Overlapping genes in a yeast double-stranded RNA virus.

The Saccharomyces cerevisiae viruses have a large viral double-stranded RNA which encodes the major viral capsid polypeptide. We have previously shown that this RNA (L1) also encodes a putative viral RNA-dependent RNA polymerase (D. F. Pietras, M. E. Diamond, and J. A. Bruenn, Nucleic Acids Res., 16:6226, 1988). The organization and expression of the viral genome is similar to that of the gag-pol region of the retroviruses. The complete sequence of L1 demonstrates two large open reading frames on the plus strand which overlap by 129 bases. The first is the gene for the capsid polypeptide, and the second is the gene for the putative RNA polymerase. One of the products of in vitro translation of the denatured viral double-stranded RNA is a polypeptide of the size expected of a capsid-polymerase fusion protein, resulting from a -1 frameshift within the overlapping region. A polypeptide of the size expected for a capsid-polymerase fusion product was found in virions, and it was recognized in Western blots (immunoblots) by antibodies to a synthetic peptide derived from the predicted polymerase sequence.

Construction of full-length cDNA copies of viral double-stranded RNA.

A method is described for the construction of full-length cDNA clones of dsRNAs. All dsRNA viruses have a capsid-associated transcriptase that is responsible for synthesis of the plus strand that is then extruded from viral particles. We have used in vitro transcripts synthesized by the segmented Saccharomyces cerevisiae virus (ScV) as templates for first-strand cDNA synthesis. Synthesis was primed by a 33-base synthetic oligonucleotide. This contained 27 nucleotides complementary to the 3' end of the plus strand from one ScV viral dsRNA segment (S14), and 6 additional nucleotides encoding an XbaI restriction site at the 5' end. The second cDNA strand was synthesized using a similar XbaI linker-synthetic oligonucleotide and the ds cDNA was cloned by standard ligation techniques. All four cDNA plasmid isolates characterized by sequence analysis contained the complete 5' end sequence of S14. Two of these were complete at the 3' end, and one lacked a single base here. Of these four clones, one also retained the XbaI sites at either end. Preparing full-length cDNA clones with unique restriction-site linkers by the use of synthetic oligonucleotides allows for easier screening for complete cDNA clones if neither the vector nor the cDNA has the chosen restriction site. It also provides for easier sequence analysis and manipulation of the genome for later studies, such as cloning into expression vectors. This method is more efficient than any previously described for production of full-sized cDNA clones.

Physical analysis of the COR region: a cluster of six genes in Saccharomyces cerevisiae.

Six genes, CYC1, UTR1, UTR3, OSM1, tRNAGly, and RAD7, have been localized within an 8-kilobase region on chromosome X of the yeast Saccharomyces cerevisiae. The physical structures and the transcripts of these genes were identified by analyzing a normal strain and six deletion mutants by genomic blotting, transcriptional analysis, and gene disruption procedures. The well-studied CYC1 gene encodes iso-1-cytochrome c; the tRNAGly gene encodes a tRNA; deletion of OSM1 and RAD7 causes sensitivity to hypertonic medium and UV irradiation, respectively. There were no observable phenotypes in strains having deletions of the UTR1, UTR3, and tRNAGly gene. The high density of transcripts, with little or almost no intragenic regions, indicates that the chromosomal organization of S. cerevisiae resembles the chromosomal organization of procaryotes rather than higher eucaryotes.

Conserved regions in defective interfering viral double-stranded RNAs from a yeast virus.

We have completely sequenced a defective interfering viral double-stranded RNA (dsRNA) from the Saccharomyces cerevisiae virus. This RNA (S14) is a simple internal deletion of its parental dsRNA, M1, of 1.9 kilobases. The 5' 964 bases of the M1 plus strand encode the type 1 killer toxin of the yeast. S14 is 793 base pairs (bp) long, with 253 bp from the 5' region of its parental plus strand and 540 bp from the 3' region. All three defective interfering RNAs derived from M1 that have been characterized so far preserve a large 3' region, which includes five repeats of a rotationally symmetrical 11-bp consensus sequence. This 11-bp sequence is not present in the 5' 1 kilobase of the parental RNA or in any of the sequenced regions of unrelated yeast viral dsRNAs, but it is present in the 3' region of the plus strand of another yeast viral dsRNA, M2, that encodes the type 2 killer toxin. The 3' region of 550 bases of the M1 plus strand, previously only partially sequenced, reveals no large open reading frames. Hence only about half of M1 appears to have a coding function.

A mutation allowing an mRNA secondary structure diminishes translation of Saccharomyces cerevisiae iso-1-cytochrome c.

The CYC1-239-O mutation in the yeast Saccharomyces cerevisiae produces a -His-Leu- replacement of the normal -Ala-Gly- sequence at amino acid positions 5 and 6, which lie within a dispensable region of iso-1-cytochrome c; this mutation can accommodate the formation of a hairpin structure at the corresponding site in the mRNA. The amount of the altered protein was diminished to 20% of the wild-type level, whereas the amount of the mRNA remained normal. However, in contrast to the normal CYC1+ mRNA that is associated mainly with four to seven ribosomes, the bulk of the CYC1-239-O mRNA is associated with one to four ribosomes. These results suggest that the stable secondary structure within the translated region of the CYC1 mRNA diminishes translation by inhibiting elongation.

Most highly repeated dispersed DNA families in the mouse genome.

The construction of a small library of mouse repetitive DNA has been previously reported (Pietras et al., Nucleic Acids Res. 11:6965-6983, 1983). Here we report that the 35 plasmids in this library corresponding to highly repeated (greater than 30,000 copies per genome) dispersed DNA sequences can be grouped into no more than 5 distinct families. These families together comprise 8 to 10% of the mouse genome. They include the previously described small elements B1, B2, and R and the large MIF-1 element. Twelve of the 35 clones contain evolutionarily conserved (EC) sequences. One EC clone in our library mostly consists of alternating dCdT residues; another consists of tandem repeats of the sequence CCTCT. The majority of B1s and B2s in the genome appear to be homogeneous, whereas R sequences, ECs, and MIF-1s are heterogeneous. Two earlier reports showed highly repeated mammalian DNA sequences in the herpesvirus genome (Peden et al., Cell 31:71-80, 1982; Puga et al., Cell 31:81-87, 1982). We show that sequences homologous to our EC clones are present in the herpesvirus genome, although these polypyrimidine stretches are not detected in poxvirus, adenovirus, and simian virus 40 genomes. We detect transcripts containing homology to all of these sequences in a nuclear transcription assay. Also, we show that small, polyadenylated RNA molecules homologous to B2 sequences are expressed in undifferentiated embryonal carcinoma cells but not in their differentiated derivatives. The significance of these findings is discussed.

Differential regulation of the duplicated isocytochrome c genes in yeast.

The two unlinked genes CYC1 and CYC7 encode iso-1-cytochrome c and iso-2-cytochrome c, respectively, in the yeast Saccharomyces cerevisiae. An examination of the steady-state level of CYC1 and CYC7 mRNAs in normal and mutant strains grown under different conditions, along with previous results of apoprotein levels, demonstrate that CYC1 and CYC7 have similar and different modes of regulation. Both CYC1 and CYC7 mRNAs are diminished after anaerobic growth. In contrast, CYC1 mRNA but not CYC7 mRNA is decreased by heme deficiency in hem1 mutants. Although both CYC1 and CYC7 mRNAs are substantially lowered after growth in glucose medium, there is a difference in the kinetics of glucose derepression. CYC1 mRNA levels rise in the early logarithmic phase of growth before complete exhaustion of glucose, whereas CYC7 mRNA levels rise in the late logarithmic phase when the level of CYC1 mRNA has plateaued. For a brief period before cessation of growth, the level of CYC7 mRNA attains a level corresponding to the high derepressed level of CYC1 mRNA. The high amount of CYC7 mRNA is surprising because iso-2-cytochrome c constitutes only 5% of the total cytochrome c complement in derepressed cells. We suggest that iso-2-cytochrome c has the potential to comprise a major proportion of cytochrome c under certain physiologic conditions that have not been experimentally defined. The cyc3 mutant, which lacks the ability to attach heme groups to apocytochromes c, contains both CYC1 and CYC7 mRNAs in normal amounts. Yet, cyc3 mutants contain only apoiso-2-cytochrome c and not apoiso-1-cytochrome c. The lack of accumulation of apoiso-1-cytochrome c in cyc3 mutants, which contain CYC1 mRNA, suggests that apoiso-1-cytochrome c is extensively regulated by a post-transcriptional process.

Construction of a small Mus musculus repetitive DNA library: identification of a new satellite sequence in Mus musculus.

We report the construction of a small library of recombinant plasmids containing Mus musculus repetitive DNA inserts. The repetitive cloned fraction was derived from denatured genomic DNA by reassociation to a Cot value at which repetitive, but not unique, sequences have reannealed followed by exhaustive S1 nuclease treatment to degrade single stranded DNA. Initial characterizations of this library by colony filter hybridizations have led to the identification of a previously undetected M. musculus minor satellite as well as to clones containing M. musculus major satellite sequences. This new satellite is repeated 10-20 times less than the major satellite in the M. musculus genome. It has a repeat length of 130 nucleotides compared with the M. musculus major satellite with a repeat length of 234 nucleotides. Sequence analysis of the minor satellite has shown that it has a 29 base pair region with extensive homology to one of the major satellite repeating subunits. We also show by in situ hybridization that this minor satellite sequence is located at the centromeres and possibly the arms of at least half the M musculus chromosomes. Sequences related to the minor satellite have been found in the DNA of a related Mus species, Mus spretus, and may represent the major satellite of that species.

Use of repetitive DNA sequences to distinguish Mus musculus and Mus caroli cells by in situ hybridization.

Mammalian chimaeras have proved useful for investigating early steps in embryonic development. However, a complete clonal analysis of cell lineages has been limited by the lack of a marker which is ubiquitous and can distinguish parental cell types in situ. We have developed a cell marker system which fulfils these criteria. Chimaeric mice were successfully produced from two mouse species which possess sufficient genetic differences to allow unequivocal identification of parental cell types. DNA-DNA in situ hybridization with cloned, species-specific sequences was performed to distinguish the parental cell types. We have identified a cloned, Mus musculus satellite DNA sequence which shows hybridization differences between Mus musculus and Mus caroli DNA. This clone was used a a probe in in situ hybridizations to bone marrow chromosomes from Mus musculus, Mus caroli, and an interspecific F1 hybrid. The clone could qualitatively distinguish Mus musculus from Mus caroli chromosomes after in situ hybridization, even when they were derived from the same F1 hybrid cell. Quantitation of this hybridization to interphase nuclei from bone marrow spreads indicates that the probe can successfully distinguish Mus musculus from Mus caroli cells and can determine the percentage contribution of Mus musculus in mixtures of bone marrow cells of these species and in chimaeric bone marrow cell preparations.

Evolution of defective-interfering double-stranded RNAs of the yeast killer virus.

We have characterized by T1 fingerprint analysis several defective interfering (DI) double-stranded RNAs of the simple yeast virus ScV. A common sequence of about 0.5 to 0.6 kilobase pairs, including both 3' termini of the parental RNA, was present in each DI RNA. Several DI RNAs had novel T1 oligonucleotides not present in their parental RNA.