James E. Darnell

Transcription units for mRNA production in eukaryotic cells and their DNA viruses.

Publisher Summary This chapter reviews that in bacteria the rate of protein synthesis for any specific protein is controlled by the supply of mRNA for that protein. In turn, the supply of any one mRNA is governed mainly by the decision to promote or restrict t transcription of the gene corresponding to that particular mRNA. The chapter discusses that in a number of instances where qualitative or quantitative changes occur in protein synthesis in eukaryotic cells, corresponding shifts of transcription also appear to occur. However, the assumption of a complete parallel in the possible mechanisms of gene control between prokaryotic and eukaryotic cells seems unwarranted when the details of mRNA production and utilization are considered. The chapter also reviews that the new developments in mRNA formation make it possible to define in molecular terms that are not wholly speculative, how and at what level eukaryotic gene expression may be regulated. Moreover, since individual primary transcripts can be measured, experiments can now settle whether process vs. discard decisions occur or whether differential use of multiple mRNAs of complex primary transcripts occurs. Once the manner of regulation is settled for several eukaryotic genes a much more coherent plan to search for regulatory proteins can be designed. Success in the identification and mode of action of regulatory proteins for eukaryotic genes is therefore not too much to hope for in the next few years.

Mapping of RNA initiation sites by high doses of uv irradiation: Evidence for three independent promoters within the left 11% of the Ad-2 genome☆

Abstract Cells infected with Ad-2 virus were irradiated so that uv-induced lesions were introduced every 500–1000 nucleotides in the genome, consequently leading to the premature termination of RNA transcription. Such cells when labeled with [ 3 H]uridine accumulate labeled promoter proximal RNA. Hybridization of this RNA after size fractionation to restriction fragments of the Ad-2 genome allowed the identification of DNA sequences containing active RNA initiation sites. Early during the infectious cycle two active RNA initiation sites were found within the left 11% of the Ad-2 genome within the 0–3.0 and 4.4–8.0 restriction fragments. During late infection (15 hr) an additional uv resistant transcript was detected indicating that a newly activated RNA initiation site, presumably for protein IX, resides within the fragment 8.0–11.,2.

Early Ad-2 transcription units: Only promoter-proximal RNA continues to be made in the presence of DRB

Abstract The nucleoside analog 5,6-dichloro-1-β-D-ribofuranosylbenzirnidazole (DRB) causes premature termination of adenovirus type 2 (Ad-2)-specific RNA chains close to their initiation sites late in virus infection of HeLa cells ( N. W. Fraser, P. B. Sehgal, and J. E. Darnell, 1978 , Nature (London) 272 , 590–593). The same phenomenon appears to occur for most of the early Ad-2 transcription units as well. In the presence of DRS, [ 3 H]uridine continued to be incorporated into short 4 S to 10 S RNA that hybridized to DNA restriction fragments previously shown to contain promoter sites for RNA synthesis (rightward reading) Sma I j (0.0–3.0), Eco RI D (75.9–83.10; and leftward reading Eco Rl C (89.7–100), Eco RI F (70.7–75.9). In addition a smaller amount of pulse-labeled RNA was found to hybridize to Sma l f (11.6–18.2) (leftward reading) than in the above regions and labeling of a portion of this RNA also resisted DRB treatment. Finally the region from 3.0–11.1 ( Sma I e) may contain one or more early rightward reading promoter sites. It is also possible that this region contains a DRB-resistant transcription unit. Since the accumulation of promoter-proximal labeled RNA chains in DRB could be observed after a 10-min [3H]-uridine label, the drug may be useful in allowing accumulation of promoter-proximal RNA sequences for analysis.

Murine chromosomal location of four hepatocyte-enriched transcription factors: HNf-3α, HNF-3β, HNF-3γ, and HNF-4

Abstract The genes for rat hepatocyte nuclear factors 3 and 4 (HNF-3α, HNF-3β, HNF-3γ, and HNF-4) have been mapped in mouse by analysis of restriction fragment length polymorphisms in interspecific backcross mice. These hepatocyte-enriched transcription factors are positive-acting transcription factors with binding sites in regulatory regions of many genes expressed in hepatocytes. Both HNF-3α, β, and γ and HNF-4 are also expressed in intestine. They have recently been implicated as potential participants in endodermal development from early gut cells because of their close homology to Drosophila genes, which themselves are expressed in the developing gut. Despite having similar functional roles and highly conserved DNA binding domains, the three loci from the Hnf-3 family of genes mapped to three different mouse chromosomes, suggesting that the Hnf-3 family has become widely dispersed during evolution and implying the necessity for independent activation of each member of the HNF-3 family.