Several properties of the viral RNA-dependent DNA polymerases and of rabbit globin mRNA make it possible to consider synthesis of the globin gene in vitro. These enzymes copy an RNA template using a short sequence of complementary nucleotides as a primer. Furthermore, globin mRNA has a 3'-terminal sequence of adenylic acid residues that make it particularly suitable as a template, since oligo(dT) can be annealed to a specific site on the mRNA. This small primer could phase the DNA polymerase, possibly ensuring that replication is initiated from that end of the globin message. We have used this approach and find that purified mRNA is an efficient template for the polymerase enzyme. The reaction requires the RNA template and the four deoxyribonucleoside triphosphates, and it is markedly stimulated by the addition of oligo(dT). Consistent with the expectation that the oligo(dT) uniquely phases the polymerase at an adenine-rich region in the globin message, oligo(dG), oligo(dC), and oligo(dA) fail to serve as primers. The product has a density intermediate between that of DNA and RNA, and shifts to a lighter DNA density after treatment with base. Further, it is specifically complementary to globin mRNA and sediments slightly faster in an alkaline sucrose gradient than a DNA standard that has a molecular weight of 129,000. The data suggest that a major portion of the DNA product is a sequence of at least 500 bases, about 50 more than would be necessary to encode rabbit globin. The potential usefulness of this interesting product is discussed.

Animals↗

PubMed1972

Globin messenger-RNA induction during erythroid differentiation of cultured leukemia cells.

A cloned line of murine proerythroblastoid cells (T-3-Cl-2), transformed by Friend leukemia virus, undergoes changes associated with erythroid differentiation when treated with dimethylsulfoxide in culture. This line, which does not undergo spontaneous differentiation, develops specific erythrocyte-membrane antigen and accumulates detectable amounts of heme within four days of dimethylsulfoxide treatment. In the present study, we have followed the phenotypic expression of the globin genes by measuring globin mRNA in differentiating cells. Our hybridization probe for this purpose is [(3)H]DNA, which is complementary to purified globin mRNA, synthesized by viral RNA-directed DNA polymerase. This probe is sufficiently sensitive to detect less than 1 ng of globin mRNA. Using it, we find little or no hybridizable globin mRNA in either uninduced cells or in treated control lymphoid cells. In contrast, globin mRNA can be detected in T-3-Cl-2 cell 2 days after induction by dimethylsulfoxide; it reaches a maximum concentration four days after induction. At this time, cells that stain positively for heme appear. The hybridizable cytoplasmic RNA induced in these cells has the sedimentation properties of 9S globin mRNA. Considering the stable character of globin mRNA, our results are most readily explained in terms of a transcriptional activation of the globin genes.

Animals↗

PubMed1972

Immunological relationships of reverse transcriptases from ribonucleic acid tumor viruses.

Antiserum to partially purified reverse transcriptase from the Schmidt-Ruppin strain of Rous sarcoma virus has been prepared and characterized. Antibody to the avian polymerase inhibited the reverse transcriptase activity of avian C-type viruses but had no effect on the polymerase activity from C-type viruses of other classes. The known mammalian C-type viral polymerases were significantly inhibited only by the antiserum to murine C-type viral polymerases; reverse transcriptases from four other mammalian viruses were immunologically distinct from both avian and mammalian C-type viral polymerases. Partially purified murine leukemia viral DNA polymerase activity was comparably reduced by specific antibody regardless of the template used for enzyme detection.

Animals↗

PubMed1972