importantly, single-leader RNAs are underrepresented in both the cytoplasm and the nucleus, suggesting that single-leader primary transcripts are preferentially degraded in the nucleus. In addition, the average number of leaders on late RNAs increases in the presence of DNA replication. Taken together with previous work from our laboratory, the results presented here are consistent with a model for the control of late gene expression at the level of RNA splicing and stability which is in turn controlled by the efficiency of transcription termination.
Animals↗
Replication-dependent transactivation of the polyomavirus late promoter.
When a plasmid containing the wild-type polyomavirus intergenic regulatory region fused to the bacterial cat gene was introduced into mouse NIH 3T3 cells along with a plasmid coding for the early viral proteins (T antigens), chloramphenicol transacetylase enzyme activity and mRNA levels were increased about 10-fold over levels observed in the absence of early proteins. To investigate this transactivation phenomenon further, 11 specific deletion mutant derivatives of the wild-type parent plasmid were constructed and studied. One mutant (NAL) with a minimal level of chloramphenicol transacetylase expression in the absence of T antigens was capable of being transactivated more than 40-fold. A number of other mutants, however, had little capacity for transactivation. Each of these mutants had in common a defect in large T-antigen-mediated DNA replication. Interestingly, one of the transactivation-defective mutants showed a basal late promoter activity fivefold higher than that of wild type and replicated in mouse cells in the absence of large T antigen. Subsequently, a small deletion abolishing viral DNA replication was introduced into those mutants capable of transactivation. The effect of the second deletion was to eliminate both replication and transactivation. Finally, wild-type and mutant constructs were transfected into Fisher rat F-111 cells in the presence or absence of early proteins. No transactivation or replication was ever observed in these cells. We concluded from these studies that the observed transactivation of the polyomavirus late promoter by one or more of the viral early proteins was due to either higher template concentration resulting from DNA replication or replication-associated changes in template conformation.
Animals↗
Enhancer and promoter elements from simian virus 40 and polyomavirus can substitute for an upstream activation sequence in Saccharomyces cerevisiae.
Ten fragments of higher eucaryotic DNA were tested for upstream activation sequence activity in Saccharomyces cerevisiae by inserting them upstream of a CYC1::lacZ promoter lacking an upstream activation sequence. Fragments containing the 21-base-pair repeat region, the enhancer of simian virus 40 or both strongly stimulated beta-galactosidase synthesis, and three fragments from the polyomavirus enhancer region stimulated moderate levels. Three of the four controls of random DNA sequences failed to stimulate significant levels, and the fourth stimulated moderate levels. The stimulation in all cases was independent of the orientation of the inserted fragment. Two series of clones were examined in which between one and six tandemly arranged copies of a fragment were inserted into the XhoI site of the vector. Very interestingly, we detected an apparent exponential relationship between the number of copies of a fragment and the amount of beta-galactosidase produced. Southern analysis showed that increases in enzyme activity were not a result of increased plasmid copy number. Rather, quantitative S1 nuclease analysis demonstrated that the increases were correlated with steady-state levels of lacZ-specific mRNA. We suggest that there may be an evolutionary relationship between some transcriptional activation sequences in yeast cells and the higher eucaryotic regulatory elements that we tested.
DNA, Recombinant↗
Translational efficiencies of polyomavirus late mRNA molecules that differ in the sequences of their 5' noncoding late leader exons.
Mouse NIH 3T6 cells were coinfected with two strains of polyomavirus that differ only in the sequences of their 5' noncoding late leader exons. Polysomes were isolated at late times after infection and probed with oligonucleotides specific for each strain. Results indicate that the sequence of the late leader does not play a role in the translational efficiency of late polyomavirus messages.
Animals↗
Leader-to-leader splicing is required for efficient production and accumulation of polyomavirus late mRNAs.
Polyomavirus late mRNA molecules contain multiple, tandem copies of a noncoding 57-base "late leader" exon at their 5' ends. This exon is encoded only once in the genome. Leader multiplicity arises from leader-leader splicing in giant primary transcripts, which are the result of multiple circuits of the viral genome by RNA polymerase II. We have been interested in learning more about the role of the leader exon in late viral gene expression. We recently showed that an abbreviated-leader mutant virus (ALM) with a 9-base leader exon is nonviable (G. R. Adami and G. G. Carmichael, Nucleic Acids Res. 15:2593-2610, 1987) and has a severe defect in both late pre-mRNA splicing and stability. However, a mutant virus with a different, substituted leader sequence of 51 nucleotides (SLM/MP8) is viable and has no apparent defects. Here we examined further the role of the late leader exon in late pre-mRNA processing. When the leader exon length was gradually reduced from 51 nucleotides to 9 nucleotides in a series of mutants, RNA splicing and stability defects were coupled. In this system there was a minimum exon size of between 33 and 27 nucleotides. Next, a number of mutations were introduced into the 3' splice site which precedes the late leader. Such mutations blocked leader-leader splicing. Surprisingly, they also interfered with leader-mVP1 body splicing and resulted in unstable primary transcripts. Thus, polyomavirus leader-leader splicing appears to be important for the efficient accumulation of late viral mRNA molecules.
Animals↗
Deletion analysis of the polyomavirus late promoter: evidence for both positive and negative elements in the absence of early proteins.
We have been interested in understanding more about the sequences that constitute the polyomavirus late promoter. Our approach has been to target specific deletions to the viral intergenic region by oligonucleotide-directed mutagenesis. Wild-type and mutant promoter cassettes with defined deletions were then inserted into a promoterless expression vector containing the bacterial chloramphenicol acetyltransferase (CAT) gene (cat). Plasmids were introduced into mouse NIH 3T3 cells by transfection, and promoter activities were assessed by quantitation of both CAT enzyme and cat mRNA levels. In this report, we present the results of experiments designed to map promoter elements which affect late transcription in the absence of early viral proteins and viral DNA replication. Using this approach, we mapped two major cis-acting elements (a positive and a negative one) which affect transcription in our transient expression system. The first, positive, element coincided with the enhancer A element, which is known to be important for early transcription and viral DNA replication. Removal of this element reduced late transcription by 50- to 100-fold. The second element was a negative one; removal of 89 base pairs that included two high-affinity large-T-antigen-binding sites just to the early side of the inverted repeat structure within the replication origin resulted in a 5- to 10-fold increase in late promoter activity. The implications of these findings for late promoter function and regulation are discussed.
Animals↗
Polyomavirus early-late switch is not regulated at the level of transcription initiation and is associated with changes in RNA processing.
Polyoma gene expression is temporally regulated during productive infection of mouse cells. Early genes are expressed throughout the viral life cycle, but late mRNAs are not detected until after the onset of DNA replication. At late times, late-strand transcripts represent the great majority of viral-specific RNA in the cell. To learn more about the mechanism by which the early-late switch is regulated, we have carried out a detailed analysis of polyomavirus transcription in mouse NIH 3T6 cells. Nuclei were isolated from cells infected for 6, 12, 18, or 24 hr, and run-on assays were performed. The resulting RNAs were then hybridized to a number of immobilized early- and late-strand-specific probes, which represent the entire polyoma genome. Results indicate that the late promoter is always on, even in the absence of DNA replication. Even though the early-late switch is characterized by a greater than 300-fold difference in the ratio of steady-state early- and late-strand RNAs, there is only a 2-fold effect at the level of transcription initiation. Furthermore, the efficiency of termination for late transcripts is very high at early times during infection (greater than 90%) but drops drastically at late times (less than 40%). In other experiments, we have found an increase in splicing efficiency of late pre-mRNA molecules that parallels the decrease in termination efficiency. These results, taken together with other studies from our laboratory, have led us to propose two possible models for the temporal control of polyomavirus late gene expression.
Animals↗
The length but not the sequence of the polyoma virus late leader exon is important for both late RNA splicing and stability.
Polyoma virus late RNA processing provides a convenient model system in which to study the mechanics of splicing in vivo. In order to understand further the role of the untranslated "late leader" unit in late RNA processing we have constructed a group of polyoma viruses with deletions and substitutions in the leader exon. This has allowed us to determine that there is a minimum exon size required for both pre-mRNA splicing and stability in this system. We show here that the non-viability of a mutant (ALM) with a 9 base late leader unit is due to a general defect in late RNA splicing. In addition, ALM-infected cells show at least 40-fold depression in the accumulation of late nuclear RNA (spliced or unspliced). The ALM late promoter, however, functions nearly normally. Substituted leader variants with 51- to 96-base long exons of unrelated sequence are viable (G. Adami and G. Carmichael, J. Virol. 58, 417-425, 1986). We show here that late RNA from one of these substituted leader mutants (containing a 51-base leader exon) is spliced at wild type levels, with virtually no defect in accumulation. Thus, in the polyoma system, splice sites separated by only 9 bases can inhibit each others usage, presumably by steric interference. We suggest that this type of inhibition leads to extreme RNA instability.
Animals↗
Polyomavirus tumor induction in mice: influences of viral coding and noncoding sequences on tumor profiles.
We determined the DNA sequences of the noncoding regions of two polyomavirus strains that differ profoundly in their abilities to induce tumors in mice. Differences between strains were found, both on the late side of the replication origin in the region containing known enhancer elements and on the early side of the origin, affecting the number and location of large-T-antigen-binding sites. By constructing and analyzing recombinant viruses between these high- and low-tumor strains, we attempted to localize determinants which affect the frequency and histotype of tumors. Seven recombinants were constructed and propagated in vitro, and the tumor profile of each was established by inoculation into newborn C3H mice. Recombinants containing noncoding sequences from the high-tumor strain and coding sequences from the low-tumor strain behaved like the latter, inducing tumors at a low frequency and strictly of mesenchymal origin. Reciprocal recombinants with noncoding sequences of the low-tumor strain linked to structural determinants from the high-tumor strain induced several types of epithelial tumors typical of the high-tumor strain but at reduced frequency, in addition to mesenchymal tumors. A high frequency and full diversity of epithelial tumors required, in addition to structural regions from the high-tumor strain, noncoding sequences on the early side of the origin also present in this strain. A high-tumor profile thus resulted from the combined effects of structural and regulatory determinants in the high-tumor strain, with the former affecting primarily the tissue tropism and the latter affecting the frequency of tumors. No differential effects of the enhancer regions from the late side of the origin in the two virus strains were seen in this study.
Animals↗
A method for constructing multiple tandem repeats of specific DNA fragments.
We describe a new method for constructing tandem repeats of DNA fragments that allows one to control the number of tandem copies appearing in a final recombinant DNA clone. The principle of the method is to prepare DNA fragments with predetermined cohesive ends and to ligate them together in such a way that unique multimers are generated. First, the fragment of interest is inserted into a vector with a multisite cloning region. Clones are picked with the fragment in both orientations, and the fragments are excised using different pairs of restriction enzymes. The resulting fragments with preprogrammed cohesive ends are mixed, ligated, and digested with one enzyme whose site appears in the cloning region of the vector. From this material multimeric fragments containing specific ends can be isolated and recloned into appropriate vectors. As an example of the method, we show the construction of clones containing either three or six tandem copies of the origin of DNA replication of the mouse polyoma virus. The prospect of using this technique to construct inverted repeat structures is discussed. As part of this work we have constructed a modified M13mp8 vector which contains a unique Xho I cloning site.
DNA Replication↗
Polyomavirus late leader region serves an essential spacer function necessary for viability and late gene expression.
All three polyomavirus late mRNAs contain multiple tandem copies of the same nontranslated 57-nucleotide sequence, the late leader, at their 5' ends. We show here that a polyoma variant (ALM) lacking 48 central bases of the 57-base leader unit is nonviable by plaque assay and by a new method for testing virus viability, an immunofluorescence burst assay. ALM is, however, unaffected in early gene expression as measured both by indirect immunofluorescence of large T antigen and by transformation levels of rat F-111 cells. DNA replication in mouse cells is also as wild type, and the defect in ALM is complemented by an early-defective helper virus DNA. ALM does not make detectable levels of late viral proteins and is minimally 200-fold depressed in the accumulation of cytoplasmic polyadenylated late RNA. When the deleted leader sequence of ALM is replaced by a variety of procaryotic sequences, viability almost always returns. Some of the substituted leader variants produce plaques with the same apparent kinetics as wild-type viral DNA. The indication is that the sequence of the polyoma late leader is not important for late gene expression but that it has an essential spacer function on the RNA or DNA level. This spacer function is apparently necessary for late viral RNA transcription, processing, or stability.
Animals↗
Residual transforming activity of PY1178T, a mutant lacking the principal in vitro tyrosine phosphorylation site, is not affected by removal of the secondary tyrosine phosphorylation site at residue 322.
Polyoma virus mutants lacking one or both tyrosines at position 315 and 322 of wild-type middle T antigen have been constructed. The effects of the removal of these tyrosines are additive for middle T phosphorylation in immune complexes, with tyrosine 315 being the major acceptor site and 322 a secondary site. Previous studies have shown little or no effect of deletion of tyrosine 322 on transforming ability, whereas a strong effect has been seen by substitution of phenylalanine for tyrosine 315. In contrast to the phosphokinase results, there is no additive effect of combining these mutations on the viruses' transforming ability. Thus the double mutant lacking both tyrosines has the same weak transforming activity as the single mutant containing tyrosine 322 and phenylalanine 315. Phosphorylation of middle T antigen at tyrosine 322 by pp60c-src or other tyrosine-specific cellular protein kinase is therefore unimportant for transformation.
Antigens, Polyomavirus Transforming↗
DNA sequence alterations in Hr-t deletion mutants of polyoma virus.
We have investigated the DNA sequence alterations in several hr-t mutants of polyoma virus. These mutants are defective in one of the two known viral functions essential for transformation and are altered with respect to several minor T antigen species. The lesions in some of these mutants have been mapped previously by marker rescue experiments to Hpa II fragment 4 (Hpa II-4, 78.4--91.7 map units) in the proximal part of the early region of the viral DNA. Thirteen of sixteen hr-t mutants examined carry deletions 2 to 5 map units (100--250 bp) long in Hpa 11-4. Three mutants carry either point mutations or very small deletions/insertions. Eight of the deletion mutants were mapped closely with restriction enzymes. Seven of them have deletions located entirely within the Hae III subfragment A of Hpa II-4 (the Hae A subfragment, 78.4--85.2 map units), and one extends just beyond this subfragment, ending at 85.5 map units. The complete sequence of the wild-type Hae A subfragment was determined and compared with those of four deletion mutants, NG-18, A-8, 6B5 and B-2. The deletion in each of these mutants is out-of-phase: NG-18, 187 bp; A-8, 127 bp; 6B-5, 179 bp; B-2, 241 bp. All are expected to remove protein sequences in the C terminal part of the small t antigen.
Amino Acid Sequence↗
Isolation of Escherichia coli 30 S ribosomal protein S1 by poly(rC)-cellulose chromatography.
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Chromatography, Affinity↗