Structural relationship between tRNALys2 and tRNALys4 from mouse lymphoma cells.

Mouse lymphoma cells have three major isoaccepting lysine tRNAs. Two of these isoacceptors, tRNALys2 and tRNALys4, were sequenced by rapid gel or chromatogram readout methods. They have the same primary sequence but differ in two modified nucleotides. tRNALys4 has an unmodified uridine replacing one dihydrouridine and an unidentified nucleotide, t6A*, replacing t6A. This unidentified nucleotide is not a hypomodified form of t6A. Thus, tRNALys4 is not a simple precursor of tRNALys2. Both tRNAs have an unidentified nucleotide, U**, in the third position of the anticodon. Also, partial sequences of minor homologs of tRNALys2 and tRNALys4 were obtained. The distinctions between tRNALys2 and tRNALys4 may be part of significant cellular roles as illustrated by the differential effects of these isoacceptors on the synthesis by lysyl-tRNA synthetase of diadenosine-5',5'''-P1,P4-tetraphosphate, a putative signal in DNA replication.

New plasma membrane proteins expressed by a high metastatic variant of a chemically induced tumor.

The composition of plasma membrane proteins of related low and high metastatic tumor cells were compared in order to elucidate possible genetic changes which may occur during progression of tumor cells towards metastatic capacity. For this purpose a procedure was worked out which allows the purification of mouse plasma membrane (PM) proteins from biosynthetically labeled cells. PM isolated from the two related T lymphoma lines Eb and ESb, which differ greatly in metastatic capacity, as well as from Con A-activated T cell blasts displayed similar degrees of purification as shown by marker enzyme analysis. When [35S]-methionine-labeled proteins of such PM preparations from cloned Eb and ESb cells were compared by 2-D gel electrophoresis it was found that maps of the highly metastatic variant ESb cells permitted detection of about 50 new protein spots which were not found on the 2-D gel maps of the parental Eb cells. Approximately 80% of these new spots were also found on maps derived from syngeneic splenic Con A blasts. This observation indicated that most of the newly expressed membrane proteins of ESb cells were possible differentiation or cell activation-related and not tumor cell-specific.

Factors affecting expression of glycolipid tumor antigens: influence of ceramide composition and coexisting glycolipid on the antigenicity of gangliotriaosylceramide in murine lymphoma cells.

Gangliotriaosylceramide (Gg3Cer) was previously described as a tumor-associated antigen in murine L5178Y lymphoma [Young, W. W., Jr., and Hakomori, S., Science (Wash. D.C.), 211: 487-489, 1981]. This paper describes the major factors affecting the expression of Gg3Cer at the surface of various clones of L5178Y lymphoma. Of 26 sublines that were recloned, six cell lines showing different degrees of Gg3Cer expression at the cell surface were used for analysis of the glycolipid composition as related to its cell surface antigenicity. Three remarkable correlations between glycolipid composition and the antigenicity of Gg3Cer have been found: (a) high-expressor sublines were characterized by a large proportion of a unique molecular species of Gg3Cer having alpha-hydroxypalmitic acid in its ceramide moiety in striking contrast to low expressors which did not contain this molecular species; (b) low expressors contained a large quantity of ganglio-N-tetraosylceramide (Gg4Cer) and NeuAc alpha 2 leads to 3Gal beta 1 leads to 3GalNAc beta 1 leads to 4Gal beta 1 leads to 4Glc beta 1 leads to 1 Cer (GM1b) gangliosides, whereas these glycolipids were almost absent in high-expressor clones; and (c) nonexpressors, which were converted from the high expressors in vivo through immunotherapy with the monoclonal antibodies to Gg3Cer, contained a large quantity of ganglio-N-tetraosylceramide and NeuAc alpha 2 leads to 3Gal beta 1 leads to 3GalNAc beta 1 leads to 4Gal beta 1 leads to 4Glc beta 1 leads to 1Cer. The nonexpressors should have an induced enzyme system to metabolize Gg3Cer to ganglio-N-tetraosylceramide and NeuAc alpha 2 leads to 3Gal beta 1 leads to 3GalNAc beta 1 leads to 4Gal beta 1 leads to 4Glc beta 1 leads to 1Cer. Three factors, i.e., ceramide composition, coexisting glycolipids, and an antibody-dependent glycolipid change, are therefore important in determination of glycolipid antigenicity and antigen modulation by antibodies. The ceramide composition may affect glycolipid organization in membranes, and the coexisting glycolipid having a longer carbohydrate chain may mask the accessibility of antibody to the antigenic glycolipid. The antigenic modulation by the action of the antibody in vivo may be based on activation of a new glycosyltransferase.

Characterization of tumor-associated ganglio-N-triaosylceramide in mouse lymphoma and the dependency of its exposure and antigenicity on the sialosyl residues of a second glycoconjugate.

Ganglio-N-triaosylceramide (GalNAc beta 1 leads to 4Gal beta 1 leads to 4Glc beta 1 leads to 1Cer), a tumor-associated marker for L5178 cells, was previously reported to separate on thin layer chromatography into three distinct bands (bands a, b, and c). The present paper describes the characterization of these bands and the factor that determines the degree of glycolipid exposure at the cell surface and its antigenicity. 1) The resolution of ganglio-N-triaosylceramide into three bands was found to be due to molecules having different fatty acid compositions. Band a contained nervonic (C24:1) and lignoceric (C24:0) acids, band b contained palmitic acid (C16:0), and band c contained alpha-hydroxypalmitic acid. 2) Surface labeling of L5178c127 cells with galactose oxidase/sodium borotritide, followed by fluorography of the isolated glycolipids, revealed that all three bands were exposed on the surface of the cell. However, treatment of cells with sialidase before treatment with galactose oxidase resulted in a 10-fold increase of label incorporated into ganglio-N-triaosylceramide. Since no sialylated form of ganglio-N-triaosylceramide was detected on these cells, and no change in the chemical amount of this glycolipid could be detected, the increase of label into this molecule was due to the exposure by sialidase of a normally cryptic glycolipid. The exposure of ganglio-N-triaosylceramide after sialidase treatment was also reflected by the increased sensitivity of these cells to monoclonal antibodies to the glycolipid and complement after enzyme treatment. Thus, the results provide clear evidence that crypticity, as well as antigenicity, of a membrane glycolipid is determined by the degree of sialylation in a second membrane glycoconjugate.

Artificial structure of chromatin derived in the preparation process.

Nuclei were isolated from mouse lymphoma L5178Y cells in the exponential growth phase, and chromatin was prepared by mechanical treatment of the nuclei. The nuclei and the chromatin were then digested to various extents with micrococcal nuclease and the resulting mono- and dinucleosome fractions of the two preparations were compared. During progressive digestion mononucleosomes from chromatin retained H1 histone and a DNA length of 165 base pairs, whereas those from nuclei released H1 histone and the length of their DNA decreased to 140 base pairs at an early stage of digestion. These nucleosomal preparations were always associated with nonhistone proteins. The dinucleosomes from nuclei contained larger amounts of nonhistone proteins than those from chromatin, but half of these proteins was released during the process of cleavage into mononucleosomes. The final mononucleosome preparation from nuclei retained 20% less nonhistone proteins than that from chromatin. The contents of nonhistone proteins in mono- and dinucleosomes from chromatin were the same. The electrophoretical distributions of molecular species of nonhistone proteins in mononucleosomes from nuclei and chromatin were different from each other: during digestion the profile of the former changed, whereas that of the latter remained constant. It is tentatively concluded that both H1 histone and nonhistone proteins were bound to nucleosomes more or less loosely in intact nuclei in situ, but that when the nuclear structure was disrupted these proteins became more tightly bound.

Irradiation-induced changes in nuclear shape and cell cycle.

Using human uterine cervical carcinoma cells transplanted in nude mice and mice leukemia L5178Y cells, changes in the cell cycle following irradiation were observed by flow cytometry (FCM), and changes in the cell nuclei during the course of irradiation were measured by FCM. Experiments in vivo as well as in vitro caused accumulation of cells in the G2 to M populations, resulting in the so-called G2 block phenomenon as revealed by FCM analysis of DNA distributions. The radiation-induced changes of nuclear shapes were dependent on abnormal mitoses, which occurred more frequently in the G2 to M phases. Therefore it is suggested that the G2 block phenomenon plays an important role in radiation-induced cell death because the process of cell death by irradiation has been shown to proceed via these abnormal mitoses.

The interaction of chromatin with alkylating agents. The monofunctional action of bis(2-chloroethyl)methylamine.

The reaction of L5178Y lymphoblast cell chromatin with the alkylating agent bis(2-chloroethyl)methylamine has been studied as a function of time, pH and reagent concentration. The reaction with DNA of chromatin from which the proteins were dissociated, as well as with purified calf thymus DNA, was studied in parallel. The extent of alkylation of DNA in intact chromatin was 4--5 times as much as in parallel free DNA samples; up to 4% of nucleotide base pairs were substituted. The extent of monofunctional substitution of the proteins was similar, on a weight basis, to that of DNA. Chromatographic analysis of the depurinated products showed that in chromatin, as in DNA, position N-7 of guanine is the major site of reaction. Up to 25% of the reaction products were guanines cross-linked as bis(2-guanin-7-yl-ethyl)methylamine, indicating a considerable degree of DNA-DNA cross linking. Column analysis shows that up to 40% of the nuclear proteins are cross-linked to DNA at 10 mM bis(2-chloroethyl)methylamine. The increased reactivity of intact chromatin is interpreted in terms of a conformational change in the position of the DNA bases when in the organized nucleohistone complex.

Intragenomic distribution of 5-methylcytosine in various forms of human and murine leukemic cells.

The degree of 5-methylcytosine formation in DNA sequences differing in reassociation rate and susceptibility to DNase II digestion has been investigated in human chronic myelogenic leukemia and acute leukemia leukocytes, human PHA-stimulated lymphocytes and murine L5178Y lymphoblasts cultured in various phases of growth. The results indicate that in all forms of cells studied by us the general pattern of intragenomic 5-methylcytosine distribution is similar, with two preferentially methylated regions: the sequences fast reassociating and rendered Mg++-soluble after DNase II digestion of nuclei. The most variable fraction as regards the level of methylation seemed to be DNA of Mg++-soluble fraction of DNase II digest, which in acute leukemia leukocytes, PHA-stimulated lymphocytes and exponentially growing L5178Y cells revealed about twice greater relative proportion of methylated cytosines than in leukocytes of chronic myelogenic leukemia and L5178Y cells maintained at saturation density.

Some problems in using density gradient centrifugation for synchronization of L5178Y-S cells.

The application of sucrose gradient sedimentation for synchronization of murine lymphoma L5178Y-S cells was investigated. Centrifugation of the cells in a linear 2--10% sucrose gradient in Fischer's medium allows to obtain populations enriched in (I) young cells, (II) DNA-synthesizing cells, and (III) old cells. Population I showed highest degree of synchrony and contained at least 70% G1 cells. However, only a proportion of cells from population I progressed normally (approximately 30% of cells from this population remained in G1 during post-separation incubation). It was shown that the sucrose concentrations used for separation did not affect growth of L5178Y-S cells. On the other hand, relatively gentle mechanical treatment severely inhibited proliferation of these cells. It is suggested that excellent adaptation of cells to the culture medium and complete stability of the medium's composition are the prerequisites for a successful synchronization of L5178Y cells by gradient centrifugation.

Heterogeneity of DNA methylation in murine L5178Y lymphoblasts.

Comparison of the extent of methylation in mouse DNA fragments rendered MgCl2 soluble after mild DNase II digestion of nuclei, with different reassociation rate and nucleoli-bound, revealed the existence of 3 regions of the genome particularly 5-methylcytosine-rich: the sequences considered to be related to the transcriptionally active chromatin with the highest content of this base and fast reassociating, as well as nucleolar DNA with somewhat lower proportion of the methylated cytosines.

Studies on the distribution of ribosomal proteins in mammalian ribosomal subunits.

Murine L5178Y cell ribosomes were dissociated into subunits either with potassium chloride in the presence of puromycin or with the chelating agent EDTA. The proteins of ribosomal subunits obtained by these different methods were compared by means of bidimensional polyacrylamide gel electrophoresis. KCl-derived 60S and 40S subunits were shown to contain 38 and 31 proteins respectively, 3 of them having identical electrophoretic mobilities. Preparations of EDTA-dissociated ribosomal subparticles contained different proportions of these proteins, and 11 major spots were shared between the EDTA-derived large and small ribosomal subunits. Furthermore, 10 proteins absent from subunits treated by high concentrations of KCl were reproducibly found in EDTA-treated ribosomal subparticles.