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.

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. II. Kinetic studies on the entry of newly made ribosomal subunits into the free and the membrane-bound ribosomal particles.

1975 ◽  
Vol 67 (1) ◽  
pp. 16-24 ◽  
Author(s):  
B Mechler ◽  
P Vassalli
Keyword(s):  
Messenger Rna ◽  
Specific Radioactivity ◽  
Kinetic Studies ◽  
Chain Termination ◽  
High Turnover ◽  
Ribosomal Subunits ◽  
Myeloma Cells ◽  
High Specific Radioactivity ◽  
Membrane Bound ◽  
Kinetics Of

The kinetics of appearance of newly made 60S and 40S ribosomal subunits in the free and membrane-bound ribosomal particles of P3K cells were explored by determining the specific radioactivities of their 18S and 28S RNA after various lengths of [3H]uridine pulse. Both 40S and 60S subunits enter free and membrane-bound polyribosomes at comparable rates from the cytoplasmic pool of newly made, free native subunits, the 40S subunits entering the native subunit pool and the polyribosomes slightly earlier than the 60S subunits. At all times, the specific radioactivity of the membrane-bound native 60S subunits was slightly lower than that of the polyribosomal 60S subunits. This indicates that the membrane-bound native 60S subunits are not precursors destined to enter membrane-bound polyribosomes and suggests that they result from the dissociation of ribosomes after chain termination. The results observed also suggest that the membrane-bound native 60S subunits are not reutilized before their release from the membranes, which probably takes place shortly after dissociation from their 40S subunits. The monoribosomes, both free and membrane-bound, had the lowest specific radioactivities in their subunits. Finally, a small amount of newly made native 40S subunits, containing 18S RNA of high specific radioactivity, and apparently also newly made messenger RNA were detected on the membranes. The high turnover of these membrane-bound native 40S subunits suggests that they may represent initiation complexes formed with mRNA which has just reached the membranes and which has not yet given rise to polyribosomes.

scholarly journals Membrane-bound ribosomes of myeloma cells. III. The role of the messenger RNA and the nascent polypeptide chain in the binding of ribosomes to membranes.

1975 ◽  
Vol 67 (1) ◽  
pp. 25-37 ◽  
Author(s):  
B Mechler ◽  
P Vassalli
Keyword(s):  
Protein Synthesis ◽  
Messenger Rna ◽  
Membrane Fraction ◽  
Polypeptide Chain ◽  
Salt Treatment ◽  
Ribosomal Subunits ◽  
Myeloma Cells ◽  
Membrane Bound ◽  
Made In

Mild ribonuclease treatment of the membrane fraction of P3K cells released three types of membrane-bound ribosomal particles: (a) all the newly made native 40S subunits detected after 2 h of [3H]uridine pulse. Since after a 3-min pulse with [35S]methionine these membrane native subunits appear to contain at least sevenfold more Met-tRNA per particle than the free native subunits, they may all be initiation complexes with mRNA molecules which have just become associated with the membranes; (b) about 50% of the ribosomes present in polyribosomes. Evidence is presented that the released ribosomes carry nascent chains about two and a half to three times shorter than those present on the ribosomes remaining bound to the membranes. It is proposed that in the membrane-bound polyribosomes of P3K cells, only the ribosomes closer to the 3' end of the mRNA molecules are directly bound, while the latest ribosomes to enter the polyribosomal structures are indirectly bound through the mRNA molecules; (c) a small number of 40S subunits of polyribosomal origin, presumably initiation complexes attached at the 5' end of mRNA molecules of polyribosomes. When the P3K cells were incubated with inhibitors acting at different steps of protein synthesis, it was found that puromycin and pactamycin decreased by about 40% the proportion of ribosomes in the membrane fraction, while cycloheximide and anisomycin had no such effect. The ribosomes remaining on the membrane fraction of puromycin-treated cells consisted of a few polyribosomes, and of an accumulation of 80S and 60S particles, which were almost entirely released by high salt treatment of the membranes. The membrane-bound ribosomes found after pactamycin treatment consisted of a few polyribosomes, with a striking accumulation of native 60S subunits and an increased number of native 40S subunits. On the basis of the observations made in this and the preceding papers, a model for the binding of ribosomes to membranes and for the ribosomal cycle on the membranes is proposed. It is suggested that ribosomal subunits exchange between free and membrane-bound polyribosomes through the cytoplasmic pool of free native subunits, and that their entry into membrane-bound ribosomes is mediated by mRNA molecules associated with membranes.

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.

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