Binding sites of HeLa cell nuclear proteins on the upstream region of adenovirus type 5 E1A gene.

Twenty one binding sites of HeLa cell nuclear proteins were identified on the upstream region of adenovirus type 5 E1A gene using DNase I footprint assay. The proximal promoter region contained five binding sites that overlapped the cap site, TATA box, TATA-like sequence, CCAAT box, and -100 region relative to the E1A cap site(+1). The -190 region was a potential site for octamer-motif binding proteins, such as NFIII and OBP100. An upstream copy of the E1A enhancer element 1 was the site for a factor (E1A-F) with the binding specificity of XGGAYGT (X = A, C; Y = A, T). E1A-F factor also bound to three other sites, one of which coincided with the distal E1A enhancer element. The distal element also contained a potential site for ATF factor. The adenovirus minimal origin of DNA replication competed for DNA-protein complex formation on the CCAAT and TATA box region and the -190 region, suggesting that these regions interacted with a common or related factor.     

Pre-early adenovirus 5 gene product regulates synthesis of early viral messenger RNAs.

The studies described here demonstrate that the expression of many early adenovirus mRNAs is dependent upon the activity of a pre-early viral product. This viral gene product is defective in adenovirus 5 host range (Ad hr) group I mutants. Adenovirus 5 host range mutants were previously isolated by their ability to replicate in the adenovirus 5-transformed human embryonic cell line 293 and by their inability to replicate efficiently in HeLa cells (Harrison, Graham and Williams, 1977). The group I complementation class of host range mutants has been mapped by marker rescue between 0 and 4.4 units (Frost and Williams, 1978). We have used the S1 nuclease gel technique to examine the expression of early mRNA after infection of HeLa cells with Ad5 hr group I and II mutants. The Ad5 hr group II mutants stimulate the synthesis of a wild-type pattern of early mRNAs. In contrast, infection of HeLa cells with Ad5 hr group I mutants gives rise to only two early mRNAs. These mRNAs map from 1.5–4.4 units, or in the same region as the Ad5 hr group I mutations. Since infection of HeLa cells with Ad5 hr group I mutants was defective for synthesis of cytoplasmic mRNAs complementary to three early regions in the right half of the genome and to the early region 4.5–11.0 units, we also analyzed nuclear RNA from these cells by the S1 nuclease gel technique for the presence of precursor RNA chains. Nuclear precursors were not detected in Ad5 hr group I-infected HeLa cells, suggesting that the gene product defective in these mutants is required for synthesis of stable nuclear RNA from the three early regions in the right half of the genome and from the early region 4.5–11.0 units.     

The DAN1 gene of S. cerevisiae is regulated in parallel with the hypoxic genes,but by a different mechanism

The DAN1 gene is expressed under anaerobic conditions in yeast and completely repressed during aerobic growth. The function of the gene is unknown, and genetic disruption had no effect on fitness which could be detected, even upon prolonged anaerobic growth. Expression of DAN1 was constitutive in a heme-deficient strain, indicating that heme participates in repression. Expression was blocked by heme in anaerobic medium, suggesting that heme acts as a negative co-effector rather than through its metabolic functions, i.e., in the production of a co-effector. Expression of DAN1 was regulated in parallel with the hypoxic gene ANB1, showing identical kinetics of induction and dose response to heme. However, unlike ANB1, DAN1 is not regulated by the repressor of the hypoxic regulon, ROX1, as shown by observation of normal aerobic repression of DAN1 in a strain carrying a deletion of ROX1. These results indicate the existence of a parallel regulatory system which produces an identical response to oxygen by a different mechanism than that controlling the hypoxic regulon.     

Cellular transformation by adenovirus type 5 is influenced by the viral DNA polymerase.

Early region 2B (E2B) of the group C adenoviruses encodes a number of proteins, including the 140-kilodalton DNA polymerase, which plays a role in the initiation of viral DNA replication. Temperature-sensitive (ts) mutants with mutations mapping to E2B are conditionally defective for both DNA replication in human cells and transformation of rat cells. Nucleotide sequence analysis shows that the E2B mutant ts36 possesses a single point mutation specific to the viral DNA polymerase; this transition of a C to a T at position 7623 changes leucine residue 249 in the polymerase to a phenylalanine. A wild-type (ts+) revertant possesses a codon specifying the original leucine at position 249. Phenotypic analysis of revertant and wild-type viruses derived by marker rescue from ts36 shows that these variants are wild type for both viral DNA replication and transformation. Thus, the single point mutation in the polymerase gene of ts36 is responsible for both defects.