Isolation of messenger-like RNA from immunochemically separated polyribosomes (original) (raw)
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Nucleic Acids Research, 1986
RNA blotting was employed to examine polyadenylated imunoglobulin a chain RNAs in a B lymphoma synthesizing membrane-bound and secretory IgA and in a hybridoma which synthesizes predominantly secretory IgA. Both cell lines were derived from the I.29 lymphoma and expressed the identical heavy chain variable region gene. In addition to the predicted mRNA precursors, four novel species of polyadenylated a RNAs were detected. (1) The presence of a RNA species which was too large to have the same 3' end as the largest mRNA for membrane-bound a chain (am) implied that transcription continued past the am poly(A) site, and that such transcripts could be polyadenylated. Alternatively, transcription of this aRNA was initiated 5' to the normal cap site. (2) Two species of RNA were detected which encoded the am domain and the intervening sequence between the a constant (Ca) and am domains but not the Ca domain. These RNA molecules were of sizes appropriate for their derivation by endonucleolytic cleavage of a precursor for am mRNA at the poly(A) site of the mRNA for secreted a chains (as). The presence of these three a RNA species suggested that alternative and successive cleavage/polyadenylation events could occur on a single transcript to produce either am or as mRNAs. (3) An additional novel species of RNA was detected which indicated that the order of removal of the large IVSs did not always proceed in the 5' to 3' direction.
A major species of mammalian messenger RNA lacking a polyadenylate segment
Proceedings of the National Academy of Sciences, 1976
Translation of total polysomal RNA from sarcoma 180 ascites cells in a wheat germ cell-free system produces two major polypeptides, A and B, with molecular weights of 50,000 and 45,000, respectively. Fractionation on Millipore filters or on oligo(dT)-cellulose leads to retention of the mRNA specific for protein A in the poly(A)-containing fraction and to accumulation of the B mRNA in the unadsorbed poly(A)-deficient fraction. The mRNA for B sediments at approximately 18 S; it is released as a 50S ribonucleorprotein upon EDTA treatment of polysomes. Its translation is particularly sensitive to an inhibitor present in the polysomal RNA. The poly(A)-deficient mRNA for the 45,000 dalton polypeptide is also present in mouse myeloma MPC-11 cells, where it seems to be localized in membrane-bound polysomes.
European Journal of Biochemistry, 1975
The conformation in solution of duck and rabbit globin mRNA, and of the duck mRNA in the mRNA . protein particle, has been investigated by optical methods and also by the use of the dye ethidium bromide which becomes highly fluorescent when intercalated into the double-stranded regions of a nucleic acid. On the basis of the properties of this dye and on the ability of homopolyribonucleotides to form double-stranded structures we have, in addition, developed a simple and sensitive assay for the detection and quantitisation of sequences rich in a particular residue that may be present in an RNA chain.
Cytoplasmic RNA in immunoglobulin-secreting myeloma tumor cells
Biochimica et Biophysica Acta (BBA) - Nucleic Acids and Protein Synthesis, 1972
A set of heterodisperse RNA molecules was identified in cytoplasmic extracts of a cultured strain of mouse myeloma cells (Strain MOPC 21). The molecules range in size from lO 5 to lO s daltons, as estimated from their electrophoretic mobilities on polyacrylamide gels. Most of the species are 4" lO5-8 " lO5 daltons. Synthesis of two of these species is selectively inhibited by I/~g/ml of ethidium bromide; these correspond in size to mitochondrial ribosomal RNA in HeLa cells. Synthesis of the other RNA molecules is not preferentially inhibited by ethidium bromide, suggesting a nuclear origin. Also, they are not affected by a concentration of actinomycin D which inhibits (nuclear) ribosomal RNA synthesis.
Biochimica Et Biophysica Acta Nucleic Acids and Protein Synthesis, 1972
A set of heterodisperse RNA molecules was identified in cytoplasmic extracts of a cultured strain of mouse myeloma cells (Strain MOPC 21). The molecules range in size from lO 5 to lO s daltons, as estimated from their electrophoretic mobilities on polyacrylamide gels. Most of the species are 4" lO5-8 " lO5 daltons. Synthesis of two of these species is selectively inhibited by I/~g/ml of ethidium bromide; these correspond in size to mitochondrial ribosomal RNA in HeLa cells. Synthesis of the other RNA molecules is not preferentially inhibited by ethidium bromide, suggesting a nuclear origin. Also, they are not affected by a concentration of actinomycin D which inhibits (nuclear) ribosomal RNA synthesis.
On the RNA in cultured myeloma cells producing immunoglobulin
Biochimica et Biophysica Acta (BBA) - Nucleic Acids and Protein Synthesis, 1969
The results of an investigation of RNA in a cell strain secreting immunoglobulin (MOPC 21) growing in cell culture are reported. Most of the cytoplasmic RNA is found in the polysomes, about half of which are associated with a membrane fraction. In several cell types the amount of RNA associated with the membranes varies and is correlated with the cell's ability to produce and secrete the antibody-like protein. In MOPC 21 both classes of polysomes are only degraded slowly in the presence of 5 #g/ml of actinomycin D.
Biochemistry, 1977
The sequence preceding the 3'-terminal poly(adeny1ic acid) [poly(A)] tract of the immunoglobulin K chain mRNA from mouse myeloma MOPC 41A was studied by analysis of complementary DNA (cDNA). Short 32P-labeled cDNA was synthesized on the mRNA using DNA polymerase I of Escherichia coli and oligo(dT) as a primer. The cDNA was characterized by analyzing the oligonucleotides produced by digestion with T4 endonuclease IV. The sequence was also studied by an adaptation (Brownlee, G. G., and Cartwright, E. M. (1 977), J. Mol. Biol. (in press)) of a rapid sequencing method using gel electrophoresis (Sanger, F., and Coulson, A. R. (1975), J. Mol. Biol. 94, 441). Labeled cDNA of variable length, synthesized using reverse transcriptase of avian myeloblastosis virus, was extended with this enzyme in four reactions, each of which contained only three deoxynucleoside triphosphates (dNTPs). Displaying the M e s s e n g e r RNAs (mRNAs) of eukaryotes contain a noncoding region before the 3'-terminal poly(A)I tract (for reviews, see . Determination of more sequences in this region should help to clarify its function. Direct sequence analysis of eukaryotic mRNAs has proven difficult, because it is not generally feasible to label the molecules sufficiently in vivo. Therefore attention has turned to sequencing methods based upon analysis of radioactive cDNA made on mRNA templates. Labeled cDNA of high specific activity can be prepared with C Y -~~Plabeled dNTPs in a reaction catalyzed either by a viral reverse transcriptase or by DNA polymerase I of Escherichia coli, which gives shorter products . Since the synthesis is generally primed by oligo(dT) on the poly(A) tract of the mRNA, the cDNA sequence is complementary to the 3'-terminal portion of the mRNA.
1967
I. An RNA that we identify as the 9-S messenger RNA previously studied can be released from rabbit reticulocyte polyribosomes by EDTA or CM-cellulose treatment. 2. The criteria for this identification are: high specific radioactivity as compared with ribosomal RNA; high sensitivity to ribonuclease when polyribosomes are treated with minute quantities of this enzyme; a sedimentation velocity identical to that of 9-S messenger RNA after sodium dodecyl sulfate treatment. 3. However, this RNA liberated from reticulocyte polyribosomes by EDTA or CM-cellulose treatment is probably associated with proteins. Indeed, it sediments more rapidly than purified 9-S mRNA if sodium dodecyl sulfate treatment is omitted. 4.5-S RNA is also released from polyribosomes by the procedure used to liberate messenger RNA. Abbreviations: mRNA, messenger RNA; tRNA, transfer RNA.