scholarly journals Membrane-bound ribosomes of myeloma cells. IV. mRNA complexity of free and membrane-bound polysomes.

1981 ◽  
Vol 88 (1) ◽  
pp. 29-36 ◽  
Author(s):  
B Mechler ◽  
T H Rabbitts
Keyword(s):  
Cross Hybridization ◽  
Myeloma Cells ◽  
Mrna Species ◽  
Sequence Complexity ◽  
Polysomal Mrna ◽  
Reticulocyte Lysate ◽  
Cdna Hybridization ◽  
Membrane Bound ◽  
Mouse Myeloma

We have analyzed the sequence complexity, frequency distribution, and template activity of free (F) and membrane-bound (MB) polysomal mRNA populations of MOPC 21 (P3K) mouse myeloma cells. Using the technique of mRNA-cDNA hybridization, we find that F poly(A)+ RNA, which represent 60% of total polysomal mRNA, consists of approximately 8,000 different mRNA sequences distributed in three abundance classes, while MB poly(A)+ RNA (20% of total polysomal mRNA) includes only 230 mRNA species and almost completely lacks very infrequent mRNA species. Cross-hybridization indicates that MB mRNA sequences are also present in F mRNA, but in reduced concentrations. Translation of F and MB RNA fractions in a messenger-dependent reticulocyte lysate indicates that essentially all MB RNA contains poly(A), whereas 25% of F mRNA lacks poly(A). Furthermore, the use of a cDNA highly specific for the immunoglobulin light (Ig L) chain mRNA allows the determination of the subcellular content of this message. Ig L mRNA, representing approximately 5% of total polysomal poly(A)+ RNA, is one of the most abundant MB mRNAs. 90% of Ig L mRNA is found in MB polysomes and 10% in F polysomes.

scholarly journals Membrane-bound ribosomes of myeloma cells. V. Subcellular distribution of immunoglobulin mRNA molecules.

1981 ◽  
Vol 88 (1) ◽  
pp. 37-41 ◽  
Author(s):  
B Mechler
Keyword(s):  
Endoplasmic Reticulum ◽  
Subcellular Distribution ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Size Class ◽  
Template Activity ◽  
Myeloma Cells ◽  
Reticulocyte Lysate ◽  
Membrane Bound ◽  
Mouse Myeloma

The subcellular distribution of the most abundant mRNA sequences, particularly those of the immunoglobulin heavy (Ig H) and light (IG L) chain mRNA sequences, of MOPC 21 (P3K) mouse myeloma cells has been examined by translating the mRNA of various subcellular fractions in a messenger-dependent reticulocyte lysate (MDL) and by identifying Ig products with the use of a specific antiserum. Analyses of the distribution of the mRNA template activity and the translation products by SDS polyacrylamide gel electrophoresis reveal that approximately 85% of the mRNA present in the free ribosomal fraction is incorporated into polysomes and that the remainder is present as mRNP particles. On the endoplasmic reticulum (ER) the mRNA is found entirely in polysomes. In general, the size class of free (F) and membrane-bound (MB) polysomes corresponds to the size of their translation products. Thus, mRNAs coding Ig H (5.0 x 10(5) daltons in size) and Ig L (2.5 x 10(5) daltons in size) are incorporated into polysomes formed of 12 and 6 ribosomes, respectively. About 10% of the Ig mRNAs are not bound to membranes. A third of these are associated with mRNPs and the remainder incorporated into F polysomes of the same size as the Ig-synthesizing MB polysomes.

scholarly journals Electrical determination of viability in saline-treated mouse myeloma cells

1982 ◽  
Vol 39 (1) ◽  
pp. 41-47 ◽  
Author(s):  
T. Matsushita ◽  
A.M. Brendzel ◽  
M.A. Shotola ◽  
K.R. Groh
Keyword(s):  
Treated Mouse ◽  
Myeloma Cells ◽  
Mouse Myeloma

scholarly journals Membrane-bound ribosomes of myeloma cells. VI. Initiation of immunoglobulin mRNA translation occurs on free ribosomes.

1981 ◽  
Vol 88 (1) ◽  
pp. 42-50 ◽  
Author(s):  
B Mechler
Keyword(s):  
Polypeptide Chain ◽  
Mrna Translation ◽  
Normal Growth ◽  
Kinetic Studies ◽  
Myeloma Cells ◽  
Nascent Polypeptide ◽  
Membrane Attachment ◽  
Membrane Bound ◽  
Terminal Amino ◽  
Mouse Myeloma

Immunoglobulin heavy (Ig H) and light (Ig L) chain mRNA molecules have been released from the endoplasmic reticulum (ER) membranes as free (F) mRNP particles when MOPC 21 (P3K) mouse myeloma cells are exposed to a hypertonic initiation block (HIB). The subsequent fate of these mRNA sequences has been examined when the cells are returned to normal growth medium. Upon return to isotonicity, all previously translated mRNA molecules reassociate with ribosomes and form functional polysomes. Ig H mRNA is found incorporated first into F polysomes and then into membrane-bound (MB) polysomes. Kinetic studies indicate that the time of passage of Ig H mRNA in F polysomes is approximately 30 s, during which a nascent polypeptide chain of approximately 80 amino acids would have been completed. When the rate of polypeptide elongation is depressed with emetine during the recovery from HIB, both Ig H and L mRNA molecules accumulate in small F polysomes. These results indicate that the formation of Ig-synthesizing polysomes proceeds in the sequence: mRNA leads to F polysomes leads to MB polysomes. With the additional observation that during HIB recovery puromycin completely prevents the reassociation of Ig mRNA with the ER, these findings support a model of MB polysome formation in which the specificity of membrane attachment is determined by the nature of the N-terminal amino acid sequence of the nascent polypeptide chain.

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