ABSTRACT
AIM:To clarify the impact of heart shock factor 4b (Hsf4b) K65R mutation on the regulation of downstream protein expression .METHODS:Non-functional Lys mutant plasmid pWZL-blast-HA-Hsf4b/K65R was genera-ted by replacing single , homologous amino acids using KOD-Plus-Mutagenesis-Kit.Mouse lens epithelial mLEC stable cell lines expressing Hsf4b or Hsf4b/K65R were constructed by lentivirus infection .The expression of Hsf4b in the mutant and the wildtype mLEC cells was confirmed by Western blotting .The expression of Hsf4b downstream proteins such as heat shock protein ( Hsp)70, Hsp90, Hsp27 and CryAB was examined by Western blotting and real-time PCR.RESULTS:The results of PCR and DNA sequencing confirmed the successful construction of mLEC Hsf 4b/K65R mutant.The K65R mutation didn’t influence Hsf4b expression in the mLEC cells.After K65R mutation in Hsf4b, the expression levels of Hsp27 and CryAB were down-regulated and the expression of Hsp 70i and Hsp90a upregulated.CONCLUSION: pWZL-blast-HA-Hsf4b/K65R can be used to construct a stable cell line by infecting with lentivirus .Hsf4b/K65R mutation influ-ences the regulation of downstream heat shock proteins .
ABSTRACT
Molecular biological methods were used to construct the eukaryotic expression vectors of precore and core gene named EBO PreC/C. Then T1862, A1896, A1899 and A1896+A1899 variants were constructed by site mutagenesis in vitro. By PCR RFLP and sequencing ,T1862,A1896,A1899 and A1896+A1899 variants were obtained. Wild type and variants were transfected to HepG2 cells, and HBeAg was tested to observe the difference of HBeAg expression between wild type and variants. After stable expression in HepG2 cells, HBeAg was detected to be positive in cells transfected wild type and A1899 variants, and negative in cells transfected with T1862, A1896,A1896+A1899 variants. The construction of these variants will play an important pole in studying the relation of PreC/C mutations and HBV expression and replication of HBV genome.
ABSTRACT
The C-terminus ends of the second putative transmembrane domains of both M-1 and M-2 Muscarinic receptors contain a triplet of amino acid residues consisting of leucine (L), tyrosine (Y) and threonine (T). This triplet is repeated as LYT-TYL in M-1 receptors at the interface between the second transmembrane domain and the first extracellular loop. Interestingly, however, it is repeated in a transposed fashion (LYT-LYT) in the sequence Of M-2 receptors. In our previous work, we investigated the possible significance of this unique sequence diversity for determining the distinct differential receptor function at the two receptor subtypes. However, we found mutation of the LYTTYL sequence of M-1 receptors to the corresponding M-2 receptor LYTLYT sequence demonstrated markedly enhanced the stimulation of phosphoinositide (PI) hydrolysis by carbachol without a change in its coupling to increased cyclic AMP formation. In this work, thus, the enhanced stimulation of PI hydrolysis in the LYTLYT M-1 receptor mutant was further investigated. The stimulation of PI hydrolysis by carbachol was enhanced in the mutant M-1 receptor, and this change was not due to alterations in the rate of receptor desensitization or sequestration. The observed larger response to carbachol at mutant M-1 receptors was also not due to an artifact resulting from selection of CHO cells which express higher levels of G-proteins or phospholipase C. Our data suggest that although the LYTTYL sequence in M-1 muscarinic receptors is not involved in determining receptor pharmacology, mutation of the sequence enhanced the coupling of M-1 receptors to the stimulation of phospholipase C.
Subject(s)
Animals , Cricetinae , Humans , Artifacts , Carbachol , CHO Cells , Cyclic AMP , GTP-Binding Proteins , Hydrolysis , Leucine , Pharmacology , Phospholipases , Receptors, Muscarinic , Threonine , Trinucleotide Repeats , Triplets , Type C Phospholipases , TyrosineABSTRACT
The enzymological characterizations of site-mutagenized rat recombinant DNA polymerase?, RQ182 and RQ183 were studied The phosphocellulose column chromatographies showed that the mutant and the wild DNA polymerases were all eluted by about 0.5 mol/ L KCI, but the denatured DNA-cellulose chromatographies showed that although the wild enzyme was eluted by 0.35 mol/L KCI, RQ182 and RQ183 were eluted by 0.55 and 0.45 mol/L KCI, respectively, indicating that the binding abilities to DNA of the mutant enzymes were increased. Km values for the substrate (dTTP)of the wild enzyme, RQ182 and RQ183 were determined as 38.5, 34.5 and 111.1 ?mol/L, respectively,and the Km values for the primer (oligo(dT)) were 1.28, 1.96 and 6.58 ?g/ml, respectively. The results showed that the affinities of RQ183 to the substrate and the primer were decreased dramatically. It is suggested that Arg182 and Arg183 were involved in the active site function of DNA polymerase ?, in binding to DNA template, in recognizing of primer, and in binding to and catalyzing of substrates of the enzyme.