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A purification procedure is described for two major toxic proteins from seeds of Abrus precatorius by successive chromatography on diethylaminoethyl-Sephadex A-50, carboxymethyl-cellulose, and DEAE-cellulose. The two toxins obtained both appeared homogeneous as judged by electrophoresis and analytical ultracentrifugation. Abrin A, the more positively charged protein obtained from the DEAE-cellulose chromatography, can be crystallized as needles from ammonium sulfate solution by the free interface diffusion technique; under various similar conditions, the other fraction, abrin C, failed to yield crystals. X-ray precession photographs have indicated that abrin A crystallizes in an orthorhombic unit cell of symmetry P212121 and dimensions a = 74.9, b = 269.8, and c = 70.3 A. The amino acid composition of abrins A and C are different. Molecular weights determined by sedimentation equilibrium are 60,100 for A as compared to 63,800 for C. After A has been treated with 2-mercaptoethanol at 100° for 2 min, polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate gives rise to one strong (corresponding to apparent subunit molecular weight of 32,000) and two much weaker bands. This is in sharp contrast with the pattern for C, two bands of approximately equal intensity, corresponding to apparent subunit molecular weights of 33,000 and 28,000. The two proteins also behave differently in affinity chromatography on a Sepharose 4B column. Abrin C at 1 µg per ml concentration level agglutinates sheep erythrocytes, whereas abrin A shows no such activity even at 40 µg per ml concentration level. Abrin C exhibits a more toxic effect on mice than abrin A. These differences in physicochemical and biological properties for the two abrins are presented in detail.

Histidyl transfer ribonucleic acid (tRNA) from 17 day mouse placenta was fractionated into six components by reversed-phase plaskon chromatography. Codon specificity of three components (tRNA2His, tRNA4His and tRNA6His) was studied by binding to mouse liver ribosomes in the presence of poly(AC) and poly(ACU). All three tRNAs respond equally to each polymer indicating a dual in vitro recognition of CAC and CAU. At present the data do not support changes in coding as a rationale for the quantitative variation shown by placental His-tRNAs during pregnancy.

Different mouse plasma cell tumors have been shown to produce different homogenous species of myeloma immunoglobulin in large amounts and are currently thought to consist of monoclonal cells ( Potter et al. , 1965 ; Lennox and Cohn, 1967 ). We have previously observed multiple isoaccepting tRNA's in a tumor of this type, MOPC 31C ( Yang and Novelli, 1968a ). In the present work, comparative studies of isoaccepting tRNA's by co-chromatography on a reversed-phase chromatographic column have been performed on cell lines of transplantable mouse plasma cell tumors that produce different kinds of myeloma immunoglobulin. Similar chromatographic patterns were observed for most of the aminoacyl-tRNA's examined in these tumors. A marked difference, however, was found between the seryl-tRNA's of an immunoglobulin-A producer (MPC 62) and those of immunoglobulin-G producers (MPC 47 and MOPC 31C).

Transfer RNAs from liver and brain of young and old BC3F1 mice were compared in regard to extent of aminoacylation and cochromatographic profiles of isoaccepting tRNA species on reversed-phase columns. Homologous synthetase preparations and optimal aminoacylation conditions were employed, having been determined for each amino acid and found to be the same for those from old and young mice. Small differences were found between tRNAs from young and old mice in the extent of acceptance for arginine and tyrosine in the liver and for aspartic acid in the brain. There were no differences observed between preparations from young and old mice in any of the cochromatographic profiles for the amino acids examined in this study, which included arginyl-, aspartyl-, glutamyl-, histidyl-, leucyl-, lysyl-, phenylalanyl-, seryl-, and tyrosyl-tRNAs from liver and arginyl-, aspartyl-, histidyl-, leucyl-, lysyl-, and seryl-tRNAs from brain. Comparisons of tRNA preparations from fetal and neonatal liver with those from adult liver did reveal both qualitative and quantitative differences. These results suggest that the postulated accumulation of errors as a result of age-related alterations in the translational mechanism does not occur in tRNA or aminoacyl-tRNA synthetases of these two tissues.

A high level of RNA-directed DNA polymerase activity is found in spleens of Balb c mice infected with Rauscher murine leukemia virus. The enzyme is associated with sedimentable subcellular fractions, can be separated from other cellular DNA polymerases, and is present in useful quantity for purification purposes. Complete solubilization of the enzyme, achieved and maintained by using a combination of detergent and high salt solution, is required for a satisfactory purification both in the initial extraction procedure and in subsequent column chromatography.

The tRNA synthesized by Escherichia coli during chloramphenicol treatment has altered chromatographic properties, as analyzed by reversed-phase chromatography. The data suggest that most of the tRNAs synthesized in the presence of chloramphenicol are immature, chromatographically distinct forms, capable of being converted to mature forms upon removal of chloramphenicol. Methylation of tRNA during chloramphenicol treatment appears to be quantitatively and qualitatively normal. The tRNA synthesized during chloramphenicol treatment differs from normal tRNA mainly in that it contains 60–70% less 4-thiouridine and dihydrouridine. Preliminary experiments indicate that these two minor bases are not required for normal aminoacylation. Phenylalanyl-, valyl-, isoleucyl- and lysyl-tRNAs from untreated and chloramphenicol-treated cells are equally functional in an in vitro hemoglobin-synthesizing system.

Soluble extracts of mouse cells with Fv-1 n or Fv-1 b gene alleles specifically and reciprocally inhibit infection of B- or N-tropic mouse leukemia viruses in permissive cell cultures. NB-tropic virus infection was not inhibited by either cell extract. Extracts from Fv-1 - cells did not inhibit infection by the three virus host-range types, but N- or B-tropic virus infection of Fv-1 - cells was inhibited by extracts of the nonpermissive cells, and Fv-1 nb cell extracts inhibited both viruses. The maximum degree of inhibition was 50-80% as determined by immunofluorescent or plaque assays, with extracts containing up to 500 μg/ml of nonpermissive cell protein. The inhibitor(s) is relatively unstable since activity is lost after 2 hr at 37° or 30 min at 56°. The inhibitor(s) was most effective if added 2 hr before or within 2 hr after infection, did not react with the virus directly, inhibit virus attachment, or inhibit the normal cell functions tested. These results indicate that nonpermissive mouse cells contain a product, possibly determined by the Fv-1 gene, which inhibits some early postpenetration event(s) in leukemia virus infection.

Unique 4S RNA species from AKR mouse embryo cells hybridize with AKR murine leukemia virus and avian myeloblastosis virus 35S RNAs in vitro . Analyses by reversed-phase column chromatography indicate that the major 4S species that hybridize with the two viral RNAs are probably the same. A 4S RNA species with similar chromatographic properties is a major component of the AKR viral 4S RNA which associates with the viral 70S RNA in vivo .

It is well recognized that immunoglobulins consist of heterogeneous populations of antibody proteins which are synthesized following a complicated series of cellular events induced by antigen stimulation. Chemical studies of purified antibodies suggested that antibodies of different specificities may differ from one another in their primary protein structure (Koshland and Englberger, 1963). The mechanism by which this tremendous variety of immunoglobulins are synthesized has thus been a very interesting biological problem. Biochemical knowledge concerning transcription and translation of the genetic messages for the synthesis of these proteins is, however, extremely deficient. This is due mainly to the difficulty in obtaining for study a sufficient quantity of lymphoid tissue with a given homogeneity of antibody production.

Publisher Summary This chapter describes an application of the reversed-phase column chromatographic system (RPC); presents general procedures for preparing tRNA and aminoacyl-tRNA synthetases from the tissue and for preparing labeled aminoacyl-tRNA suitable for the chromatographic analysis; and demonstrates the analysis of iso-accepting tRNAs of an amino acid with a sample size in the range of 1 A260 unit of mixed tRNA. This method is very useful, especially in cases, where the source of material for preparing tRNA is limited, such as tissue culture cells and small animal organs. The sensitivity of this method is facilitated by the availability of radioactive amino acids with high specific activity. The isoaccepting peaks of an aminoacyl-tRNA are sufficiently well resolved in the column that quantitative comparative studies can be done by cochromatography of two samples with different labels. Because, the size of the tRNA sample is small, it becomes very important that artificial modifications of amino-tRNA should be prevented. The profiles of isoaccepting tRNAs for individual amino acids are much different between the mammalian and the bacterial sources.