Search PubMed⌕ Search

Biomedical subjects

P Leder

Publications and source records attributed to P Leder.

At least 289 records · Page 16Linked to original sources
PubMed1972

Translation of bacteriophage Q messenger RNA in a murine Krebs 2 ascites tumor cell-free system.

Qbeta is a small bacterial virus whose three genes are encoded in a single-stranded molecule of RNA. This RNA serves directly as the Qbeta message. Here we describe conditions under which RNA corresponding to the coat cistron of this bacterial virus is translated in a system derived from mammalian cells. Translation of the bacterial virus messenger RNA is less effective than that of mammalian globin messenger RNA, but is somewhat enhanced by mild alkali treatment of the messenger. The synthesized product when subjected to electrophoresis migrates with authentic Qbeta coat protein and yields tryptic peptides that correspond to those derived from the Qbeta coat protein.

Amino Acids↗
PubMed1972

In vitro synthesis of DNA complementary to purified rabbit globin mRNA (RNA-dependent DNA polymerase-reticulocyte-hemoglobin-density gradient centrifugation-oligo(dT) primer).

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

Purification of biologically active globin messenger RNA by chromatography on oligothymidylic acid-cellulose.

A convenient technique for the partial purification of large quantities of functional, poly(adenylic acid)-rich mRNA is described. The method depends upon annealing poly(adenylic acid)-rich mRNA to oligothymidylic acid-cellulose columns and its elution with buffers of low ionic strength. Biologically active rabbit globin mRNA has been purified by this procedure and assayed for its ability to direct the synthesis of rabbit globin in a cell-free extract of ascites tumor. Inasmuch as various mammalian mRNAs appear to be rich in poly(adenylic acid) and can likely be translated in the ascites cell-free extract, this approach should prove generally useful as an initial step in the isolation of specific mRNAs.

Acrylamides↗
PubMed1972

Purification and properties of biologically active messenger RNA for a myeloma light chain.

A cell-free system derived from Krebs II ascites tumor has been used to assay biologically active mRNA for myeloma (MOPC-41) light chain during its purification by oligothymidylate-cellulose chromatography and sucrose gradient centrifugation. The purified mRNA directs the synthesis of a product that yields tryptic peptides corresponding to those derived from authentic myeloma protein and that forms a specific immunoprecipitate with antibody directed against the MOPC-41 protein. The fact that the light-chain mRNA anneals to oligothymidylic acid-cellulose suggests that it, like several other eukaryotic mRNAs, contains a region rich in adenylic acid residues. The most active fractions of light-chain mRNA, representing about 0.1% of the RNA originally extracted from membrane-bound myeloma polysomes, sediment as a discrete peak with an s(20,w) of about 13, roughly corresponding to an RNA molecule containing 850 bases. The results suggest that the light-chain mRNA is monocistronic and that it contains about 200 more bases than would be necessary to encode the variable and constant regions of a single light-chain molecule.

Adenine Nucleotides↗
PubMed1971

Characterization of the initial peptide of Q-beta RNA polymerase and control of its synthesis.

Bacteriophage Qbeta RNA directs the cell-free synthesis of two fMet-containing dipeptides prior to the first round of ribosomal translocation. One of these, fMet-Ala, corresponds to the initial segment of the Qbeta coat protein. In the present work we take advantage of the fact that translation of the Qbeta RNA polymerase cistron is repressed by its coat protein to correlate the other peptide, fMet-Ser, with the Qbeta RNA polymerase gene. Our experiments show that repression affects translation of the first two codons of the polymerase cistron, thereby isolating the effect of Qbeta coat repressor. Further studies, using single rounds of translocation of the Qbeta mRNA and codon-specific tRNAs, allow us to predict the first three amino acids of the Qbeta RNA polymerase protein, fMet-Ser-Lys, and to suggest that the initiation region of the Qbeta RNA polymerase cistron has the sequence ...AUG.UC[unk].AA[unk]...

Bacteriophages↗
PubMed1971

Protein synthesis directed by encephalomyocarditis virus RNA: properties of a transfer RNA-dependent system.

Small amounts of encephalomyocarditis virus RNA direct a 50-fold increase in amino acid incorporation, in appropriately supplemented ascites tumor cell extracts, under conditions that give rise to authentic viral polypeptides. Incorporation in these crude extracts has a novel characteristic, namely, that it is almost entirely dependent upon the addition of exogenous tRNA. Further, this incorporation is restricted in that tRNA derived from ascites tumor cells or from rat liver permits translation of viral RNA, whereas tRNA from yeast or Escherichia coli does not. These translational barriers are due, at least in part, to an incompatibility between the tRNA of yeast and E. coli and the aminoacyl-tRNA synthetases of the ascites tumor cell. A more extensive basis for this incompatibility is suggested, however, by the failure of the E. coli aminoacyl-tRNA synthetases to restore viral RNA-directed protein synthesis in the presence of tRNA from E. coli, although the coli synthetases fully restore the poly(U)-directed synthesis of polyphenyl-alanine. The possible role that unique or favored codon classes might play in this restriction is considered, together with the implications of the observed requirement for tRNA.

Alanine↗