JBC, Vol. 252, Issue 8, 2592-2598, Apr, 1977
Initiation and translation in vitro of mRNA for MOPC 315 immunoglobulin heavy chain and characterization of translation product
D. L. Bedard and R. C. Huang
An initiation study of mineral oil-induced plasmacytoma (MOPC) 315 heavy
chain immunoglobulin (H315) in vitro has been conducted using
formyl-[35S]methionyl-tRNAfMet and a highly purified 18 S message from MOPC
315 solid tumor in a crude rabbit reticulocyte lysate system. The product
was specifically precipitated by antibodies directed against MOPC 315
immunoglobulin and H315. The in vitro H315 products terminally labeled with
formyl-[35S]methionine or internally labeled with [3H]leucine were
electrophoretically identical with in vivo H315 on sodium dodecyl
sulfate-polyacrylamide gels. All of the [35S]-methionine was incorporated
at the NH2 terminus, not internally, since there is a near complete
recovery of [35S]methionine following one cycle of Edman degradation. The
NH2-terminal cyanogen bromide peptide, CN2, of in vivo and in vitro H315
co-migrated exactly on gel electrophoresis under conditions which
completely resolved two proteins differing in size by only 14 amino acids.
These data strongly suggest that there is no NH2-terminal precursor of H315
in this system. Cyanogen bromide peptide profiles of in vivo and in vitro
H315 were chromatographically indistinguishable. Three peptides, CN1, CN2,
and CN4, which represent approximately 85% of the total amino acids of H315
were isolated and further characterized by electrophoresis and paper
chromatography. All were very similar to the corresponding peptides of
authentic H315. We conclude that the fidelity of H315 translation is
preserved in vitro.
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