Biochemical studies on bovine adenovirus type 3. III. Cleavage maps of viral DNA by restriction endoncleases EcoRI, BamHI, and HindIII

Abstract

Cleavage of bovine adenovirus type 3 (BAV3) DNA by restriction endonucleases EcoRI, BamHI, and HindIII yielded 7 (A to G), 5 (A to E), and 12 (A to L) fragments, respectively. The order of these fragments has been determined to be GDACBFE for EcoRI fragments, AEBDC for BamHI fragments, and JEBKAC-DHFGIL for HindIlI fragments, and cleavage sites of these enzymes have been mapped on the genome of BAV3. BAV3 preparation contains incomplete virus whose genome has a deletion of about 13% of complete virus genome. Restriction endonuclease digestion of the incomplete virus DNA revealed that EcoRI E and F, BamHI C and HindIll G, I, and L fragments were deleted. Therefore, the deleted region of incomplete virus DNA is located near the right-hand end of the BAV3 DNA molecule, a result consistent with our previous electron-microscopic observations on heteroduplex molecules formed between complete and incomplete BAV3 DNA.

Biochemical Studies on Bovine Adenovirus Type 3 IV. Transformation by Viral DNA and DNA Fragments

“…In the preceding paper (16), we reported that the DNA of bovine adenovirus type 3 (BAV3), one of the DNA tumor viruses, is cleaved into 7, 5, and 12 fragments with restriction endonucleases EcoRI, BamHI, and HindIII, respectively, and the order of these fragments on the viral genome was determined. The transformation with BAV3 DNA fragments suggests the possibility of isolating the specific DNA fragment involved in transformation and of determining the location of the transforming gene(s) on the BAV3 genome.…”

“…DNA fragments. Specific fragments of viral DNA were prepared with restriction endonucleases (16). Restriction endonuclease EcoRI was isolated as described by Greene et al (13), and endonuclease HindIII was kindly provided by K. Fujinaga and K. Sekikawa.…”

“…Restriction endonuclease EcoRI was isolated as described by Greene et al (13), and endonuclease HindIII was kindly provided by K. Fujinaga and K. Sekikawa. The digestion with these enzymes was performed as described previously (16). DNA fragments were purified by electrophoresis in cylindrical gels of 0.9 or 1.2% agarose (Seakem) (20).…”

“…The DNA bands were monitored by staining the gels with ethidium bromide (0.5 ,tg/ml) in gels and electrophoresis buffer during electrophoresis and illuminating with a UV lamp. The procedure for extraction of DNA from agarose gel was described elsewhere (16).…”

“…Joining of DNA fragments. HindIII E and J fragments of BAV3 DNA were prepared from the DNA of BAV3-0 incomplete virus (14), because this incomplet virus DNA lacks HindIII G, I, and L fragments (16), allowing the isolation of HindIII J free from any contamination with I fragment. HindIII E and J fragments were joined by ligase reaction.…”