A recombinant plasmid for expression of rat DNA polymerase beta was constructed in a plasmid/phage chimeric vector, pUC118, by an oligonucleotide-directed mutagenesis technique. The insert contained a 1005 bp coding sequence for the whole rat DNA polymerase beta. The recombinant plasmid was designed to use the regulatory sequence of Escherichia coli lac operon and the initiation ATG codon for beta-galactosidase as those for DNA polymerase beta. The recombinant clone, JMp beta 5, obtained by transfection of E. coli JM109 with the plasmid, produced high levels of DNA polymerase activity and a 40-kDa polypeptide that were not detected in JM109 cell extract. Inducing this recombinant E. coli with isopropyl beta-thiogalactopyranoside (IPTG) yielded amounts of 40-kDa polypeptide as high as 19.3% of total protein. Another recombinant clone, JMp beta 2-1, which was constructed by an oligonucleotide-directed mutagenesis to use the second ATG codon for the initiation codon, thus deleting the first 17 amino acid residues from the amino terminus, produced neither high DNA polymerase activity nor the 40-kDa polypeptide. The evidence suggests that this amino-terminal structure is important for stability of this enzyme in E. coli. The DNA polymerase was purified to homogeneity from the IPTG-induced JMp beta 5 cells by fewer steps than the procedure for purification of DNA polymerase beta from animal cells. The properties of this enzyme in activity, chromatographic behavior, size, antigenicity, and also lack of associated nuclease activity were indistinguishable from those of DNA polymerase beta purified from rat cells, indicating the identity of the overproduced DNA polymerase in the JMp beta 5 and the rat DNA polymerase beta.

The 3' noncoding region (3' NCR) of the hepatitis C virus (HCV) genome contained in viral particles was analyzed by an RNA linker ligation followed by reverse transcription-polymerase chain reaction. Sequence analysis of the amplified fragment from four strains, including different genotypes 1b, 2b, 3a, and 3b indicated that the 3' NCR is composed of between 200 and 235 nts. The sequence of the 3' NCR consists of a type-specific region (immediately following the termination codon), a poly(U) stretch, a C(U)n-repeat, and highly conserved region termed the core element. The poly(U) stretch and C(U)n-repeat regions varied in length and in sequence among different genotypes. Core elements having putative secondary structure consisted of 98 or 100 nts and were highly conserved in all genotypes. Most of the nt changes found in different genotypes did not affect the secondary structure of the core elements, suggesting that this region may play an important role in replication, stabilization of the HCV RNA, and/or packaging of the genome. Most of the HCV-1b strains carried two U residues at the 3' end of the core element, while the minor HCV-1b strains had no U residues, demonstrating that there are two variants in type 1b strains. Amplification of the core element using linker-primed cDNA was comparable with that using the 3' proximal core element-primed cDNA, indicating that the 3' end of HCV genome was terminated by an OH group.

Hepatitis C virus (HCV) type K3a (type 3a), which represents a minor genotype in Europe, the U.S.A. and Asia, appears to be significantly distributed throughout Australia and Brazil. We amplified the HCV-K3a/650 genome by reverse transcription polymerase chain reaction in ten overlapping fragments and determined the nucleotide sequences. The total sequence was 9454 bases in length and contained an open reading frame of 3021 amino acids, which is 10 or 11 amino acids longer than in HCV type 1 and 12 amino acids shorter than the sequence of type 2. These differences were due to the different lengths of both the putative envelope protein E2 and the NS5A regions, whose nucleotide lengths differ between types 1 and 2 also. Phylogenetic analysis of the putative core region and a portion of NS5B encoding the Gly-Asp-Asp motif indicated that HCVK3a closely matched the corresponding type 3a group. The deletion and addition of amino acids in both E2 and NS5A may be associated with their pathobiological features.

The sequence Gly-Asp-Met-Asp, spanning positions 189-192 of rat DNA polymerase beta, is similar to the sequence motif Gly-Asp-Thr-Asp that is highly conserved in a number of replicative DNA polymerases from eukaryotic cells, viruses, and phages. The role of this sequence in the catalytic function of rat DNA polymerase beta was investigated by individually changing each amino acid in this region by site-directed mutagenesis. The mutant enzymes DE190 and DE192, in which aspartic acid residues at positions 190 and 192, respectively, were replaced by glutamic acid, showed about 0.1% activity of the wild-type enzyme. On the other hand, the replacement of Gly-189 by alanine or Met-191 by isoleucine or threonine only slightly affected the enzyme activity. A gel mobility shift assay showed that DNA complexes with enzyme DE190 and especially with DE192 were less stable than the corresponding complex with the wild-type enzyme. Kinetic analysis with these mutant enzymes indicate that their Km's for primer DNA were about 10-fold higher than that of the wild type, while Km's for deoxyribonucleoside triphosphate were not changed. Since neither DE190 nor DE192 had any significant alteration in secondary structure, our results suggest that both Asp-190 and Asp-192 are located in the active site and are involved in the interaction of DNA polymerase beta with primer.

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