ABSTRACT
A modification of the hydroxylamine cleavage of proteins is presented in which proteins were cleaved while immobilized in the matrix of a polyacrylamide gel. The reaction under these conditions retains its high specificity for Asn-Gly bonds and has the advantage that the gel matrix, acting as a carrier, facilitates simultaneous treatment of many samples, and contributes to a high recovery efficiency (60-90%) of the cleavage products. The cleavage is performed with individual protein bands excised from dried slab gels after detection by staining, autoradiography, or fluorography. The procedure can be easily combined with other techniques to further characterize the cleavage fragments. Also a two-dimensional version of the cleavage method was developed, which allows rapid recognition of interrelationships between proteins in a complicated mixture. The versatility of the procedure is demonstrated in a number of applications. Highly related strains of murine leukemia viruses were easily distinguished from one another by the unique cleavage patterns of their gag- and env-precursor polypeptides. Comparing the env-precursor gPr82env synthesized in the presence or absence of tunicamycin with its cell-free synthesized counterpart, revealed the presence of an amino-terminal signal sequence. Cleavage patterns of pro-opiomelanocortin (POMC) from three different species revealed a high degree of homology between rat and mouse POMC, whereas Xenopus POMC was very different. Regions to which carbohydrates are attached could be identified by comparing glycosylated and unglycosylated forms of POMC. Combining the hydroxylamine cleavage procedure with immunological characterization of the fragments showed a small but significant difference between the amino-terminal sequences of the recombinant transforming protein P120 of Abelson murine leukemia virus and of its parent molecule Pr65gag of Moloney murine leukemia virus.
Subject(s)
Electrophoresis, Polyacrylamide Gel/methods , Hydroxylamines , Peptides/analysis , Proteins/isolation & purification , Animals , Mice , Molecular Weight , Pituitary Hormones, Anterior/isolation & purification , Pro-Opiomelanocortin , Protein Biosynthesis , Protein Precursors/isolation & purification , Rats , Viral Proteins/isolation & purification , Virion/genetics , XenopusABSTRACT
Precursor polyproteins containing translational products of the gag gene of Moloney murine leukemia virus were purified by gel electrophoresis and cleaved into large fragments by hydroxylamine, mild acid hydrolysis, or cyanogen bromide. The hydroxylamine cleavage method (specific for asparagine-glycine bonds) was adapted especially for this study. The electrophoretic mobility and antigenicity of the fragments and, in some cases, the presence or absence of [35S]methionine revealed detailed information on the structure of Pr65gag, gPr80gag, and Pr75gag (the unglycosylated variant of gPr80gag formed in vivo in the presence of tunicamycin or in vitro in a reticulocyte cell-free system). When compared with Pr65gag, gPr80gag contains 7,000 daltons of additional amino acids, presumably as, or as part of, a leader sequence at or very close to its N terminus. We present evidence that this leader may have replaced part of the p15 sequence. Furthermore, gPr80gag contains three separate carbohydrate groups. One is attached to the presumed leader sequence or to the p15 domain, and two are attached to the p30 domain. Each of the Moloney murine leukemia virus gag precursor proteins Pr65gag, gPr80gag, and Pr75gag corresponds with a read-through product into the pol gene. We designated these products Pr180gag-pol, gPr200gag-pol, and Pr190gag-pol (the unglycosylated variant of gPr200gag-pol), respectively. gPr200gag-pol contains all of the extra amino acids and carbohydrate groups present in gPr80gag and at least one carbohydrate group in its pol sequences.
Subject(s)
Moloney murine leukemia virus/analysis , Protein Precursors/analysis , Viral Proteins/analysis , Amino Acid Sequence , Carbohydrates/analysis , Cyanogen Bromide , Electrophoresis, Polyacrylamide Gel , Gene Products, gag , Hydrolysis , Hydroxylamine , Hydroxylamines , Protein Precursors/isolation & purification , Tunicamycin/pharmacology , Viral Proteins/isolation & purificationABSTRACT
We present the preparation of an ion exchange paper which can be used as a solid carrier in the transfer of RNA from gels. In addition to detection by blot hybridization to specific probes, transferred RNA can be characterized by cell-free translation in situ. Preparation of the paper is simple, inexpensive and reproducible. Examples of applications are shown and possible other applications are discussed.
Subject(s)
Moloney murine leukemia virus/genetics , Nucleic Acid Hybridization , Protein Biosynthesis , RNA, Messenger/genetics , RNA, Viral/genetics , Animals , Cell-Free System , Chromatography, Ion Exchange/methods , Reticulocytes/metabolismABSTRACT
Treatment of Ehrlich ascites cells with anisomycin induces an almost threefold increase in the level of native 60S ribosomal subunits. This increase is not the result of an increase in rate of synthesis or transport of these subunits but is caused by a defect in the joining of the 60S subunits to the smaller initiation complex to form an 80S complex. Experimental evidence for such a blocking of the "joining reaction" could be found in the formation of "half-mer"-type oligosomes and by the release of extra 40S subunits when these oligosomes were treated with ribonuclease. Cycloheximide, an inhibitor of the translocation reaction, and inhibitors of the initiation prevent the increase of native 60S subunits induced by anisomycin. Our results imply that the increse of 60S subunits induced by anisomycin may be helpful in estimating the amount of initiating mRNAs in the cell.
