Cloning in Escherichia coli and physical structure of hepatitis B virion DNA.

Abstract

A restriction map of hepatitis B virion DNA was established after cloning of the whole viral genome in Escherichia coli. By use of EcoRI, Xho I, Bgl II, Xba I, BamHI, HincII, and Hae III endonucleases, a total of 28 restriction sites were mapped. The single-stranded region was localized on the restriction map and the 5' end of the short strand was mapped at a fixed position. Hepatitis B is a frequent and sometimes severe disease. In some areas of Asia and tropical Africa, 10% of the population carry the viral surface antigen (1). The Dane particle (2), found in the sera of patients, is considered to be the etiological agent (3). It contains a circular DNA molecule, hepatitis B virion (HBV) DNA, with a nick (or a short gap) and a single-stranded region (4-6). HBV DNA is the smallest known mammalian viral genome. These unique characteristics of the genome, the frequency of hepatitis B, and the likely relationship between this virus and primary liver carcinoma (7,8) justify an accurate investigation of the structure of the HBV genome.In this article we present a physical map of the HBV genome. Twenty eight restriction sites are located, and new information concerning the physical structure of HBV DNA is also established. In particular, the single-stranded region is localized on the restriction map. The position of the nick is discussed. MATERIALS AND METHODSDNA Preparations and Cloning. HBV were purified as described by Summers et al. (4) and their DNA was labeled with the endogenous DNA polymerase (6), with [32P]dTTP (22 Ci/mmol, 1 Ci = 3.7 X 1010 becquerels) as the radioactive precursor. After purification of the HBV DNA (4), it appeared from electron microscope observation that, under the action of the endogenous DNA polymerase, the single-stranded region of about 10% of the DNA molecules was totally repaired.The purified HBV DNA was used for cloning. The circular HBV genome was cleaved by EcoRI endonuclease at its unique restriction site (J. Summers, personal communication) and inserted in vitro into the DNA of bacteriophage Xgt WES. XB as described (9). After transfection of C600 recBC bacteria with the hybrid DNA, recombinant phage clones were purified three times. For each recombinant clone a first phage stock was made and, in order to avoid any genetic drift, all subsequent stocks were made from this initial sample. The cloned EcoRI DNA fragment will be referred to as Eco HBV DNA.After digestion of the DNA of a recombinant clone with EcoRI endonuclease, Eco HBV DNA was purified from the two vector arms by zone centrifugation in a 5-30% sucrose gradient.Enzymatic Digestions and Chemicals. Digestions by the following restriction nucleases (New England BioLabs), EcoRI, BamHI, Sma I,Xho I, Pst I, Xba I, Bgl II, Sst I, Sal I, Kpn I, HincII, and Hae III, were performed in the buffers recommended by New England BioLabs. Alkaline phosphatase (P-L Biochemicals) was used in 10 mM Tris-HCI, pH 8.5/10 mM MgCl2. The 5' ends were labeled by means of [y-32P]ATP (New England Nuclear, 3000 Ci/mmol) and polynucleoti...

Multiple chemical forms of hepatitis B surface antigen produced in yeast.

“…Purified, formaldehyde-treated antigen, isolated from human plasma, is an effective vaccine for the prevention of hepatitis B infection (7). Recently, the HBsAg gene has been cloned in yeast (8)(9)(10)(11) and a vaccine has been made that is safe and immunogenic in humans (12,13) and is effective in preventing hepatitis B infection in chimpanzees (14).…”

Plasmid-directed synthesis of hepatitis B surface antigen in monkey cells.

“…This ignorance is largely a consequence of the inability of HBV to propagate in any tissue culture system or to infect convenient experimental animals (for reviews, see references 47 and 55). Recently, the 3,200-base pair (bp) viral genome has been molecularly cloned from infectious virus isolated from serum (8,50), and the determination of the complete nucleotide sequence of the cloned DNA has provided definitive evidence that at least two viral antigens are encoded by the genome of the virus (7,15,42,57). These include the core antigen, a basic polypeptide of 19,000 daltons which represents the major viral nucleocapsid protein, and the surface antigen (HBsAg).…”

Detection of Hepatitis B Virus DNA in Pancreas, Kidney and Skin of Two Human Carriers of the Virus

“…DNA was then transferred to a nitrocellulose filter by a modification of the Southern technique described by Wahl et al (1979). The filter was hybridized with 3'P-labelled, nick-translated cloned HBV DNA (Charnay et al, 1979); the specific activity was > 2 x 108 ct/min/~g. After extensive washing under stringent conditions (0.1 x SSC, 50 °C), the filter was autoradiographed using Kodak XAR-5 film with a DuPont Lightning Plus screen for 1 to 3 days at -70 °C.…”

“…It was therefore of interest to investigate the possibility of HBV infection in non-hepatic tissues. For this purpose we have used the Southern blot technique (Southern, 1975;Wahl et al, 1979) with cloned viral DNA (Charnay et al, 1979) as a probe to detect HBV sequences in the DNA of various organs.Tissue samples were obtained at autopsy from two patients. Patient A was an 80 year old woman who died of cirrhosis with HCC.…”