Differences in the incorporation of [3H]-glucosamine into nascent polypeptide chains on free polysomes and two fractions of membrane-bound polysomes in mouse myeloma cells

1978 ◽  
Vol 4 (4) ◽  
pp. 223-228 ◽  
Author(s):  
Ian F. Pryme ◽  
Asbj�rn M. Svardal
Keyword(s):  
Myeloma Cells ◽  
Nascent Polypeptide ◽  
Membrane Bound ◽  
Polypeptide Chains ◽  
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.

Extinction of expression of immunoglobulin genes in myeloma X fibroblast somatic cell hybrids

1987 ◽  
Vol 7 (2) ◽  
pp. 936-939
Author(s):  
A Greenberg ◽  
R Ber ◽  
Z Kra-Oz ◽  
R Laskov
Keyword(s):  
Light Chain ◽  
Immunoglobulin Genes ◽  
Somatic Cell Hybrids ◽  
Immunoglobulin Production ◽  
Myeloma Cells ◽  
Rna Transcripts ◽  
Processing Level ◽  
Early Processing ◽  
Polypeptide Chains ◽  
Mouse Myeloma

Adherent hybrids between immunoglobulin-producing mouse myeloma cells and fibroblasts do not produce immunoglobulin polypeptide chains. These hybrids retained the actively rearranged immunoglobulin genes of the myeloma parental cells but lacked immunoglobulin heavy- and light-chain RNA transcripts. We conclude that the shutoff of immunoglobulin production in these hybrids occurs at the transcription or early processing level.

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 IMMUNOGLOBULIN SYNTHESIS AND SECRETION

1970 ◽  
Vol 46 (1) ◽  
pp. 42-51 ◽  
Author(s):  
Isaac Schenkein ◽  
Jonathan W. Uhr
Keyword(s):  
Immunoglobulin G ◽  
Plasma Cells ◽  
Nascent Polypeptide ◽  
Immunoglobulin Synthesis ◽  
Polypeptide Chains ◽  
Mouse Myeloma

This study was designed to determine the time in the intracellular life of immunoglobulin when the carbohydrate moieties are added. Plasma cells from a mouse myeloma tumor were exposed to glucosamine-3H (a "bridge" sugar), galactose-3H, or leucine-3H. With each of the above isotopes, the percentage of total radioactive immunoglobulin that has been secreted after different periods of labeling and the extent to which puromycin prevented incorporation into immunoglobulin were determined. The results indicate that both galactose and glucosamine (in its N-acetyl form) become covalently incorporated into immunoglobulin G late in its intracellular life and suggest that glucosamine is also added onto nascent polypeptide chains (i.e., on polyribosomes).

scholarly journals SYNTHESIS, ASSEMBLY, AND SECRETION OF GAMMA GLOBULIN BY MOUSE MYELOMA CELLS

1971 ◽  
Vol 134 (5) ◽  
pp. 1316-1334 ◽  
Author(s):  
Reuben Baumal ◽  
Michael Potter ◽  
Matthew D. Scharff
Keyword(s):  
Lymph Nodes ◽  
Cell Lines ◽  
Gamma Globulin ◽  
Myeloma Cells ◽  
Cultured Cell ◽  
Cytoplasmic Proteins ◽  
Popliteal Lymph Nodes ◽  
Polypeptide Chains ◽  
Mouse Myeloma

The synthesis, assembly, and secretion of the three major subclasses of mouse IgG has been examined in 14 myeloma tumors and two cultured cell lines as well as in the cells from the popliteal lymph nodes of immunized mice. The total amount of IgG synthesized was between 15 and 43% of the cytoplasmic proteins made during a 15 min period. H2 and H2L were the major precursors of IgG2a and IgG1 but, in all of the tumors, HL was also an intermediate. In contrast, HL was a major precursor of IgG2b. Most of the noncovalent and covalent assembly of IgG occurred after release of the newly synthesized H and L chains from the polyribosomes and assembly was not completed until 10 min or more after the synthesis of the polypeptide chains.

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 Active translocon complexes labeled with GFP–Dad1 diffuse slowly as large polysome arrays in the endoplasmic reticulum

2002 ◽  
Vol 158 (3) ◽  
pp. 497-506 ◽  
Author(s):  
Andrei V. Nikonov ◽  
Erik Snapp ◽  
Jennifer Lippincott-Schwartz ◽  
Gert Kreibich
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Synthesis ◽  
Membrane Proteins ◽  
Effective Diffusion ◽  
Diffusion Constant ◽  
Temperature Sensitive ◽  
Lateral Mobility ◽  
Nascent Polypeptide ◽  
Membrane Bound ◽  
Polypeptide Chains

In the ER, the translocon complex (TC) functions in the translocation and cotranslational modification of proteins made on membrane-bound ribosomes. The oligosaccharyltransferase (OST) complex is associated with the TC, and performs the cotranslational N-glycosylation of nascent polypeptide chains. Here we use a GFP-tagged subunit of the OST complex (GFP–Dad1) that rescues the temperature-sensitive (ts) phenotype of tsBN7 cells, where Dad1 is degraded and N-glycosylation is inhibited, to study the lateral mobility of the TC by FRAP. GFP–Dad1 that is functionally incorporated into TCs diffuses extremely slow, exhibiting an effective diffusion constant (Deff) about seven times lower than that of GFP-tagged ER membrane proteins unhindered in their lateral mobility. Termination of protein synthesis significantly increases the lateral mobility of GFP–Dad1 in the ER membranes, but to a level that is still lower than that of free GFP–Dad1. This suggests that GFP–Dad1 as part of the OST remains associated with inactive TCs. Our findings that TCs assembled into membrane-bound polysomes diffuse slowly within the ER have mechanistic implications for the segregation of the ER into smooth and rough domains.

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