Oligonucleotide primers were used to amplify DNA sequences from a plasma membrane cation transporting ATPase gene and a transcription factor IID (TFIID) gene from Pneumocystis carinii genomic DNA. The entire P. carinii ATPase gene was cloned from a genomic library by hybridization to the PCR-amplified DNA product. The nucleotide sequence of the gene contained a 2,799 base-pair open reading frame that encoded a 102,274 dalton protein composed of 933 amino acids. The P. carinii ATPase protein was 69-74% identical to four fungal proton pumps but less than 35% identical to protozoan and mammalian cation transporting ATPase genes or the Ca++ ATPases of Saccharomyces. The nucleotide sequence of a portion of the TFIID gene could be translated to produce a peptide of 53 amino acids in two regions of the sequence, interrupted by a 45 bp intron. The predicted TFIID amino acid sequence was identical to yeast TFIID genes in this region.

A 90-base-pair tract of a simple sequence composed of alternating guanosine and thymidine nucleotide residues (poly[d(GT) . d(CA)]) was inserted into the simian virus 40 genome at nucleotide 2666 (0.17 map units). The poly[d(GT) . d(CA)] insertion was stably maintained in the viral genome, but the variant virus grew more slowly than simian virus 40.

A mutant was isolated which demonstrates that the transforming activity of simian virus 40 large T-antigen is separable from its function in viral DNA replication. The mutant, SVR9D, is nonconditionally defective for viral DNA synthesis, but competent at wild-type level for morphological transformation of cultured rat cells. The lytic growth defect in SVR9D is complemented by the simian virus 40 A gene product present in the transformed CV1 cell line, COS1. The lesion in SVR9D DNA was mapped genetically by marker rescue of plaque formation and localized to a 214-base-pair segment of the viral genome bounded by nucleotide numbers 4100 and 4314. DNA sequence analysis showed the mutation to be an adenine-to-guanine transition at nucleotide number 4178. This change predicts a lysine-to-glutamic acid amino acid change at residue number 214 of the mutant large T-antigen polypeptide.

DNA fragments containing the integrated viral DNA present in the simian virus 40 (SV40)-transformed rat cell lines SVRE9 and SVRE17 were cloned in procaryotic vectors, and the DNA sequences linking SV40 and cell DNA were determined. Comparison of the DNA sequences at the SV40-cell junctions in SVRE9 and SVRE17 cells with those of a previously characterized viral insertion from SV14B cells shows that no specific viral or cellular sequences occur at SV40-cell junctions and that the cellular DNA sequences adjacent to integrated SV40 DNA do not display the direct repeat structure characteristic of transposons and retrovirus proviruses.

Herpes simplex virus type 1 (HSV-1) DNA covalently bound to cellulose was used as a reagent to isolate viral RNA transcripts for size analysis on denaturing agarose gels. Nuclear and polyribosomal RNA isolated at 2 h postinfection (p.i.) migrated with sizes between 1,500 and 5,500 nucleotides. At 6 h p.i. (when viral DNA synthesis is underway), viral polyribosome-associated polyadenylated RNA showed different discrete sizes of species predominating, with RNA larger than 5,500 nucleotides clearly present. Nearly 50% of the newly made viral RNA found in the nucleus at 6 h p.i. was from 5,000 to 10,000 nucleotides in length. A high-resolution transcription map of the viral mRNA abundant at 2 h p.i. was compiled from the hybridization of Southern blots of HSV-1 DNA restriction fragments to both sizes of fractionated polyribosomal polyadenylated RNA and 3' complementary DNA probe made to this size of fractionated RNA. We have identified and mapped 16 mRNA species abundant at 2 h p.i. These RNAs range in size from 1,500 to 5,300 nucleotides and map throughout the HSV-1 genome. In some instances, a direction of transcription can be suggested. Further, about one-third of this number of mRNA's has been found in cells infected with a DNA-negative temperature-sensitive mutant (tsB2) and grown at the nonpermissive temperature (39 degrees C).

RNA displacement loop patterns in intact herpes simplex virus DNA and herpes simplex virus DNA restriction fragments indicate that viral RNA associated with polyribosomes early after infection is transcribed from three major areas of the genome. One area of early transcription is in the short segment of the viral DNA and is roughly delineated by the inverted repeat sequences bounding this segment. The other two areas of early mRNA transcription map in the long segment. Each of three major areas of early mRNA transcription can be further resolved into several regions of freqent looping bordered by regions in which RNA displacement loops are rare. These regions range in size from about 1.5 kilobases to about 9 kilobases with a mean size of about 3.5 kilobases. Although the data do not allow precise assignment of individual early gene locations, it is seen, even at the lowest level of resolution, that the early genes are not completely contiguous but are distributed along the length of the herpes simplex type 1 viral genome.

RNA labeled with [methyl-3H]methionine and/or [32P]orthophosphate was isolated from the polyribosomes of herpes simplex virus (HSV) types 1-infected cells and separated into polyadenylylated [poly(A+)]and non-polyadenylylated [poly(A-)] fractions. Virus-specific RNA was obtained by hybridization in liquid to either excess HSV DNA or filters containing immobilized HSV DNA. Analysis in denaturing sucrose gradients indicated that HSV-specific poly(A+) RNA sedimented in a broad peak, with a modal S value of 20. The ratio of [3H]methyl to 32P decreased with increasing size of RNA, suggesting that each RNA chain contains a similar sumber of methyl groups. Further analysis indicated an average of one RNase-resistant structure of the type m7G(5')pppNmpNp or m7G(5')pppNmpNmpNp per 2,780 nucleotides. The following components were identified in the 5'-terminal oligonucleotides of polyribosome-associated HSV-specific poly(A+) and poly(A-) RNA: 7-methylguanosine, N6,2'-O-dimethyladenosine, and the 2'-O-methyl derivatives of guanosine, adenosine, uridine, and denosine, and the 2'-O-methyl derivatives of guanosine, adenosine, uridine, and cytidine. The most common 5'-terminal sequences were m7G(5')pppm6Am and m7G(5')pppGm. An additional modified nucleoside, N6-methyladenosine, was present in an internal position of HSV-specific RNA.

We have quantitatively analyzed the size and amount of herpes simplex virus (HSV)-specific RNA synthesized in HeLa cells using DNA and RNA excess hybridization. At 2 h after infection (early), transcripts from 20% of the total HSV DNA are present on polyribosomes as poly(A+) RNA. At this time, viral poly(A+) RNA comprises 60 to 75% of the newly synthesized poly(a+) mRNA on polyribosomes. By 6 h after infection (late), poly(A+) HSV RNA transcribed from 35 to 40% of the viral DNA is found on polyribosomes. These viral poly(A+) transcripts comprised as much as 90% of newly synthesized poly(A+) mRNA and are measurably larger than viral poly(A+) transcripts isolated early. Some but not all of this size difference is due to the fact that the poly(A) tails on early transcripts are shorter than those found on transcripts made late. Even late after infection, a small but readily measurable amount of cellular poly(A+) RNA is still being made and entering polyribosome complexes. In the nucleus, late after infection, poly(A+) HSV RNA is complementary to 50% of the total HSV DNA. Both early and late after infection, total nuclear viral transcripts are, on the average, larger than viral transcripts found on polyribosomes; however, nuclear HSV poly(A+) RNA is not measureably larger than the corresponding cytoplasmic viral poly(A+) sequences at either time. A major portion (30 to 40%) of the polyribosomal HSV RNA made either early or late after infection is not polyadenylated. This HSV poly (A-) RNA is transcribed from the same sequences as HSV poly(A+) RNA but, when labeled and isolated either early or late after infection, both nuclear and polyribosomal viral poly(A-) RNA molecules sediment faster in sucrose-formaldehyde gradients than their polyadenylated counterparts.