Asp537, Asp812 are essential and Lys631, His811 are catalytically significant in bacteriophage T7 RNA polymerase activity.

To define catalytically essential residues of bacteriophage T7 RNA polymerase, we have generated five mutants of the polymerase, D537N, K631M, Y639F, H811Q and D812N, by site-directed mutagenesis and purified them to homogeneity. The choice of specific amino acids for mutagenesis was based upon photoaffinity-labeling studies with 8-azido-ATP and homology comparisons with the Klenow fragment and other DNA/RNA polymerases. Secondary structural analysis by circular dichroism indicates that the protein folding is intact in these mutants. The mutants D537N and D812N are totally inactive. The mutant K631M has 1% activity, confined to short oligonucleotide synthesis. The mutant H811Q has 25% activity for synthesis of both short and long oligonucleotides. The mutant Y639F retains full enzymatic activity although individual kinetic parameters are somewhat different. Kinetic parameters, (kcat)app and (Km)app for the nucleotides, reveal that the mutation of Lys to Met has a much more drastic effect on (kcat)app than on (Km)app, indicating the involvement of K631 primarily in phosphodiester bond formation. The mutation of His to Gln has effects on both (kcat)app and (Km)app; namely, three- to fivefold reduction in (kcat)app and two- to threefold increase in (Km)app, implying that His811 may be involved in both nucleotide binding and phosphodiester bond formation. The ability of the mutant T7 RNA polymerases to bind template has not been greatly impaired. We have shown that amino acids D537 and D812 are essential, that amino acids K631 and H811 play significant roles in catalysis, and that the active site of T7 RNA polymerase is composed of different regions of the polypeptide chain. Possible roles for these catalytically significant residues in the polymerase mechanism are discussed.     

Construction of a human cytochrome c gene and its functional expression in Saccharomyces cerevisiae

The nucleotide sequences of a partial cDNA and three pseudogenes of human cytochrome c were determined. The complete nucleotide sequences which encode human cytochrome c were constructed on the basis of one of the pseudogenes by in vitro mutagenesis. The constructed human cytochrome c was functionally expressed in Saccharomyces cerevisiae. The recombinant human cytochrome c was purified and characterized.     

Heterozygote advantage in Tay-Sachs carriers?

Chi-square analyses of new data as well as data previously reported by Myrianthopoulos have shown that grandparents of Tay-Sachs carriers die from proportionally the same causes as grandparents of noncarriers. It is unlikely that there is any advantage to being a Tay-Sachs carrier insofar as resistance to tuberculosis is concerned. Our results are further evidence to support Fraikor's claim that the high carrier frequency of the allele in Ashkenazi Jews is probably caused by a combination of founder effect, genetic drift, and differential immigration patterns.     

DIFFERENTIAL INCORPORATION OF [3H]LYSINE INTO VISUAL CORTEX PROTEIN FRACTIONS DURING FIRST EXPOSURE TO LIGHT

—A resolution of the enhancement of protein synthesis in the visual cortex of rats during first exposure to light (Richardson and Rose , 1972) was achieved by polyacrylamide gel electrophoresis using a double-labelling technique. Differential incorporation of lysine was established between exposed and control animals in two fractions of the soluble proteins and seven fractions of the insoluble proteins. This suggests that exposure to a new experience of this type involves a specific effect on protein synthesis, rather than a general stimulation across all fractions.     

Activated lymphocytes trigger lymphoblast extravasation.

Intraperitoneal stimulation of adoptively sensitized rats with bacterial antigen promotes the localization of lymphoblasts at the site of antigen deposition. Lymphoblast extravasation activity (LEA) is generated only when specifically immune donor lymphocytes and the recipients of these cells share at least on Ag-B haplotype. However, if the specificity criteria for its formation are satisfied, LEA promotes the local development of lymphoblasts of all available specificities and irrespective of their Ag-B genotype. Allogeneic lymphoblasts do not participate actively in the delayed inflammatory reaction even when they are passively recruited into exudates. The results suggest that LEA is a T cell-derived mediator that amplifies the delayed type hypersensitivity reaction by directing recently activated lymphocytes into lesions.