Sune Frederiksen

Synthesis of low molecular weight RNA components A, C and D by polymerase II in alpha-amanitin-resistant hamster cells.

In an attempt to establish which RNA polymerase catalyzes the synthesis of the low molecular weight RNA components A, C and D, Ama 1 cells (mutant Chinese hamster cells) were used in experiments with addition of alpha-amanitin. Ama 1 cells contain an altered RNA polymerase II which is 800 times more resistant towards inhibition by alpha-amanitin than the wild type enzyme. Alpha-amanitin (up to 200 microgram/ml) added to these cells does not affect the synthesis of the low molecular weight RNAs A, C and D. These data together with our previous data showing that alpha-amanitin (0.5 - 5.0 microgram/ml) preferentially inhibits the synthesis of A, C and D in normal cells indicate that RNA polymerase II catalyzes the synthesis of the low molecular weight RNA components A, C and D.

Fine mapping of human 5S rRNA genes to chromosome 1q42.11→q42.13

The human 5S rRNA genes have been localized by in situ hybridization to metaphase chromosomes. Tritiated RNA probes were made by transcription from 2.300-bp and 638-bp DNA fragments containing an isolated human 5S rRNA gene. Hybridization to metaphase spreads from a balanced reciprocal translocation carrier, 46,XX.t(1:7)(q42.13;p11.1), showed that the 5S rRNA genes were entirely localized on the normal and the derivative chromosome 1. This narrows the chromosome position of the major fraction of 5S rRNA genes and pseudogenes to the region 1q42.11----q42.13.

Precursors to small molecular weight RNA components

Metabolically long-lived RNA components such as tRNA and rRNA are formed from unstable larger precursors [ 1,2] . Baby Hamster Kidney cells (BHK-2 1 cells) contain four small mol. wt RNA components (sm RNA) with a metabolic half-life of 5-7 days [3] . The size of these components (D, C, A, and L) was determined to be from 145 to 235 nucleotides based on their migration in polyacrylamide gels [4]. The present paper demonstrates the presence of larger unstable precursors for some of the sm RNA components.

Lysosomes: a possible target for psoralen photodamage.

Treatment in vitro of Ehrlich ascites tumor cells or human fibroblasts with 8-methoxypsoralen (8-MOP, 2.4 microM) and UVA irradiation results in a 30% and 60% respectively reduction in lysosomal beta-galactosidase activity in situ. Under identical conditions one 8-MOP adduct was formed per 2 X 10(4) bases of DNA, one 8-MOP adduct was formed per approximately 10(4) tRNA molecules and one per approximately 100 ribosomes. It is suggested that the decrease in lysosomal beta-galactosidase activity is a result of leakage through the lysosomal membrane caused by psoralen-UVA damage of the lipids in the membrane, since no effect was found on beta-galactosidase in vitro. These results indicate that the lysosomes may also be a target for cellular photodamage by 8-methoxy-psoralen.

Isolation of two small molecular weight RNA components by MAK column chromatography

Abstract RNA was extracted at 0 ° with potassium phosphate buffer and phenol but without detergents and chromatographed on methylated albumin kieselguhr columns. Two small molecular weight RNA components—X1 and X2—were found in extracts from Ehrlich ascites tumor cells, Yoshida ascites tumor cells, and L 5178 Y lymphoblast ascites cells. When Ehrlich cells were incubated with 3H-uridine or 3H-methyl-methionine, the specific activity of X1 and X2 RNA was different from that of tRNA, 5S RNA, and rib-RNA. The incorporation of 3H-uridine into the five RNA components was inhibited to a different extent by 3′-deoxyadenosine and by actinomycin. The base composition and/or the content of pseudouridylic acid in X1 and X2 are different from those of tRNA, 5S RNA, and rib-RNA. X1 RNA and X2 RNA from Ehrlich cells sediment in a sucrose gradient in the 4-7S position. X1 RNA amounts to 1.0% and 1.1% of the total RNA in Ehrlich cells and Yoshida cells, respectively. X2 RNA amounts to 0.5% and 0.6%, respectively. The percentage of X1 RNA in the extract of different fractions of Ehrlich cells was, in whole cells: 1.5%, nuclei: 3.4%, cytoplasm: 0.7%, and nuclear residue: 4.7%. The percentage of X2 RNA was, in whole cells: 0.7%, nuclei: 2.0%, cytoplasm: 0.5%, and nuclear residue: 4.7%.

PNA as Specific Probe for In Situ Hybridization to Metaphase Chromosomes

The present protocol concerns a sensitive detection of biotinylated PNA probes on metaphase chromosomes by FISH (fluorescent in situ hybridization). The mono-and dibiotinylated PNA probes used are different 18-mers complementary to sequences within the 5S rRNA gene.