Albert van Ooyen

Structure and nucleotide sequence of the putative mammary oncogene int-1; proviral insertions leave the protein-encoding domain intact

Many mammary tumors induced by mouse mammary tumor virus (MMTV) contain a provirus in the same region of the host-cell genome, leading to expression of a putative cellular oncogene called int-1. Here we present the structure and nucleotide sequence of int-1. We have established several proviral insertion sites exactly by nuclease S1 analysis or by molecular cloning and DNA sequencing. The protein-encoding domain of int-1 is distributed over four exons. At the 5 end of the gene two overlapping exons were detected, one of which is preceded by a TATA box. The deduced int-1-encoded protein has 370 amino acids, with a preponderance of hydrophobic residues at the NH2 terminus. Proviruses are found at both sides of the gene, usually oriented away from the gene. Downstream integrations occur frequently in the long 3 untranslated region of the last exon. One upstream provirus is inserted in the 5 untranslated region and, unlike the other upstream insertions, in the same orientation as the int-1 gene. Proviral integrations always leave the protein-encoding domain intact, providing further evidence that the int-1 protein contributes an essential step in mammary tumorigenesis.

Specific inhibition of ribosomal RNA synthesis in vitro by guanosine 3' diphosphate, 5' diphosphate.

CONSIBERABLE evidence has accumulated that guanosine 3′-diphosphate, 5′-diphosphate (ppGpp)1 is involved in the regulation of ribosomal RNA (rRNA) synthesis in Escherichia coli in vivo (see ref. 2). The involvement of ppGpp is most convincingly indicated by results obtained under conditions of amino acid starvation. A strong increase in ppGpp accumulation is always accompanied by a concomitant decrease in stable RNA accumulation3. In vitro, a specific effect of ppGpp has not been found, neither in a purified4, nor in a crude system5. Travers et al.6,7 have proposed that ppGpp specifically affects rRNA synthesis by counteracting the effects of the protein elongation factor EF-Tu. Here, we show that ppGpp can specifically inhibit rRNA synthesis in vitro using purified RNA polymerase with two templates—DNA prepared by phenol extraction, and nucleoids prepared using methods developed by Pettijohn et al.8.

Ribosomal and non-ribosomal RNA synthesis in vitro

The synthesis of total and ribosomal RNA using nucleoids of Escherichia coli as template was measured; of the total RNA synthesized by endogenous RNA polymerase which only completes chains, and added RNA polymerase which initiates new chains, 50-70 and 3-5%, repectively, was rRNA. Total RNA synthesis by added enzyme, however, was 10-20 times higher than endogenous RNA synthesis; thus rRNA was synthesized at the same rate by the endogenous and the added enzyme. We conclude that the percentage rRNA in vitro is no measure of the rate of rRNA synthesis. Furthermore, it follows that the added enzyme, like the endogenous one, is packed at the physical limit on the ribosomal cistrons. Consequently, initiation of ribosomal cistrons by added enzyme was at or near the maximal rate possible for this system in which the elongation rate is 10-20% of that in vitro. When RNA synthesis was assayed at various ratios of RNA polymerase to phenol-extracted DNA, the amount of rRNA made per DNA, which is a measure of the frequency of transcription of ribosomal cistrons, varied. The ratio of rRNA synthesis relative to total RNA synthesis also varied, but in a different way, again leading to the conclusion that this ratio, as determined in vitro, does not reflect the efficiency of transcription of the ribosomal cistrons.

Heterogeneity in Subregions of the Terminal Repeats of Herpes Simplex Virus Type 2 DNA

We constructed recombinant plasmids from the 0.5 molar terminal fragments of herpes simplex virus type 2 (HSV2) DNA. Physical mapping of three different L-terminal clones demonstrated one, two or three copies of the terminally redundant a repeat. The nucleotide sequence at the end of the terminal a repeats was shown to be identical in these three cloned HSV2 genomic L-termini and closely resembled the internal a repeat sequence reported previously by Davison and Wilkie. Virtually all clones that were obtained from the S-termini differed in a specific subregion of the inserts. This heterogeneous region of the HSV2 genome, that varied in size from 0.2 to 0.85 M daltons, seemed to result from local deletions or amplifications and mapped near the junctions between US and c repeats.