[Construction of point mutation plasmids expressing HCV NS3/4A with different secondary structures at amino-terminal and their expressions in Huh 7 cells].

OBJECTIVE: To construct point mutation plasmids expressing HCV NS3/4A with different secondary structures at amino-terminal, and express the constructs in Huh 7 cells. METHODS: Using pSG5/M-H05-5/4A as the template (A1-1) and primers designed according to the typing criteria, 4 single point mutation plasmids, namely pSG5/M-H05-5(A1-2)/4A(A1-2) (Y56F), pSG5/M-H05-5(B1-1)/4A(B1-1) (L80Q), pSG5/M-H05-5(B2-1)/4A(B2-1) (V51A), and pSG5/M-H05-5(B2-2)/4A(B2-2) (S61A), were constructed. With A1-2, B2-1, and B2-2 as the templates, the leucine to glutamine mutation at position 80 (L80Q) was induced to construct another 3 double point mutation plasmids pSG5/M-H05-5(B1-2)/4A(B1-2), pSG5/M-H05-5(A2-1)/4A(A2-1), and pSG5/M-H05-5(A2-2)/4A(A2-2), respectively. DNA sequencing was performed for confirmation of the mutations. Huh 7 cells were transfected with the constructs using FuGene 6 transfection reagents. Indirect immunofluorescence staining and Western blotting were used to detect the expression of the constructs. RESULTS: Indirect immunofluorescence assay revealed 4 subcellular localization patterns of NS3 protein, including dot-like staining, diffuse staining, doughnut-like staining, and rod-shape staining. Western blotting also demonstrated successful expression of the constructs and weak in cis and in trans NS3 serine protease activities of subtypes A2-1 and B2-1 in comparison with other subtypes. CONCLUSION: The point mutation plasmids expressing HCV NS3/4A with different secondary structures at amino-terminal are constructed successfully, which provides the basis for further study of different subtypes of HCV.

Multiple Variant mRNAs with Different Length Tandem Repeats of (CAYYCC)n Produced from Bovine Selenoprotein P-like Protein Gene

In contrast to selenoprotein Ps (SelPs) from other animal species, bovine selenoprotein P-like-protein (SelPLP) was found to contain a tandem repeat of (CAYYCC)11. During an investigation into whether SelPLP was a bovine substitute for SelP or uniquely bovine, its mRNA was found to consist of multiple variants with different length tandem repeat, namely p(0) with (CAYYCC)11, p(-4) lacking (CAYYCC)4, p(-8) lacking (CAYYCC)8, and p(-9) lacking (CAYYCC)9. Although they were encoded on a single gene locus, neither classical GT-AG nor minor class AT-AC donator-acceptor sequences for alternative splicing were identified. A subsequent S1 protection assay using oligonucleotides, whose sequence may occur as variants, performed against bovine poly(A)+RNA identified a total of nine variants. Judging from the sequence of these variants and the branch point mapping, the consensus sequence for recognition of the donator was CACCCCCAC and of the acceptor and the branch point A nucleotide, ACCCCCAT or ACCCCCATCCCCAT. Furthermore, when the p(0) insert mRNA was expressed in COS-7 cells derived from an African green monkey kidney, cDNAs corresponding to p(-8) and p(-9) could be isolated. Therefore, the bovine SelPLP mRNAs consisted of multiple variants probably due to a novel splicing mechanism which was not bovine-specific but common to other mammals.

Multiple variant mRNAs with different length tandem repeats of (CAYYCC)n produced from bovine selenoprotein P-like protein gene

In contrast to selenoprotein Ps (SeIPs) from other animal species, bovine selenoprotein P-like-protein (SeIPLP) was found to contain a tandem repeat of (CAYYCC)(11). During an investigation into whether SeIPLP was a bovine substitute for SeIP or uniquely bovine, its mRNA was found to consist of multiple variants with different length tandem repeat, namely p(0) with (CAYYCC)(11), p(-4) lacking (CAYYCC)(4), p(-8) lacking (CAYYCC)(8), and p(-9) lacking (CAYYCC)(9). Although they were encoded on a single gene locus, neither classicalGT-AG: nor minor classAT-AC: donator-acceptor sequences for alternative splicing were identified. A subsequent S1 protection assay using oligonucleotides, whose sequence may occur as variants, performed against bovine poly(A)(+)RNA identified a total of nine variants. Judging from the sequence of these variants and the branch point mapping, the consensus sequence for recognition of the donator was CACCCCCAC: and of the acceptor and the branch point A nucleotide,ACCCC: CAT orACCCC: CATCCCCAT. Furthermore, when the p(0) insert mRNA was expressed in COS-7 cells derived from an African green monkey kidney, cDNAs corresponding to p(-8) and p(-9) could be isolated. Therefore, the bovine SeIPLP mRNAs consisted of multiple variants probably due to a novel splicing mechanism which was not bovine-specific but common to other mammals.