Three segments from the monkey genome that hybridize to simian virus 40 have common structural elements.

Three cloned segments that hybridize to a region of simian virus 40 (SV40) deoxyribonucleic acid including the origin of replication have been isolated from a monkey genomic library. The primary structure of one segment was previously reported (T. McCutchan and M. Singer, Proc. Natl. Acad. Sci. U.S.A. 78:95-99, 1981). We report here the sequences of the other two segments and a comparison of all three. The SV 40-hybridizing region in each segment is limited to several hundred base pairs. All of the segments contain multiple and disconnected sequences homologous to the region of SV40 directly surrounding the viral replication origin. The number and arrangement of the homologous sequences is different in the three segments. However, the segments have the following features in common: (i) each contains multiple copies of the sequence GGGCGGPuPu, which also appears six times near the origin of SV40; (ii) each contains several strong homologies to the central dyad symmetry of SV40; (iii) each contains a long internal repeat, as does the origin region of SV40. The three SV40-hybridizing segments are members of a larger family of genomic sequences that hybridize well to each other, but not necessarily to SV40.     

Chromatin structure of simian virus 40-pBR322 recombinant plasmids in COS-1 cells.

To study the nucleoprotein structure formed by recombinant plasmid DNA in mammalian cells, nuclei were isolated from COS-1 cells after transfection with a recombinant (pJI1) containing pBR322 sequences and a segment of simian virus 40 containing information for a nuclease-sensitive chromatin structure. The nuclei were incubated with DNase I. DNA fragments which were the size of linear pJI1 DNA were isolated, redigested with restriction enzymes, fractionated by electrophoresis, and detected by hybridization with nick-translated segments prepared from the plasmid DNA. Two DNase I-sensitive sites were detected in the simian virus 40 portion of the plasmid at the same sites that were DNase I sensitive in simian virus 40 chromatin prepared late after infection of African green monkey kidney (BSC-1) cells. One site extended from the viral origin of replication to approximately nucleotide 40. The 21-base pair repeated sequences were relatively DNase I resistant. A second site occurred over the single copy of the 72-base pair segment present in this plasmid. These results indicate that the nuclease-sensitive chromatin structure does not depend on the presence of viral structural proteins. In addition, late viral proteins added to pJI1-transfected COS-1 cells by superinfection with simian virus 40 caused no change in the distribution of DNase I-sensitive sites in plasmid chromatin. Analysis of transfected plasmid DNA may provide a general method applicable to the study of the chromatin structure of cloned segments of DNA.     

Structure of simian virus 40 recombinants that contain both host and viral DNA sequences. II. The structure of variant 1103 and its comparison to variant CVPS/1P2 (EcoRI res).

In an effort to characterize sites of recombination between SV40 and monkey DNA, we have determined the primary sequence of a large portion of the SV40 variant, designated 1103. This virus contains DNA sequences derived both from the wild type SV40 genome and from the permissive monkey cell in which the virus was propagated. Further, the monkey sequences included in the defective genome are homologous to both highly repeated monkey DNA (alpha component) and sequences that are infrequently repeated in the monkey genome. The regions of the 1103 genome where DNA sequences were determined include 1) the segments of the variant that surround joints connecting SV40 and monkey sequences, 2) the segment that contains the joint between monkey sequences of high and low reiteration frequency, and 3) the DNA segment of the variant that is homologous to monkey alpha component DNA. Comparison of the data obtained from the sequences analysis of the SV40 variants 1103 and CVP8/1/P2 (EcoRI res) (described in Wakamiya, T., McCutchan, T., Rosenberg, M., and Singer, M. (1979) J. Biol. Chem 254, 3584-3591) reveals certain similarities between the two that may be involved in eukaryotic recombination and defective variant formation.     

Simian agent 12 is a BK virus-like papovavirus which replicates in monkey cells.

We have begun to characterize the genomic structure and replication of the baboon papovavirus simian agent 12 (SA12). We have defined a wild-type clone of SA12 (SA12 wt100) by plaque purification from a heterogeneous stock. The functional map of SA12 wt100 can be aligned with those of the other primate papovaviruses by assigning one of the two EcoRI sites as 0/1.0 map units. The origin of bidirectional viral DNA replication maps near 0.67 map units, consistent with the limits of sequences homologous to origin sequences in the other papovaviruses. DNA sequence analysis shows that the organization of the SA12 genome is similar to that of the other primate papovaviruses studied. The arrangement and sequence of functional elements in the origin of replication region, as well as the sequences of the N-terminal regions of early protein products, indicate that SA12 is most closely related to the human virus BK, next most closely related to JC virus, and less closely related to simian virus 40. Unlike BK virus, SA12 is capable of productive infection of African green monkey kidney cells.     

Topological requirements for homologous recombination among DNA molecules transfected into mammalian cells.

Cultured animal cells rearrange foreign DNA very efficiently by homologous recombination. The individual steps that constitute the mechanism(s) of homologous recombination in transfected DNA are as yet undefined. In this study, we examined the topological requirements by using the genome of simian virus 40 (SV40) as a probe. By assaying homologous recombination between defective SV40 genomes after transfection into CV1 monkey cells, we showed that linear molecules are preferred substrates for homologous exchanges, exchanges are distributed around the SV40 genome, and the frequency of exchange is not diminished significantly by the presence of short stretches of non-SV40 DNA at the ends. These observations are considered in relation to current models of homologous recombination in mammalian cells, and a new model is proposed. The function of somatic cell recombination is discussed.     

Rescue of chromosomal T-antigen sequences onto extrachromosomally replicating, defective simian virus 40 DNA by homologous recombination.

Recombination between chromosomal and extrachromosomal DNA sequences was analyzed by investigation of the recombinational rescue of a 1,018-base-pair (bp) segment of the T-antigen gene of simian virus 40 from the chromosome of monkey COS cells to two different, extrachromosomally replicating, simian virus 40 DNA molecules lacking this 1,018-bp sequence. The ratio of rescued to unrecombined virus was as high as 10(-3). The rescued molecules, detected optimally 5 to 9 days after transfection of COS cells, had completely recovered the 1,018-bp DNA segment from the chromosome. The recombination event is proposed to occur either by double reciprocal recombination or by gene conversion between the chromosomal T-antigen gene and the extrachromosomal molecules missing the 1,018-bp sequence.     

Structure of a newly isolated variant of Simian virus 40 DNA containing monkey DNA and its similarity to previously isolated variants

The structure of a newly and independently isolated defective variant of simian virus 40 that contains covalently linked monkey and SV40 DNA sequences is described. This variant, termed 290, has a structure essentially identical with a previously isolated and characterized variant named CVP8/1/P2 (Eco RI res). The structural similarities include the monkey (host) DNA segment that is combined with viral DNA sequences, the particular viral DNA segment that is present, and the arrangement of these within the defective genome. The monkey DNA segment contains sequences derived from both low and high reiteration frequency monkey DNA. The viral sequences include the origin of replication. The separate isolation of essentially identical variants suggests a high level of specificity in the events leading to the formation and amplification of this type of defective genome.     

Structure of a defective simian virus 40 genome bearing an operator from bacteriophage lambda.

We examined further the physical structure of the simian virus 40 (SV40) and bacteriophage lambda DNA sequences in an SV40-lambda hybrid that had been propagated in monkey kidney cells. The SV40 vector portion of the hybrid, which was a small fragment isolated from a reiteration mutant of SV40, contained the site for initiation of SV40 DNA replication. Electron microscope heteroduplex and restriction endonuclease analyses revealed a tandem duplication of the SV40 vector segment linked to a 2,300-base pair portion (lambda map units 71 to 76) of the lambda immunity region. The defective hybrid genome thus harbors two origins for SV40 DNA replication in addition to the leftward operator and the N gene of lambda.     

Propagation of a segment of bacteriophage lamda-DNA in monkey cells after covalent linkage to a defective simian virus 40 genome.

A 520 base pair DNA segment was excised from the bacteriophage lamda-genome by cleavage with the bacterial restriction endonuclease, endo R. Hindll. This segment was covalently joined in vitro to an 880 base pair simian virus 40 (SV40) DNA segment which contains the initation site for SV40 DNA replication. The latter segment was derived from the genome of a defective reiteration mutant of SV40 also by endo R. Hindlll cleavage. When the recombinant molecule, together with wild-type SV40 DNA as helper, was introduced into monkey cells by DNA infection, replication of the lamda-DNA sequences was observed, and hybrid genomes were encapsidated into progeny SV40 virions. The structure of the lamda-DNA segment after serial passage in monkey cells was examined by use of restriction endonucleases and electron microscopic heteroduplex analysis.     

Characterization of tau antigens isolated from uninfected and simian virus 40-infected monkey cells and papovavirus-transformed cells.

Tau antigens (also known as cellular or nonviral tumor antigens) were detected in uninfected and simian virus 40-infected monkey cells after immunoprecipitation with serum from hamsters bearing simian virus 40-induced tumours (anti-T serum). These two proteins (56,000 daltons) were digested to similarly sized peptides with various amounts of Staphylococcus aureus V8 protease. The Tau antigen isolated from infected monkey cells was closely related but was not identical to the corresponding protein from human cells transformed by simian virus 40, as determined by two-dimensional mapping of their methionine-labeled tryptic peptides. Hamster cells transformed by various primate papovaviruses (simian virus 40, BK virus, and JC virus) synthesized indistinguishable Tau antigens, as determined by two-dimensional peptide mapping. When tested by the same procedure, these proteins and the ones made in monkey and human cells were found to be related to the Tau antigens isolated from simian virus 40-transformed mouse and rat cells. Based on these results, an "evolutionary tree" was constructed to show the relationship among the methionine-containing tryptic peptides of all of these proteins.     

The minimum amount of homology required for homologous recombination in mammalian cells.

Although DNA sequence homology is believed to be a prerequisite for homologous recombination events in procaryotes and eucaryotes, no systematic study has been done on the minimum amount of homology required for homologous recombination in mammalian cells. We have used simian virus 40-pBR322 hybrid plasmids constructed in vitro as substrates to quantitate intramolecular homologous recombination in cultured monkey cells. Excision of wild-type simian virus 40 DNA by homologous recombination was scored by the viral plaque assay. Using a series of plasmids containing 0 to 243 base pairs of homology, we have shown that the recombination frequency decreases as the homology is reduced, with the sharpest drop in recombination frequency occurring when the homology was reduced from 214 to 163 base pairs. However, low recombination frequencies were also observed with as little as 14 base pairs of homology.     

Mutagenesis in UV-irradiated simian virus 40 occurs predominantly at pyrimidine doublets

Phenotypic wild-type revertants from a UV-irradiated temperature-sensitive late mutant (ts BC245) of simian virus 40 (SV40) were isolated after replication in monkey cells at the nonpermissive temperature. The mutations occurring in 7 revertants were identified by DNA sequence analysis of the entire gene involved. All 10 mutations identified constituted single base substitutions, 7 of which occurred opposite pyrimidine doublets. Transitions were 3 times more abundant than transversions. Three base changes did not occur opposite pyrimidine-pyrimidine sequences. Exchange of a DNA fragment harbouring the altered base from a revertant with the corresponding fragment from the parental virus, showed that the base substitution was indeed responsible for the reversions to the wild-type phenotype (growth at the restrictive temperature). The data suggest that most base substitutions in highly UV-irradiated simian virus 40 are targeted at sites comprising two adjacent pyrimidines.     

Genomic arrangement of an adenovirus-simian virus 40 hybrid virus, Ad2+ND4del.

Ad2+ND4del is an adenovirus type 2-simian virus 40 hybrid virus nondefective for growth in human cells. The virus was first observed when stocks of Ad2+ND4, a hybrid isolated from primary monkey kidney cells, were propagated in human cells. This paper describes the DNA sequence at two sites of DNA recombination, the site of the left adenovirus type 2-simian virus 40 junction and the site of a deletion of internal simian virus 40 sequences. Since the deletion was observed when the virus was switched from monkey to human cells, an analysis of gene expression in the region of DNA rearrangement may prove useful for the elucidation of molecular events that accompany virus growth in different hosts.     

Measurements of the molecular size of the simian virus 40 large T antigen.

A measure of the molecular weight of the large simian virus 40 T antigen was sought by SDS-polyacrylamide gel electrophoresis, random-coil chromatography, and sedimentation-velocity analysis in a density gradient. Large T antigen obtained from a simian virus 40-transformed human cell line either by immunoprecipitation or by standard preparatory methods migrated like a 94,000-molecular-weight (approximately 94K) polypeptide in SDS-gels but was found to have an approximate was observed with T antigen obtained from lytically infected monkey cells. In view of the strong theoretical basis for the guanidine method and the agreement with the sedimentation data, these findings suggest that the molecular weight of this protein is approximately 75 to 80K as opposed to 94 to 100K and, therefore, that considerably less than the entire early region of simian virus 40 is required to encode it. This size estimate is in keeping with earlier results which revealed a normal-size T antigen in cells infected with viable deletion mutants lacking as much as 10% of the early region.