Supercoiled DNA molecules were used for the molecular cloning of full-length avian sarcoma virus (ASV) DNA. Viral DNA produced by the Schmidt-Ruppin A (SR-A) strain of ASV was isolated from acutely infected transformed quail cells. Supercoiled DNA was separated from linear and open circular DNA by acid phenol extraction, opened into a full-length linear form by cleavage with the restriction endonuclease Sacd, and cloned into XgtWES * AB. Four different cloned viral DNA molecules were isolated: SRA-1 contains two copies of the 330-base pair terminal redundancy normally found at each end of the linear DNA molecules, but harbors a 63-base pair deletion that spans the site at which the two copies of the terminal redundancy are joined in circular DNA molecules; SRA-2 contains two complete copies of the terminal redundancy; SRA-3 probably contains only one copy of the terminal redundancy but in all other respects appears to be similar to SRA-2; SRA-4 contains a 2,500-base pair deletion that removes all of the src gene (the gene responsible for transformation by ASVs) plus additional nucleotides adjacent to the src gene whose precise locations have not been determined. Transfection of chicken embryo fibroblasts by either SRA-1 or SRA-2 resulted both in the appearance of transformed cells and in the production of infectious virus. These results demonstrate that the cloned DNA molecules are functionally identical to viral DNA produced in vivo; therefore, molecular cloning did not cause any major alterations of the DNA. The infectivity of SRA-1 DNA indicates that the 63 base pairs missing from that molecule are not required for the initiation of viral RNA synthesis, even though the deletion is located in a copy of the terminal redundancy thought to carry a promoter for RNA synthesis. This suggests that the deletion does not remove any sequences required for the initiation of transcription.

We have determined the nucleotide sequence of portions oftwo circular avian sarcoma virus (ASV) DNA molecules cloned in a prokaryotic host-vector system. The region whose sequence was determined represents the circle junction site-i.e., the site at which the ends ofthe unintegrated linear DNA are fused to form circular DNA. The sequence from one cloned molecule, SRA-2, shows that the circle junction site is the center of a 330-base-pair (bp) tandem direct repeat, presumably representing the fusion ofthe long terminal repeat (LTR) units known to be present at the ends of the linear DNA. The circle junction site is also the center ofa 15-bp imperfect inverted repeat, which thus appears at the boundaries of the LTR. The structure of ASV DNA-unique coding region flanked by a direct repeat that is, in turn, terminated with a short inverted repeat-is very similar to the structure ofcertain transposable elements. Several features ofthe sequence imply that circularization to form the SRA-2 molecule occurred without loss of information from the linear DNA precursor. Circularization of another cloned viral DNA molecule, SRA-1, probably occurred by a different mechanism. The circle junction site of the SRA-1 molecule has a 63-bp deletion, which may have arisen by a mechanism that is analogous to the integration of viral DNA into the host genome. Flanking one side of the tandem direct repeat is the binding site for tRNAT"P, the previously described primer for synthesis of the first strand of viral DNA. The other side of the direct repeat is flanked by a polypurine tract, A-G-G-G-A-G-G-G-G-G-A, which may represent the position of the primer for synthesis of the second strand of viral DNA. An A+T-rich region, upstream from the RNA capping site, and the sequence A-A-T-A-A-A are present within the direct repeat sequence. These sequences may serve as a promoter site and poly(A) addition signal, respectively, as proposed for other eukaryotic transcription units.Three major types of virus-specific DNA have been identified in cells after infection with retroviruses: linear duplex (form III) DNA, the initial product of synthesis by RNA-directed DNA polymerase; covalently closed circular (form I) DNA, derived from linear precursors; and DNA covalently integrated into the host genome (proviral DNA) (1, 2).Physical maps of the unintegrated and integrated forms of avian sarcoma virus (ASV) DNA have helped to clarify the structural relationships among these forms (cf. Fig. 1). Form III and proviral DNA are coextensive with a subunit of the RNA genome (10-13). These forms also contain a long terminal repeat (LTR) ofabout 300 base pairs (bp) that is not present in the RNA genome (10-13). The repeated domain is composed of sequences unique to each end of the RNA (U5 and U3) joined by a short sequence, R, that is present as a terminal repeat in the RNA (7). Two principal classes ofcircular DNA have been characterized (10, 11). One class bears a single copy of the LTR sequence and presumably arises by recombination between the LTRs at...

SUMMARYWe have used cloned fragments of Marek's disease virus (MDV) DNA and in situ hybridization to search for virus DNA and study its expression in infected chick embryo fibroblasts (CEF), lymphoblastoid cell lines, tumours and neural lesions.DNA from the HPRS 16/att strain of MDV was cleaved with EcoRI endonuclease and several fragments were cloned in Escherichia coli using the vector PBR322.Seven fragments ranging in size from 2.6 to 11 kbp representing approx. 25 % of the MDV genome were labelled in vitro and annealed to EcoRI digests of DNA from infected cells and tumours following separation and transfer according to the Southern blotting procedure. Most of the selected MDV DNA fragments hybridized to fragments of corresponding sizes in EcoRI digests of DNA from cell lines and tumours and failed to hybridize to digests of uninfected chick cell DNA. In situ hybridization using 3H-labelled DNA with specific activity of 108 d/min//dg as probe showed intranuclear MDV DNA in infected CEF, in every celt of two lymphoblastoid cell lines and in the majority of infiltrating or proliferating lymphoid cells found in type 'A' lesions of grossly enlarged peripheral nerves. Both intranuclear and cytoplasmic RNA were detected in cells that contained virus DNA. However, comparatively little virus RNA appears to be transcribed in cell lines and in infected tissues from the regions of virus DNA (25 % of genome) used as probe in this study. Our results favour the hypothesis that the accumulation of lymphoid cells in nerves is not the result of an inflammatory response to infected nerve cells but is rather the consequence of proliferating transformed cells.

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The genomes of numerous avian retroviruses contain at their 3' termini a conserved domain denoted "c". The precise boundaries and function of "c" have been enigmas. In an effort to resolve these issues, we determined the sequence of over 900 nucleotides at the 3' end of the genome of the Schmidt-Ruppin subgroup A strain of avian sarcoma virus (ASV). We obtained the sequence from a suitable fragment of ASV DNA that had cloned into the single-stranded DNA phage M13mp2. Computer-assisted analysis of the sequence revealed the following structural features: i) the length of "c" - 473 nucleotides; ii) the 3' terminal domain of src, ending in an amber codon at the 5'boundary of "c"; iii) terminator codons that preclude continuous translation from "c"; iv) suitably located sequences that may serve as signals for the initiation of viral RNA synthesis and for the processing and/or polyadenylation of viral mRNA; v) a repeated sequence that flanks src and that could facilitate deletion of this gene; vi) repeated sequences within "c"; and vii) unexplained homologies between sequences in "c" and sequences in several other nucleic acids, including the 5' terminal domain of the ASV genome, tRNATrp and its inversion, the complement of tRNATrp and its inversion, and the 18S RNA of eukaryotic ribosomes. We conclude that "c" probably does not encode a protein, but its sequence may nevertheless serve several essential functions in viral replication.