No abstract

The nucleotide sequence of an EcoRI duck hepatitis B virus (DHBV) clone was elucidated by using the Maxam and Gilbert method. This sequence, which is 3,021 nucleotides long, was compared with the two previously analyzed hepatitis B-like viruses (human and woodchuck). From this comparison, it was shown that DHBV is derived from an ancestor common to the two others but has a slightly different genomic organization. There was no intergenic region between genes 5 and 8, which were fused into a single open reading frame in DHBV. Genes for the surface and core proteins were assigned to open reading frames 7 and 5/8. Amino acid comparisons showed some structural relationship between gene 6 product and avian reverse transcriptase, suggesting either evolution from a common ancestor or convergence to some particular structure to fulfill a specific function. This should be correlated with the synthesis of an RNA intermediate during DNA replication. This is also taken as an argument in favor of the hypothesis that gene 6 codes for the DNA polymerase that is found within the virion. DNA sequence comparison also showed that the two mammalian hepatitis B viruses are more homologous to each other than they are to DHBV, indicating that DHBV starts to evolve on its own earlier than the two other viruses, as do birds compared with mammals. From this it is proposed that the viruses evolved in a fashion parallel to the species they infect.

The complete nucleotide sequence of a woodchuck hepatitis virus genome cloned in Escherichia coli was determined by the method of Maxam and Gilbert. This sequence was found to be 3,308 nucleotides long. Potential ATG initiator triplets and nonsense codons were identified and used to locate regions with a substantial coding capacity. A striking similarity was observed between the organization of human hepatitis B virus and woodchuck hepatitis virus. Nucleotide sequences of these open regions in the woodchuck virus were compared with corresponding regions present in hepatitis B virus. This allowed the location of four viral genes on the L strand and indicated the absence of protein coded by the S strand. Evolution rates of the various parts of the genome as well as of the four different proteins coded by hepatitis B virus and woodchuck hepatitis virus were compared. These results indicated that: (i) the core protein has evolved slightly less rapidly than the other proteins; and (ii) when a region of DNA codes for two different proteins, there is less freedom for the DNA to evolve and, moreover, one of the proteins can evolve more rapidly than the other. A hairpin structure, very well conserved in the two genomes, was located in the only region devoid of coding function, suggesting the location of the origin of replication of the viral DNA.

We have cloned the X gene (HBx) and the HBc antigen (HBc Ag) gene of human hepatitis B virus (HBV) in Escherichia coli as fusion products with beta‐galactosidase. Both HBV genes are expressed in E. coli strain CSR 603. Expression is detected by u.v. irradiation of the bacteria, metabolic labelling and electrophoresis of the labelled extracts on SDS‐polyacrylamide gels. The HBc Ag protein produced in bacteria can be recognised by anti‐HBc sera and peptides derived from the protein are also recognised by anti‐HBe sera. The HBx protein is recognised by some, but not all, sera which are anti‐HBe positive. HBx Ag is also recognised by a woodchuck antibody similar to anti‐HBe (anti‐WHe). These results constitute the first proof that the open reading frame X is a true viral gene and is expressed during HBV (and WHV) infection and that an HBx/anti‐HBx system, which may have important biological implications, can exist in parallel with the classic HBe/anti‐HBe system.

The RNA14 and RNA15 gene products have been implicated in a variety of cellular processes. Mutations in these genes lead to faster decay of some mRNAs and yield extracts that are deficient in cleavage and polyadenylation in vitro. These results suggest that the RNA14 and RNA15 gene products may be involved in both adenylation and deadenylation in vivo. To explore the roles of these gene products in vivo, we examined the site of adenylation and the rate of deadenylation for individual mRNAs in rna14 and rna15 mutant strains. We observed that the rates of deadenylation are not affected by lesions in either the RNA14 or the RNA15 gene. This result suggests that the proteins encoded by these genes are not involved in regulation of the deadenylation rate. In contrast, we observed that the site of adenylation for the ACT1 transcript can be altered in these mutants. Interestingly, we also observed that mutation of the poly(A) polymerase gene altered the site of ACT1 polyadenylation. These observations suggest that the RNA14, RNA15, and PAP1 proteins are involved in poly(A) site choice. This alteration in poly(A) site choice in the rna14 mutant can be corrected by the ssm4 suppressor, indicating that this suppression acts at the level of polyadenylation and not by slowing mRNA degradation.Most eukaryotic mRNAs have a polyadenylate sequence at the 3Ј end which is added in a posttranscriptional process. This poly(A) tail is important for the proper stability of transcripts (for a review, see reference 9), for efficient initiation of translation (11,17,32), and for efficient nuclear-cytoplasmic transport (25, 34). In addition, the site at which the poly(A) tail is added can be regulated, thereby leading to the production of alternative gene products from a single transcriptional unit.Examination of the process of polyadenylation in mammalian cell extracts has led to substantial progress in understanding the mechanism by which the poly(A) tail is added (for reviews, see references 18, 38, and 40). Addition of a poly(A) tail is initiated by assembly of a complex of proteins on the sequences that specify a polyadenylation site. This complex cleaves the pre-mRNA and then adds a short poly(A) tail. The addition of a nuclear poly(A) binding protein, PABII, then alters the adenylation reaction to allow rapid elongation of the short poly(A) tail to its full length (3). In Saccharomyces cerevisiae, the use of an in vitro system has also shown that mRNA 3Ј end maturation is similar in that the precursor mRNA is also cleaved at a specific site to which the poly(A) tail is added (5). However, there are clearly differences in the sequences that specify a polyadenylation site in S. cerevisiae and mammals (for reviews, see references 18, 38, and 40).To understand the process of 3Ј end formation, the proteins involved in cleavage and polyadenylation need to be identified and their functions need to be determined. In mammalian cells, fractionation of extracts has identified several factors required for adenylation, in addition to the poly(...