Host DNA Sequences Research Articles

Early events in the replication of Mu prophage DNA

To determine whether the early replication of Mu prophage DNA proceeds beyond the termini of the prophage into hose DNA, the amounts of both Mu DNA and the prophage-adjacent host DNA sequences were measured using a DNA-DNA annealing assay after induction of the Mu vegetative cycle. Whereas Mu-specific DNA synthesis began 6 to 8 min after induction, no amplification of the adjacent DNA sequences was observed. These data suggest that early Mu-induced DNA synthesis is constrained within the boundaries of the Mu prophage. Since prophage Mu DNA does not undergo a prophage lambda-like excision from its original site after induction (E. Ljungquist and A. I. Bukhari, Proc. Natl. Acad. Sci. U.S.A. 74:3143--3147, 1977), we propose the existence of a control mechanism which excludes prophage-adjacent sequences from the initial mu prophage replication. The frequencies of the Mu prophage-adjacent DNA sequences, relative to other Escherichia coli genes, were not observed to change after the onset of Mu-specific DNA replication. This suggests that these regions remain associated with the host chromosome and continue to be replicated by the chromosomal replication fork. Therefore, we conclude that both the Mu prophage and adjacent host sequences are maintained in the host chromosome, rather than on an extrachromosomal form containing Mu and host DNA.

The structure of a cloned substituted SV40 genome

The structure of the DNA of a cloned host-substituted SV40 variant was investigated by restriction endonuclease cleavage mapping and hybridization of the cleavage products to monkey and wild-type SV40 DNAs. The circular DNA of this variant was shown to consist of four tandemly repeated segments, each of which consists of covalently linked viral and monkey DNA sequences. Only two of the repeated subunits are of equal size. A detailed cleavage map of the variant genome is presented. The viral sequences in each of the four subunits are derived from within a portion of the wild-type SV40 genome (0.59–0.73 map units), which contains the origin of replication. Monkey DNA sequences of the reiterated type are linked to monkey DNA sequences of the less-reiterated or nonreiterated type in three of the subunits. The monkey DNA sequences in the fourth subunit are predominantly of the reiterated type. Hybridization experiments to clarify the relationship between the host DNA sequences in the cloned variant and those in other independently derived substituted SV40 populations indicate that some populations share some of the same reiterated and nonreiterated host DNA sequences, whereas other populations share only the reiterated host DNA sequences.

Polyoma virus defective DNAs: I. Physical maps of a related set of defective molecules (D76, D91, D92)

Three related polyoma virus species, designated D92 (92% the size of full-length polyoma virus DNA), D91 (91%) and D76 (76%) have been analysed and their structures compared with that of polyoma virus A2 DNA. Three independent methods (restriction endonuclease cleavage, depurination fingerprinting and DNA-DNA hybridization) were used in the analysis. The defective DNAs appear to be: (1) entirely composed of viral sequences (no host DNA sequences were detected): (2) made up in part of long continuous sequences of DNA which appear identical to sequences of A2 DNA (D92 contains continuous sequences from 1 to 72 map units on the physical map of A2 DNA; that is, it contains the entire late region and part of the early region of the viral DNA. D91 and D76 contain those same sequences except for a 1% deletion around 18 map units): (3) made up in part of rearranged viral sequences. Several interesting features were noted about the rearranged sequences present in the defective DNAs. Sequences from the region around 67 map units were found linked to other (non-contiguous) regions of the DNA. Sequences from about 72 map units were linked to sequences from about 1 map unit. Multiple copies of sequences from 67 to 72 map units (from around the origin of DNA replication) were found (4 copies in D91 and D92, and 2 copies in D76).

Polyoma virus defective DNAs: II. Physical map of a molecule with rearranged and reiterated sequences (D74)

The structure of the polyoma virus defective species D74 (74% the size of full-length polyoma virus DNA) has been determined and compared with that of polyoma virus A2 DNA. D74 appears to be composed entirely of viral DNA sequences. (No host DNA sequences have been detected.) It is made up of three DNA segments, each about 24, 24 and 27% in size. The two 24% segments appear to be identical and the 27% segment contains one copy of all the sequences found in the 24% fragments as well as a duplication of some of the sequences. When related to the physical map of A2 DNA, each segment is found to be composed of viral sequences from 1 to about 19 map units, 67 to 69 map units and 70 to 72 map units. Three features found in other polyoma virus defective species (Lund et al., 1977) are also present in D74. (1) Sequences from the region around 67 map units are linked to other (non-contiguous) viral sequences. (2) Sequences at about 72 map units are linked to sequences at 1 map unit. (3) Multiple copies of sequences from around the origin of viral DNA replication are present. From studies on other polyoma defective molecules (Griffin & Fried, 1975; Lund et al., 1977), the origin of DNA replication for polyoma virus has been defined to lie within the sequences from 67 to 72 map units. Since D74 replicates efficiently but lacks the sequences between 69 to 70 map units, the origin of DNA replication appears to be further defined as lying within 67 and 69 map units and/or 70 to 72 map units.

Variations in integration site of avian oncornaviruses in different hosts

We examined the integration site of avian oncornaviruses in the genome of different hosts with respect to the repetitive frequency of the cellular DNA sequences adjacent to the integrated proviral DNA. The following systems were studied: avian sarcoma virus (B-77) and avian leukosis virus (Rous-associated virus-61) in cultured duck embryonic cells and B-77 in cultured mouse 3T3 cells. These systems represent different host responses to viral infection, i.e., one in which both cellular transformation and viral replication occur (B-77-infected duck cells), one in which viral replication, but not transformation, occurs (Rous-associated virus-61-infected duck cells), and one in which transformation, but not viral replication, occurs (B-77-infected 3T3 cells). Two sequential hybridizations were used. First, large denatured DNA fragments (2.8 X 10(6) daltons) were reassociated to different C0t (mole-seconds per liter) values. Next, DNA remaining single stranded at different C0t values was isolated by hydroxylapatite column chromatography, immobilized on nitrocellulose filters, and hybridized with an excess of 3H-labeled 35S viral RNA to titrate the concentration of proviral DNA. Results show that B-77 sarcoma virus and Rous-associated virus-61 integrate in the unique region of duck DNA, whereas B-77 proviral DNA is associated with both repeated and unique host DNA sequences in transformed mouse 3T3 cells.

Size and distribution of SV 40 DNA sequences covalently linked with the DNA of permissive mammalian cells

CV-1 cells productively infected with SV 40 contain viral DNA which is covalently linked with the host cell DNA. These linear duplex viral-host DNA molecules are replicated during the infectious cycle. They can be selectively isolated and purified by two successive cycles of DNA-DNA hybridization and elution steps using first CV-1 cell and then SV 40-DNA immobilized on filters. In an attempt to clarify the nature of the host DNA sequences neighbouring the viral DNA it was found that reiterated host DNA must be within the range of 800 bases from the viral sequences. Reassociation kinetics and treatment of the reassociated viral-host DNA sequences with single strand-specific S1 nuclease have shown that unique host DNA sequences are always present in close neighbourhood of the viral DNA. Most of the SV 40 DNA sequences are probably intergrated as fragments of subgenomic length.

The host DNA sequences in different populations of serially passaged SV40

The closed-circular SV40 DNA which evolved during 4 independent sets of high-multiplicity serial passages, initiated from plaque-purified virus, was studied by reassociation kinetics. The results indicate that the host DNA sequences covalently linked to viral sequences in such serially passaged SV40 DNA, (1) are predominantly of the nonreiterated type, (2) are similar in populations which evolved during a given set of serial passages, (3) can be different in populations which arose during different sets of serial passages starting from the same plaque isolate, and (4) do not represent a random selection of the total sequences present in the host cell genome. These results are discussed in terms of the viral recombination sites on the host genome and the consequent evolution of virus DNA molecules containing host DNA sequences.

Preferential integration of simian virus 40 deoxyribonucleic acid into a particular size class of CV-1 cell deoxyribonucleic acid

Permissive CV-1 cells were productively infected with low multiplicities (0.5 PFU/ cell) of plaque-purified Simian virus 40 (SV40). The DNA was radioactively labeled throughout the period of infection (48 hr) and then separated into a supernatant and pellet fraction by a selective extraction method. Covalent linkage of SV40 DNA sequences with host DNA sequences was shown to occur preferentially in newly replicated, small linear, double-stranded DNA molecules that can be isolated from the supernatant. These molecules have an average length of 1.5–2 times the contour length of SV40 DNA. Covalent linkage of SV40 DNA sequences with larger host DNA sequences which can be pelleted during selective extraction has also been demonstrated to occur, although to a much lesser extent. The viral-host DNA sequences which can be purified by the technique described in this paper in sizable amounts are discussed as a possible means for the study of the properties of the site of integration of tumor virus genes in mammalian DNA.

The evolution of new species of viral DNA during serial passage of simian virus 40 at high multiplicity

Viral DNA isolated during serial undiluted passage of SV40 was analysed by electrophoresis of fragments produced by H. influenzae restriction endonuclease and by DNA-DNA hybridization. Compared to parental SV40 DNA, early passage DNA showed alterations at many sites in the population of DNA molecules, although certain sites were more frequently altered than others as evidenced by the reduced yield of some original DNA fragments and the appearance of new fragments. With continued passage, new dominant species of viral DNA evolved which yielded simple restriction endonuclease digest patterns. DNA molecules from all passages retained SV40 DNA sequences; however, the percentage of total DNA sequences which corresponded to SV40 sequences diminished to about 30% or less in late passage DNA. Repetitive host cell DNA sequences were detected in a high percentage of late passage DNA molecules, but accounted for 20% or less of total sequences present. Therefore the new species of viral DNA which have evolved probably contain predominantly nonrepetitive host DNA sequences.