Physical mapping of adeno-associated virus serotype 4 DNA

Abstract

The organization of adeno-associated virus serotype 4 (AAV 4) DNA was probed by using restriction enzymes. The cleavage sites of the following enzymes were mapped: BglII, BamI, HincIl, KpnI, PstI, SaLI, SstI, and XhoI. The orientation of transcription on the physical cleavage map was deternined by locating the fragments which contain the 3' end of the mRNA. Strand separation gels were run for HinII fragments of AAV 4 DNA. By hybridizing AAV 4 mRNA to the resolved strands, the polarity of the DNA strands was identified. Restriction digestion of AAV 4 DNA sometimes produced terminal fragments which migrated in agarose gels as doublets. However, when AAV 4 DNA was prepared devoid of any single-stranded nicks, these terminal doublet bands were not observed upon subsequent restriction analysis. During these studies, the molecular weights of both AAV 4 and AAV 2 duplex DNA were measured and were found to be somewhat larger than previously reported (3.18 x 106 and 3.10 x 10', respectively). Adeno-associated virus type 4 (AAV 4) is a defective simian parvovirus requiring an adenovirus helper for a productive infection (1, 25). Although virions contain a single strand of DNA of about 1.6 x 106 molecular weight, both plus and minus strands are encapsidated separately and in approximately equal amounts (4, 7, 25, 31). Thus, isolation of DNA from AAV 4 virions can result in full-length duplex DNA molecules under proper annealing conditions. This duplex DNA is suitable for analysis by restriction endonucleases. In this report, we describe a physical map of AAV 4 duplex DNA which has been constructed by using the restriction enzymes BamHI, Sall, HincII, BgIII, PstI, SstI, KpnI, and XhoI. Cleavage of AAV 4 DNA by a majority of these enzymes produces only one terminal fragment for each end of the linear molecule analyzed by gel electrophoresis of the digests on 1.4% agarose. However, DNA which has previously sustained single-stranded nicks produces doublet bands for a terminal fragment when digested by KpnI or SstI, and sometimes by HincII, Sail, or BamHI. Doublet bands are not observed for terminal fragments larger than about 1,500 base pairs under these conditions, and never for internal fragments. When unnicked AAV 4 DNA is digested by KpnI or SstI

Cloning of adeno-associated virus type 4 (AAV4) and generation of recombinant AAV4 particles

“…DNA hybridization studies have indicated a similar level of homology for AAV1 through AAV4 (4,50). However, mRNA hybridization studies suggest that AAV4 may be organized differently from AAV2, since only one size class of mRNA has been fractionated from infectious cells (46). Therefore, to understand the nature of AAV4 and to determine its usefulness as a vector for gene transfer, the viral genome was cloned and sequenced and recombinant virus was produced with the AAV4 Rep and Cap proteins.…”

Mechanism of Action of the Endonuclease Associated with the αβ and ββ Forms of Avian RNA Tumor Virus Reverse Transcriptase

“…T4 DNA ligase and Escherichia coli DNA exonuclease III were from P-L Biochemicals, Inc., Milwaukee, Wis. DNA polymerase I of E. coli was fraction VII prepared by the method of Jovin et al (11). T4 polynucleotide kinase was prepared as described by Muster et al (19).…”