Nonrepetitive DNA transcription in normal and regenerating rat liver.

Initial RNA excess hybridization experiments employing total cell RNA and the complete complement of nonrepetitive DNA sequences showed no differences between normal and regenerating rat liver. However, when the DNA from the RNA-DNA hybrids was isolated and then reacted with homologous and heterologous RNAs the sensitivity of the assay was sufficiently improved to reveal that some of the nonrepetitive DNA transcrips present in normal liver are missing at 24 h and 48 h after a 70% partial hepatectomy. Additional experiments showed that while some of the missing sequences were common to both stages of regeneration, some were also different. The data thus suggest both quantitative and qualitative changes during liver regeneration in the population of RNA molecules transcribed from nonrepetitive DNA.

Complexity of poly(A+) and poly(A-) polysomal RNA in mouse liver and cultured mouse fibroblasts.

RNA excess hybridization experiments were used to measure the complexity of nuclear RNA, poly(A+) mRNA, poly(A-) mRNA, and EDTA-released polysomal RNA sedimenting at less than 80 S in mouse liver and in cultured mouse cells. With both cell types, poly(A-) RNA was found to contain 30-40% of the sequence diversity of total mRNA. In the case of liver this represents 5,700 poly(A-) molecules and 8,600 poly(A+) molecules for a total of approximately 14,300 different mRNAs. Comparison of the complexity of mRNA with that of nuclear RNA revealed that in liver and in cultured cells, mRNA has only 10-20% of the sequence diversity present in nuclear RNA. This latter observation is consistent with existing data on mammalian cells from this and other laboratories.

Sequence diversity of nuclear and polysomal polyadenylated and non-polyadenylated RNA in normal and regenerating rat liver.

A DNA probe purified from RNA . DNA hybrids of total sham-operated liver RNA and non-repetitive DNA was used to show that nuclear poly(A)-rich RNA from sham-operated liver and from 2.5-h and 48-h regenerating liver contains about 50% of the complexity of total liver RNA. It was further shown that the differences between normal and regenerating liver, reported earlier from this laboratory, occur in the poly(A)-free fraction of nuclear RNA. At the polysome level it was found that polysomal RNA has one-third of the sequence diversity of total RNA and of this, approximately 65% can be accounted for by poly(A)-rich and 55% by poly(A)-free molecules. When DNA probes were prepared from hybrids formed using polysomal RNA from sham-operated liver and regenerating liver at 2.5 h and 48 h post-hepatectomy and then employed in reactions with homologous and heterologous RNAs, no differences were detectable between either normal and regenerating liver or regenerating liver at times during hypertrophy and hyperplasia.

Polyoma genome transcription in transformed mouse cells growing in culture and as tumors in syngeneic mice.

The strands of the polyoma genome coding for early and late viral RNA were separated by means of asymmetric cRNA synthesized under high salt conditions by Escherichia coli RNA polymerase. Each strand was then employed in RNA-DNA hybridization experiments to determine the degree to which it is transcribed in transformed cells under several conditions. Except for a concanavalin-A-selected revertant, approximately one-quarter of the early strand was expressed in all of the situations investigated. In contrast, while no significant late strand transcription was detected in transformed cells in culture, the tumors induced by these cells contained transcripts complementary to about 12% of this strand. The results are discussed in terms of current knowledge of the amount of the virus genome required to transform cells.