High Molecular Weight Subunits Research Articles

Properties and subunit composition of RNA polymerase II from plant cell cultures

The purification of DNA-dependent RNA polymerase II (EC 2.7.7.6) from plant cell cultures of Petroselinum (parsley) is described. The procedure during which enzyme I is eliminated includes initial precipitation with (NH 4) 2SO 4, an ultracentrifugation step, gel filtration on Sepharose 4B, chromatography on DEAE-cellulose, DNA-agarose and DEAE-Sephadex. The enzyme purified almost to homogeneity exhibits maximal activity with denatured DNA, and is activated preferentially by Mn 2+; α-amanitin acts as a strong inhibitor. Electrophoresis of the enzyme in the presence of dodecylsulphate indicates that it is composed of seven subunits with mol. wts of 200 000, 180 000, 140 000, 43 000, 26 000, 25 000 and 16 000. The results of molecular weight and molar ratio determinations suggest that Petroselinum RNA polymerase II may exist in two active forms differing only in the composition of their high molecular weight subunits.

Reassociation of 4 S and 5 S RNA's with the genome of avian sarcoma virus

A tryptophan tRNA derived from the host cell serves as primer for the initiation of DNA synthesis by the reverse transcript ase of avian sarcoma virus (ASV). We have characterized the binding of primer to the viral genome by studying the reassociation of low molecular weight RNA's with high molecular weight subunits of ASV-RNA. Primer can be annealed to a limited number (two to four) of sites on the viral genome to reconstitute a template fully active with reverse transcriptase. The annealing is specific and accurate; none of the other 4 S RNA's found in ASV will reassociate with viral genome under any of the conditions tested, and the reconstructed complex denatures with the same T m as the native complex between primer and viral genome. These results further substantiate our previous conclusion that the primer in ASV is a single species of tRNA, although the normal host cell may contain other 4 S RNA's which could also serve as primer if they were included in the viral genome during assembly. Chicken ribosomal 5 S RNA also reanneals to the ASV genome, presumably by binding to the sites originally occupied by 5 S RNA in viral 70 S RNA.

Immunologic Studies of Antihemophilic Factor (AHF, Factor VIII). V. Immunologic Properties of AHF Subunits Produced by Salt Dissociation

Abstract Human antihemophilic factor (AHF, factor VIII), a large plasma protein with a molecular weight of approximately two million, is dissociated by changes in ionic strength. The immunologic properties of subunits obtained by sucrose density ultracentrifugation in 1 M NaCl and by agarose gel filtration in 0.24 M CaCl2 have been determined using human and rabbit anti-AHF. Asymmetric dissociation of AHF has been identified with formation of two subunits in these separations: a nonfunctional highmolecular-weight (HMW) subunit similar in size to plasma AHF which is identified by immunoprecipitation and radioimmunoassay for AHF antigen, and an active lowmolecular-weight (LMW) subunit which is not detected by these antigen assays. The LMW subunit retains AHF antigens, however, for it is inactivated by both human and rabbit anti-AHF. Antibody neutralization studies verify the presence of AHF antigens on the HMW subunit. These immunologic studies provide constraints which must be incorporated into models of AHF structure.

RNA-directed DNA synthesis by virions of Rous sarcoma virus: further characterization of the templates and the extent of their transcription.

Abstract Four species of RNA found in Rous sarcoma virus have been tested for their roles as templates for RNA-directed DNA synthesis by detergent-activated virions of RSV: the high molecular-weight subunits of the 70S RNA complex, 4S and 5S RNAs contained in the 70S complex, and 7S RNA extracted from purified virions. All three forms of low molecular-weight RNA are at least partially transcribed, but the principal templates for DNA synthesis are the high molecular-weight subunits of 70S RNA. DNA synthesized by virions of RSV contains nucleotide sequences representative of most or all of the viral genome, but the majority of these sequences are present in very small proportions. These results further substantiate the occurrence of preferential transcription from a limited region of the viral genome as defined previously for double-stranded enzymatic product by measurement of reassociation kinetics. By contrast, single-stranded DNA synthesized by virions of RSV in the presence of actinomycin D is a complete and relatively uniform copy of the entire viral genome. The latter observation facilitates both the study of nucleic acid homologies among RNA tumor virus genomes and the execution of competition hybridizations to determine the portion of a viral genome present in various populations of cellular RNA. An example of each of these procedures is given.