Hitomi Shibata

Nuclear factor I stimulates transcription of the adenovirus 12 E1A gene in a cell-free system

Binding to the cis-acting region of NF-I-like protein and/or NF-III-like protein was previously suggested to be responsible for the preferential stimulation of transcription from distal start-site of the adenovirus 12 E1A gene in a cell-free system. In this study, nuclear extracts of Ehrlich ascites tumor cells depleted of NF-I-like protein were found to lose activity to stimulate the E1A gene transcription. This activity was recovered when NF-I purified from HeLa cells with no contamination of NF-III was supplemented. It is thus evident that NF-I is involved in stimulating distal transcription of the adenovirus 12 E1A gene. Moreover, activities for both stimulating the E1A gene transcription and binding to a region recognized by NF-I did not apparently exist in nuclear extracts of a cell line expressing the adenovirus 12 E1A gene. These results suggest that transcription of the adenovirus 12 E1A gene may possibly be autoregulated at least in part through modulation of the activity of NF-I.

Analysis of promoters of adenovirus type 12 E1A gene in a cell-free transcription system

Transcription from the E1A gene of adenovirus type 12 initiates at two sites in vivo. In this paper we analyzed the E1A promoter(s) in a cell-free transcription system. Primer extension assay revealed that transcriptions were accurately initiated at two sites apart by 140 bp. The efficiency of transcriptions from two sites was almost equal in the standard reaction. However, when transcription reaction was done following pre-incubation of a nuclear extract and template DNA in the absence of ribonucleotides, transcription from the site proximal to E1A gene significantly decreased with little effect on that from distal site. This suggests the existence of a mechanism which controls the efficiency of transcriptions from two start sites of E1A gene.

Biological characteristics of plasmid carrying a repeated deoxyribonucleic acid sequence

It was found that a plasmid which had a foreign deoxyribonucleic acid (DNA) between two repeated sequences did not multiply in E. coli recBCsbcB, even if it multiplied in wild-type E. coli, E. coli recBC or E. coli recBCsbcBrecF when the insert was longer than 351 base pair. The multiplication of these plasmids were, however, inhibited when a plasmid expressing recF gene was introduced into E. coli recBCsbcBrecF. The inviability of the plasmid carrying the repeated sequence in E. coli recBCsbcB was discussed by the mechanism of recombination, and the functions of recF, recBC and sbcB were speculated. When E. coli recBC was transformed with pDR1 which was a derivative of pBR322 carrying a directly repeated sequence between which a DNA fragment derived from plasmid R6K with its origin was inserted, the intramolecular recombinant appeared. The recombinant recovered was, however, only the plasmid which had the replication origin of pBR322. The result suggests that pBR322 is compatible with pDR1 but R6K is not. The replication origin of R6K seems to be preferrentially used by pDR1.