Minor Transcripts Research Articles

Analysis of the major transcripts encoded by the long repeat of human cytomegalovirus strain AD169.

In this report, we describe the size and kinetics of appearance of RNAs from the long repeat of human cytomegalovirus. The most abundant RNA from this region was a 2.7-kilobase (kb) species that was detected throughout the infection and was most abundant at 27 and 72 h after infection. The 2.7-kb RNA was the only major species detected with a probe that included the terminus of the long repeat and the heterogeneous L-S junction region. Other transcripts were detected with probes from the internal portion of the long repeat, including an immediate-early RNA of 1.3 kb, early and late RNAs of 1.2 kb, and minor late transcripts of 4.4, 3.6, 3.3, and 1.8 kb. S1 nuclease and exonuclease VII protection analyses of RNA from immediate-early, early, midpoint, and late times in the infection indicated that the major 2.7-kb RNA was not spliced and that the RNA mapped within the long repeat, 1.6 kb from the heterogeneous region. No evidence for temporally regulated changes in transcription initiation, splicing, or choice of 3' end of this RNA was observed. Nuclease protection analysis also demonstrated that the second most abundant late RNA from this region, the 1.2-kb species, was not spliced and had the same polarity as the 2.7-kb RNA. The 1.2-kb also mapped entirely within the long repeat, with its 3' terminus 1.7 kb upstream from the 5' terminus of the 2.7-kb RNA.

Bidirectional transcription of maize streak virus DNA and identification of the coat protein gene

Three RNA transcripts encoded by maize streak virus DNA were detected in polyadenylated RNA from virus-infected maize leaves. Two of the transcripts, a major 0.9 kb and a minor 1.05 kb RNA, were mapped on the virion (+) sense DNA and the other minor transcript of 1.2 kb was mapped on the complementary (-) sense DNA, demonstrating that transcription of MSV DNA was bidirectional. The two virion sense transcripts were 3' coterminal at nucleotide 1114 but had 5' termini at nucleotides 2682 and 163 respectively. Virus-specific polyadenylated RNA translated in vitro to produce a 28,000 MW polypeptide, specifically immunoprecipitable by antiserum raised against whole virus. The mRNA for this protein was mapped by hybrid-arrested translation to the long open reading frame in virion sense DNA whose potential amino acid composition, calculated from nucleotide sequence data, closely agreed with that determined experimentally for the coat protein.

Efficient expression of cloned complementary DNAs for secretory proteins after injection into Xenopus oocytes

Cloned complementary DNAs encoding chicken ovalbumin, chicken prelysozyme and calf preprochymosin, prochymosin and chymosin were inserted downstream from various viral promoters in modified recombinant “shuttle” vectors. Microinjection of the ovalbumin, prelysozyme and preprochymosin constructs into the nuclei of Xenopus laevis oocytes resulted in the synthesis, segregation in membranes and secretion into the extracellular medium of ovalbumin, lysozyme and prochymosin, respectively. Judging from molecular weight estimations, lysozyme and prochymosin were correctly proteolytically processed while ovalbumin, which lacks a cleavable signal sequence, was glycosylated. Injection of the DNA construct encoding prochymosin without its signal sequence resulted in synthesis of prochymosin protein that was localized exclusively in the oocyte cytoplasm. No immunospecific protein was detected after injection of the DNA encoding mature chymosin. In terms of protein expression in oocytes, the Herpes simplex thymidine kinase (TK) promoter was up to sevenfold more effective than the simian virus 40 (SV40) early promoter, and equally as effective as the Moloney murine sarcoma virus long terminal repeat element. Where tested, protein expression in oocytes was much reduced if DNA sequences encoding the SV40 small t intron and its flanking sequences were present in the constructs. S 1 nuclease mapping of transcripts produced after injection of DNAs containing the TK promoter indicated that the majority of transcripts initiated at, or within, two bases of the known “cap” site. However, minor transcripts initiating upstream from this site were observed and one (or more) of these transcripts was responsible for the synthesis of an ovalbumin polypeptide containing a 51 amino acid N-terminal extension. This extended protein remained in the oocyte cytosol. When ovalbumin cDNA was inserted into the vectors with opposite polarity to the viral promoter, expression in oocytes resulted in the predominant synthesis and secretion of a variant ovalbumin with a 21 amino acid N-terminal extension, although some full-length ovalbumin was also synthesized and secreted. S 1 mapping revealed the presence, in these oocytes, of transcripts of predicted polarity initiating 118 bases upstream from the wild type ovalbumin initiator ATG, at a previously unreported SV40 “promoter”. No protein synthesis was detected after the injection of these reverse-orientation constructs into baby hamster kidney (BHK-21) cells.

Cottontail rabbit papillomavirus-specific transcripts in transplantable tumors with integrated DNA

The viral DNA and viral transcripts of a Vx2 and a Vx7 transplantable carcinoma lines were analyzed. Both tumor lines contain equivalent numbers of gene copies per cell of exclusively integrated viral DNA. Integration is in head-to-tail tandem repeats, in probably three sites and at least two integration sites are common. In both tumors, about half of the viral genomes share a 90-bp deletion. Common integration sites and deletions suggest that the two tumor lines were derived from the same parent tumor. The tumors contain the same major viral transcripts of 1.3 and 2.0 kb also found in both papillomas and primary carcinomas of domestic rabbits [M. Nasseri, F. O. Wettstein, and J. G. Stevens, J. Virol. 44, 263–268 (1982)]. However, the carcinoma lines differ in their minor transcripts. Vx7 has four minor transcripts of 5.2, 3.8, 3.6, and 3.0 kb and Vx2 has two of 4.8 and 3.0 kb. The 4.8-kb transcript is relatively major and appears to be identical to a major transcript present in virus-producing tumors of cottontail rabbits [M. Nasseri and F. O. Wettstein, J. Virol. 51, 706–712 (1984)].

Nucleotide sequence of the thymidine kinase gene region of monkeypox and variola viruses

Among the orthopoxviruses variola virus induces in cells a characteristic thymidine kinase (TK) activity that can be feedback inhibited in reactions with thymidine triphosphate. Northern blot analyses of variola and monkeypox virus-infected cell extracts showed RNAs of the same molecular weight as the major (590-base) and minor (2380-base) TK transcripts described for vaccinia virus. The nucleotide sequences of 1275 bp in the TK gene region of variola and monkeypox viruses have been determined. When these sequences were compared with such sequences reported for vaccinia virus, differences were observed at 41 nucleotide positions. Examination of the putative encoded TK polypeptide for the three viruses revealed variation at eight amino acid positions. Two major differences in the amino acid composition of the variola virus TK were identified that might play a role in alteration of its kinetic properties.

Tandem promoters in the gene for ribosomal protein S20.

Examination of the nucleotide sequence of the gene for ribosomal protein S20 (rpsT) of Escherichia coli suggested the presence of two promoters ("sites 1 and 2") separated by 90 base pairs (Mackie, G. A. (1981) J. Biol. Chem. 256, 8177-8182). We have investigated the properties of purified or cloned DNA fragments containing one or other or both these sites for their ability to promote transcription in vivo and in vitro. In reactions in vitro containing DNA and purified RNA polymerase as the sole macromolecular components, both sites 1 and 2 act as promoters directing the synthesis of "runoff" transcripts. The 5' termini of such transcripts have been determined by direct sequencing or by identification of the 5' terminal nucleoside 5'-triphosphate, 3'-monophosphate. In site 1, the major transcript initiates with GTP at residue 141 in the DNA sequence. A minor start occurs at residue 142 and uses CTP as the initiating nucleotide. In site 2, the major transcript (approximately 55% of all initiations in site 2) initiates with CTP at residue 232 while minor transcripts, each comprising approximately 20% of the total, initiate at residues 231 and 233 with GTP and CTP, respectively. In four methods of assay which reflect to varying extents the usage of promoters in vivo, site 1 is responsible for 10-30% of the total transcription of the gene for S20 and site 2 the remainder. Sites 1 and 2 appear to act independently and additively in assays based on the rate of synthesis of S20 in a system for coupled transcription and translation. Together, the two promoters for S20 are from 10-25-fold more active than the fully induced lac operon promoter.

The transposition unit of variant surface glycoprotein gene 118 of Trypanosoma brucei: Presence of repeated elements at its border and absence of promoter-associated sequences

At the DNA level, antigenic variation in trypanosomes is brought about by the replacement of one variant surface glycoprotein (VSG) gene by another in an expression site with a strong promoter. In several cases studied, mobilization of a VSG gene for expression involves a duplication-transposition. We have determined the DNA sequence of most of the transposed segment of one such VSG, the VSG 118. At the 3' side, the transposed segment ends within the end of the gene; at the 5' side, the transposed segment is preceded by a putative VSG gene, extending our previous conclusion that VSG genes are tightly clustered. The total length of the transposed segment is about 3.5 X 10(3) base-pairs and 1.8 X 10(3) base-pairs of this codes for the VSG 118 messenger RNA. Near the 5' border of the transposed segment we find five imperfect repeats of about 70 base-pairs that are also present in front of other VSG genes, as shown by hybridization. The termini of three minor VSG 118-specific transcripts map within these repeats. The repeats have the potential to adopt non-B-DNA conformations, and could play a role in the recombination process that exchanges VSG genes in the expression site or, less likely, in pre-mRNA processing. Comparison of the DNA and the mRNA sequence has previously revealed that a terminal exon of 35 nucleotides is spliced onto the main body of the RNA. We show here that these 35 nucleotides are not in the transposed segment and they must, therefore, be contributed by the expression site. This argues persuasively that the transposition activates VSG gene expression by promoter addition rather than by a position effect.

Nucleotide sequence of bovine parathyroid hormone messenger RNA

The sequence of bovine parathyroid hormone mRNA has been determined by sequence analysis of near full-length cloned DNA complementary to the mRNA. Restriction fragments hybridized to the mRNA and extended toward the 5' terminus with reverse transcriptase were analyzed to derive the sequence not present in cDNA. The reverse transcripts were heterogeneous in length with three major stopping points within 8 nucleotides of each other and a minor stop about 30 bases further toward the 5' terminus of the mRNA. The sequence of the gene corresponding to the minor reverse transcript begins with the sequence 5' XXXATATATAAAA which contains the consensus sequence for a TATA box, a putative eukaryotic promoter sequence. Assuming that the major reverse transcriptase stop nearest the 5' terminus of the mRNA, which is 24 bases downstream from the TATA box, represents the beginning of bovine PTH mRNA, the mRNA contains 672 nucleotides, 100 in the 5' noncoding region, 348 in the coding region and 224 in the 3' noncoding region. Bovine PTH mRNA contains 38% G and C bases. The 3' noncoding region is particularly rich in A and U bases with the last 100 nucleotides of the molecules containing 46% U and 32% A. As with other mRNAs, the sequences CO and UAG occur much less than expected. The 5' noncoding region does not contain an AUG before the initiator codon and contains two potential regions that could base-pair with sequences near the 3' terminus of 18S ribosomal RNA. The sequence AAUAAA is present 14 nucleotides from the polyadenylic acid at the 3' terminus. Bovine PTH mRNA exhibits extensive homology with human PTH mRNA.

5′ termini of polyoma virus early region transcripts synthesized in vivo by wild-type virus and viable deletion mutants

The 5′ termini of polyoma virus early region transcripts synthesized during the productive infection of permissive mouse cells by wild-type or tsa virus, and those expressed in a variety of transformed rodent cell lines, have been mapped on the viral genome. Results were obtained using the S 1 nuclease and primer extension gel mapping procedures. Principal 5′ ends of cytoplasmic polyadenylated mRNAs in every instance mapped between nucleotides 145 and 156 (numbering according to Soeda et al., 1980) in the DNA sequence, 17 to 28 base-pairs before the translational initiation codon for early proteins and 26 to 37 base-pairs after a sequence agreeing with the TATA box consensus. Our data implied a minimum of two different termini with this region of the genome, at nucleotide 147 ± 2 and at 152 ± 2. The 5′ ends at 147 ± 2 were particularly common in the mRNA overproduced after thermal inactivation of the large T protein at late times during infection. The sequences determining the principal 5′ termini, unlike the analogous sequences in the closely related simian virus 40 (SV40), are distinct from those involved in the viral origin of DNA replication. A number of minor alternative 5′ termini of cytoplasmie mRNAs. located both before and after the principal 5′ ends, were also detected. Of those downstream from the principal termini, one at nucleotide 300 ± 2 was prominent. Although this apparent 5′ end is well within the early protein coding sequence, it occurs at a position 31 ± 2 base-pairs after a second TATA box. The several minor apparent 5′ termini mapping upstream of the principal termini occurred primarily in the vicinity of the highly conserved papovavirus DNA replication origin sequence. This sequence includes a third TATA box. Two of the minor 5′ termini, at 14 ± 2 and at 20 ± 2. were near the consensus distance from this TATA box, but the others mapped within or before it. Early region mRNA extracted at late times from the cytoplasm of cells infected with wild-type virus, or with tsa virus at the permissive temperature, was usually (but not invariably) enriched for RNA species with apparent 5′ termini mapping in the replication origin region, as well as for even longer RNAs. Such RNAs were correctly spliced and had the normal polyadenylated 3′ ends. They were very minor in the cytoplasmic mRNA overproduced after thermal inactivation of the large T protein at late times during infection, but the nuclear RNA from these cells comprised giant species with highly heterogeneous apparent 5′ ends, including predominantly those in the origin region and others further upstream. Nuclear viral RNA from most transformed cell lines lacked the giant species and had principal 5′ termini in the nt145 to 156 region. We consider two models to account for the presence of the longer mRNAs in the cytoplasm of infected cells at late times during infection. The first postulates a shift in transcriptional initiation sites to upstream positions because of the repressor action of the large T protein. The second, which we favour, proposes that the longer species occur because of inefficient transcriptional termination, which leads to transcription around the entire circular genome and consequently to the eventual accumulation of long cleavage products in the cytoplasm. We further studied the mRNAs expressed by three viable deletion mutants (Bendig et al., 1980) that lack all or part of the sequence determining the principal 5′ termini and the TATA box that precedes it. These efficiently synthesized early region mRNA with slightly or highly heterogeneous 5′ ends. Two of the mutants (dl-75 and dl-17) produced mRNAs with principal alternative 5′ ends located slightly before or after the deletions. The 5′ ends of these mutant mRNAs corresponded to those of very minor transcripts of wild-type templates. These results suggest that sequence information other than the TATA box has an important role in specifying the approximate position of mRNA 5′ ends.

Regulation of herpes simplex virus gene transcription in vitro.

We used partially purified RNA polymerase II from uninfected (Pol II) and from herpes simplex virus type 1 (HSV-1) infected HEp-2 cells (Pol II-H) to transcribe HSV-1 DNA in vitro. Gel electrophoretic analysis of the products produced from native HSV-1 DNA yielded weight average chain lengths of 4.0 and 3.5 kb for the Pol II and Pol II-H products, respectively. Blot hybridization analyses of the HSV DNA transcripts showed that both enzymes transcribed RNA from essentially all regions of the genome. However, Pol II preferentially transcribed regions coding for the immediate-early or alpha mRNAs, whereas Pol II-H preferentially copied regions coding for the early (beta) and late (gamma) gene products. Transcriptional analyses of the cloned HSV-1 Bam HI-Q fragment (containing the thymidine kinase (TK) gene) and its subfragments showed that (1) the major transcripts produced by Pol II-H were distinctly different from those produced by Pol II; (2) Pol II and Pol II-H utilized different promoters for the synthesis of major transcripts; (3) both enzymes produced three minor transcripts that were partially overlapping and in opposite direction to the TK gene; and (4) only Pol II-H initiated transcription from the TK promoter. In contrast, both Pol II and Pol II-H generated an identical set of transcripts from an adenovirus 2 early region DNA fragment. The sizes of the products suggest that RNA processing may be occurring in vitro. These results show that HSV-1 infection alters the in vitro transcriptional specificity of RNA polymerase II and demonstrate that this system should be useful for studying in vitro the regulation of gene transcription.

RNA splicing is required to make the messenger RNA for a variant surface antigen in trypanosomes.

The expression of the gene for variant surface glycoprotein (VSG) 118 in Trypanosoma brucei is activated by transposing a DNA segment containing the gene and 1-2 kb in front of it to an expression site elsewhere in the genome. By S1 nuclease protection and RNA blotting experiments we show here the presence of several minor transcripts in trypanosomes synthesizing VSG 118, one of which covers the entire transposed segment. Comparison of the sequence of the 5' terminal segment of VSG 118 messenger RNA (mRNA), determined by primed reverse transcription, and the corresponding region of the 118 VSG gene, shows that the 5' terminal 34 nucleotides of the mRNA are not encoded in the 118 VSG gene contiguous with the remainder of the mRNA. We conclude that synthesis of a VSG mRNA involves splicing of a much longer primary transcript, which may start outside the transposed segment.

Deletion mapping of sequences essential for in vivo transcription of the iso-1-cytochrome c gene.

The 5' termini of yeast CYC1 RNA molecules have been mapped, by nuclease S1 digestion of mRNA . DNA duplexes, to seven locations from 29 to 93 base pairs upstream from the initiating ATG codon. When the CYC1 gene is introduced into yeast in plasmid YEp13, substantially the same transcripts are made. Using this system to study in vivo gene expression, we measured the capacity of enzymatically produced DNA deletions to form the normal set of RNAs. Four regions of 5'-flanking DNA were identified as functional. Sequences within the region -242 to -139 are required for maximal CYC1 transcript formation; their deletion reduces transcription by a factor of 15 but does not change the pattern of 5' ends observed. Deletion of the sequence between -242 and -99 does not further change the overall transcript level but does affect the specificity of CYC1 mRNA starting. A deletion that extends from -242 to -75 causes both an additional shift in the pattern of 5' ends observed and a further large decrease (factor of 10--20) in CYC1 RNA level. Deletions that extend from -242 to -43, and particularly two deletions that extend still closer to the initiating ATG, cause the appearance of an abundant transcript which starts upstream of position -1078 and of minor transcripts starting in the region -325 to -245.

Mapping of transcription units in the bacteriophage T4 tRNA gene cluster.

Abstract The transfer RNA gene cluster of bacteriophage T4 (about 1600 base-pairs long) is comprised of ten genes that code for eight tRNA and two other stable RNA species. It is arranged in two tight subclusters separated by a spacer of 600 base-pairs. We studied the transcription of these genes in vitro by using DNA templates derived from a series of T4 mutants partially deleted within the tRNA genetic region. The mixtures of primary products made by RNA polymerase on the different T4 DNA templates were fractionated by gel electrophoresis into discrete RNA species. Wildtype and deletion-specific transcripts of the tRNA gene cluster were isolated and their sizes and stable RNA sequence composition were determined. Three primary transcripts of the intact tRNA operon, one major and two minor species, were identified. The major RNA species measured about 3000 nucleotides and harboured the sequences of both subclusters of stable RNA genes. The two minor RNA species (2300 and 1700 nucleotides, respectively) lacked the sequences of the promoterdistal gene subcluster. The existence of a fourth minor primary transcript (about 3500 nucleotides) that contains the sequences of both gene subclusters is inferred from the results. On the basis of these data and the location of a promoter for the T4 tRNA gene cluster by Fukada et al. (1980b), we suggest two alternative schemes for transcriptional organization of the T4 tRNA genes. Both schemes envisage a major transcription unit defined by a promoter 1000 base-pairs upstream, and a terminator 300 base-pairs downstream from the tRNA genes. The existence of weak terminator(s) in the spacer region is also proposed. Primary transcripts of the tRNA operon were processed with S-100 extracts of Escherichia coli and nine T4-specific stable RNAs were identified among the products. This indicates that E. coli contains all the enzymes necessary for the processing of polycistronic T4 tRNA precursors. The in vitro synthesized primary transcripts provide useful substrates for the study of processing enzymes involved in the early steps of tRNA maturation.

Analysis of bacteriophage SP82 major "early" in vitro transcripts.

A restriction map was constructed for the 17.5-kilobase SalI C fragment of SP82 DNA. Unfractionated SP82 DNA, the SalI C fragment, and restriction fragments derived from SalI-C were used as templates for in vitro synthesis by the Bacillus subtilis RNA polymerase, and the resulting transcripts were analyzed by gel electrophoresis. Comparison of the RNA species obtained from SalI-C with those produced from unfractionated DNA indicated that most of the RNAs and all of the major transcripts originate from the SalI C fragment; this fragment contains one copy of the terminally redundant portion of the genome. Seven major transcripts, a bidirectional terminator site, and 5 of the 13 minor transcripts were located within the 13-kilobase redundant region. The binding of polymerase to fragments of DNA produced by digestion of SalI-C with HpaII and HhaI was used to identify promoter-bearing regions within SalI-C.

Isolation and characterization of a new family of mobile dispersed genetic elements, mdg3, in Drosophila melanogaster.

The properties of a new family of mobile dispersed genetic elements, mdg3, are described. Mdg3 is represented by a DNA fragment of about 5.5 kb long which is framed with two repetitive sequences 300-500 base pairs in length. Virtually the whole region of mdg3 including the repetitive sequences is transcribed and, as the result, 26S poly(A)+RNA is formed. The repetitive sequence is present at least at the 3'-end of a major 26S transcript. Both strands of mdg3 are transcribed, but one direction is predominant. Besides 26S RNA, a minor transcript, 15S poly(A)+RNA, can be observed. 15S RNA dose not contain sequences corresponding to the middle of mdg3. Possibly it is formed from 26S RNA by means of splicing. About 15 copies of mdg3 are present in the genome of embryonic cells and polytene chromosomes. Different copies of mdg3 are identical but surrounded with different DNA sequences. In culture cells, the gene is amplified and the number of copies is increased approximately 13 times. Mdg3 is localized in 15-17 sites on different chromosomes of D. melanogaster. Its location varies from one animal to another. The properties and possible nature of mobile dispersed genetic elements are discussed.

The beta major and beta minor globin nuclear transcripts of Friend erythroleukemia cells induced to differentiate in culture.

Friend erythroleukemia clone 745 cells were induced to differentiate by dimethyl sulfoxide (Me2SO) or by hemin treatment, and nuclear transcripts of the beta major and beta minor globin genes examined. The beta major and beta minor genomic sequences selectively labaeled in the nonhomologous part of the large intervening sequence 2 were hybridized to the nuclear RNA of Friend cells. The nuclear RNA of Me2SO-treated cells contained both beta major and beta minor nuclear transcripts, whereas hemin-treated cells contained only the beta minor transcripts. The beta minor nuclear transcripts were more abundant in the Me2SO-treated cells than in hemin-treated cells. Nuclear transcripts with the size of partially processed beta major globin mRNA precursors were also detected in the Me2SO-treated cells.

Expression of the autonomous parvovirus H1 genome: Evidence for a single transcriptional unit and multiple spliced polyadenylated transcripts

We identified viral transcripts in parvovirus H1-infected rodent cells using the S1 nuclease mapping technique of Berk and Sharp (1977, 1978). The most abundant viral transcript, present in both nucleus and cytoplasm, is approximately 2.8 kb long and represents about 56% of the viral genome. Less abundant viral transcripts of 3.0, 1.45 and 1.30 kb, and possibly other minor viral transcripts, are also detected in nuclear and cytoplasmic fractions. In contrast, a prominent 4.7 kb viral transcript which corresponds to 95% of the viral DNA is found only in the nucleus; this finding suggests that the parvovirus genome may function as a single transcription unit. Virus-infected cells pretreated with cycloheximide accumulate all these viral transcripts. Analyses of RNA-DNA hybrids (isolated from neutral agarose gels) by electrophoresis on alkaline agarose gels indicate that the 4.7, 3.0 and 2.8 kb viral transcripts are “spliced” RNAs. The nuclear-specific 4.7 kb transcript appears to be encoded by two noncontiguous DNA segments of 2.2 and 2.6 kb. The 3.0 and 2.8 kb transcripts are apparently encoded by a 2.6 kb segment of DNA and one or more much smaller noncontiguous DNA segments, one of which is approximately 170 nucleotides long.

Identification and mapping of the transcriptional and translational products of the yeast plasmid, 2μ circle

We have identified two major and approximately ten minor poly(A)-containing RNA species in S. cerevisiae which arise from in vivo transcription of the yeast plasmid, known as 2μ circle. The two major species, which are 1325 and 1275 bases in length, are transcribed from the two unique halves of the plasmid and extend into the inverted repeat sequences which separate the unique regions. The map positions of the minor transcripts, which range in length from 350 to 2600 bases, indicate that except for a small region of the genome in which no transcription is observed, both strands of the entire 2μ circle genome are transcribed. We also present evidence demonstrating that RNA transcribed from 2μ circular DNA is used to program the synthesis of specific proteins in yeast: that is, yeast RNA complementary to 2μ circle DNA can be translated in vitro to produce specific polypeptides of substantial size. Finally, the pattern of transcription of 2μ circle suggests the possibility that messenger RNA species are derived by cleavage of larger transcripts, and in addition, that the intramolecular recombination of 2μ circle which occurs in yeast functions as a genetic switch to allow separate expression of two sets of genes on the 2μ circle genome.