Marialuisa Melli

The in vitro transcription of the 7SK RNA gene by RNA polymerase III is dependent only on the presence of an upstream promoter

Deletion analysis was carried out on the human 7SK RNA gene to map regions essential for in vitro transcription by RNA polymerase III. The sequence promoting transcription is located between 37 and 3 bp upstream of the 7SK RNA coding region. RNA polymerase III transcription of adjacent plasmid sequences can be directed by this promoter in the complete absence of the 7SK RNA coding region, indicating that no internal promoter sequences are required. Transcription is terminated by a stretch of T residues, typical of RNA polymerase III transcription. The promoter contains a TATA box at position -25, mutations within which dramatically reduce the efficiency of transcription. Upstream sequences from position -37 to -243 increase the promoters efficiency. The promoter recognized by RNA polymerase III is structurally and functionally similar to the promoter of genes transcribed by RNA polymerase II.

Hybridization between rat liver DNA and complementary RNA.

Abstract The hybridization of rat liver DNA with complementary RNA has been studied using the membrane technique of Gillespie & Spiegelman (1965). Complementary RNA was synthesized in vitro using Micrococcus RNA polymerase and rat liver DNA. Hybridization was rather rapid, but only about 5% of the total DNA became involved in hybrid formation. The hybrid melted sharply with a mean temperature of 72 °C, only 3 deg. C lower than the mean melting temperature of enzymic DNA-RNA hybrid. RNA complementary to at least half of the total DNA was isolated from a 2:1 enzymic DNA-RNA hybrid. Only about 5% of the DNA hybridized with this complementary RNA under standard annealing conditions, showing that the limited hybridization of rat liver DNA is caused by factors other than limited complementarity of this RNA. It is suggested that the factor responsible is the extreme complexity of mammalian DNA. Mammalian DNA is known to contain a minor fraction which, following denaturation, renatures rapidly compared with the bulk of the DNA. Renaturing rat liver DNA reduced its capacity to hybridize with complementary RNA by two-thirds, showing that it is the rapidly renaturing DNA fraction which hybridizes with the RNA. In confirmation of this, when the rapidly renaturing and slowly renaturing DNA fractions were separated by differential adsorption to hydroxylapatite at 70 °C, preferential hybridization of complementary RNA to the rapidly renaturing DNA fraction was observed. Liver nuclear RNA competed completely against the hybridization of complementary RNA with rat liver DNA. DNA-like RNA from liver, kidney, spleen and brain all competed strongly against complementary RNA. It is concluded that the hybridization of DNA and RNA from higher organisms under the usual conditions permits mainly or only the hybridization of the minor DNA fraction which renatures rapidly. RNA complementary to most of this DMA fraction is found in several different tissues.

Evolutionary conservation of the human 7 S RNA sequences

The cloning and characterization of the cytoplasmic 7 S RNAs of HeLa cells has provided pure probes to study the organization of the corresponding genomic DNA sequences. Such analysis has shown that the 7 S L and K RNAs are derived from families of middle repetitive DNA (Ullu & Melli, 1982; Ullu et al., 1982). In this work we analyze the evolutionary conservation of these sequences in the RNA and DNA of distantly related species. Hybridization of the 7 S recombinants to the RNA of rodents, birds, amphibians and echinoderms suggests high conservation of these sequences throughout evolution. Southern blot analysis of genomic DNAs from the same species shows the presence of families of repeated sequences homologous to the 7 S recombinants and Alu DNAs in the genomes of the same species. We were unable to hybridize the 7 S probes to the RNAs of Drosophila melanogaster or Dictyostelium discoideum, although sequence(s) homologous to the 7 S L probe were found in the genome of D. discoideum and to both 7 S L and K probes in the genome of D. melanogaster.