Polypeptide composition of rat sperm outer dense fibers. A simple procedure to isolate the fibrillar complex

Treatment of caput or cauda epididymal rat sperm with a low concentration (0.05%) of the cationic detergent cetyltrimethylammonium bromide and 30 mM 2-mercaptoethanol solubilized most of the sperm structures except for the sperm head and the outer dense fiber-connecting piece complex. The latter were purified, and about 10% of these complexes are formed by nine fibers attached to the connecting piece. Of these fibers, two are shorter than the other seven and presumably correspond to fibers 3 and 8 (Fawcett, D.W. (1975) Dev. Biol. 44, 394-436). Electron microscopy confirmed the purity of the isolated outer dense fibers and revealed their characteristic irregular cross-sectional shape. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate showed six major polypeptides (Mr = 87,000, 30,400, 26,000, 18,400, 13,000, and 11,500) with a high content of serine, aspartic and glutamic acids, proline, cysteine, leucine, and tyrosine. Furthermore, several lines of evidence indicate a close structural relationship between the components of 30,400 and 26,000 Da. The six major components of the fibers are phosphorylated at serine residues. These results indicate that the major components of rat sperm outer dense fibers are a unique family of phosphoproteins.     

Properties of cloned promoters

Strong early bacteriophage T7 promoters A2 and A3 and also A2 and lac UV5 promoters with altered segments downstream the initiation of RNA start point were cloned using specially constructed plasmid vectors pBRS188 and PBRS240. The relative signal strengths of these promoters in vivo and in vitro were evaluated and the kinetic parameters of their interaction with RNA polymerase were determined. It has been shown that the nucleotide sequence of the transcribed region plays a significant role in specific promoter-RNA polymerase interaction and that the rate-limiting step of RNA synthesis initiation is different for various promoters.     

Selective alkylation of G24 residue in tRNAPhe

Alkylation of E. coli tRNAPhe with 4-(N-2-chloroethyl-N-methylamino) benzyl-5'-phosphamide of oligonucleotide d(pAACCA) was studied. G24 residue located near the sequence C17GGDA21 partially complementary to the oligonucleotide moiety of the reagent was shown to be alkylated. Oligonucleotide d(pAACCA) inhibited the alkylation. Association constant of oligonucleotide derivative with tRNAPhe (10(3) M-1) was evaluated from the dependence of the extent of tRNA modification on the concentration of the reagent. The reported method for selective alkylation of tRNA may be used for preparing photoaffinity derivatives of tRNA bearing an arylazidogroups in desired position.     

Localization of histone H1 in chromatin. Cross-linking of the N- and C-terminal halves of the molecule with bifunctional reagents

Mutual arrangement of histone H1 molecules was studied in calf thymus nuclei, extended chromatin and chromatin, isolated and kept in 8 M urea. Histone H1 dimers crosslinked with methyl 4-mercaptobutyrimidate were digested with chymotrypsin and crosslinked fragments obtained were analysed by diagonal gel electrophoresis. In all chromatins tested the N- and C-terminal parts of the H1 molecules were crosslinked in all possible combinations, i.e. C-C, C-N and N-N. These and related data obtained earlier indicate, that the proximity of histone H1 molecules in chromatin is determined by the structure of nucleosomal chain itself and not by chromatin superstructure. The results also suggest that the H1A and H1B subfractions of histone H1 are interspersed in extended nucleosomal chains.     

Ta1, a novel 105 KD human T cell activation antigen defined by a monoclonal antibody

By using a murine monoclonal antibody produced against an IL 2-dependent human T cell line, we defined a T lineage-specific molecule, termed Ta1, that is expressed strongly on activated T lymphocytes of both the T4 and T8 subsets, as well as on T cell lines and clones, but only weakly on a fraction of resting T cells. SDS-PAGE analysis of immunoprecipitates from 125I-labeled, activated T cells demonstrates a single major band of apparent m.w. 105 KD under both reducing and nonreducing conditions. Unlike anti-IL 2 receptor antibodies, anti-Ta1 does not inhibit T cell proliferative responses to mitogen, antigen, or IL 2-containing medium. Moreover, anti-Ta1 has no effect on T cell-mediated cytotoxicity. Ta1 appears to be a novel human T cell-specific activation antigen that may serve as a useful marker of T cell activation in human disease.     

Thymectomized, irradiated, and bone marrow-reconstituted chimeras have normal cytolytic T lymphocyte precursors but a defect in lymphokine production

A model system has been developed to study extrathymic T cell differentiation; mice have been thymectomized, lethally irradiated, and reconstituted with bone marrow cells depleted of Thy-1+ cells. After 8 wk, the spleen cells of these athymic, bone marrow-reconstituted chimeras contain Thy-1+ precytolytic T lymphocytes (CTL) that are able to respond to antigen only if supernatant from Con A-activated T cells is added to culture. The phenotype of these pre-CTL is similar to that of thymocytes, suggesting that they may be immature T cells. Initial evaluation of the CTL repertoire of these athymic mice demonstrated that the CTL generated to trinitrophenyl-modified syngeneic cells are H-2-restricted, and that the CTL generated to alloantigens have many of the cross-reactivities observed in normal mice but not in nude mice. In this report, we demonstrate a helper T cell defect in these thymectomized chimeras. These chimeras lack an Ly-1+ helper cell required for thymocytes to differentiate to CTL. Further studies revealed that when spleen cells from these thymectomized chimeras were stimulated with Con A, they produced normal levels of interleukin 2. However, these splenocytes were defective in the production of another factor needed for CTL differentiation.     

T cell repopulation from functionally restricted splenic progenitors: 10,000-fold expansion documented by using limiting dilution analyses

Mice depleted of T cells by thymectomy, lethal irradiation, and reconstitution with Thy-1-depleted syngeneic bone marrow were given graded doses of splenic T cells to see whether post-thymic cells had the ability to regenerate immune function in these hosts. Using limiting dilution methods to estimate the number of antigen- and mitogen-responsive cells in recipients 12 to 20 wk after reconstitution, we found that new helper and cytotoxic precursor cells were produced, but attained levels only 10 to 20% of normal. Because these repopulated mice were able to produce nearly normal levels of helper and cytotoxic activity in conventional, high density cultures, despite their relative paucity of precursors, we infer that their normal function in conventional assays may reflect a balanced deficiency of effector and regulatory cell types. Surface phenotyping of the progenitor cells responsible for repopulation showed that Lyt-2- cells were required for helper cell regeneration and that Lyt-2+ cells acted as progenitors only for the cytotoxic lineage, contrary to earlier speculation that the splenic Lyt-1+ 2+ (Ly-123) pool included cells antecedent to both effector lineages. Comparison of the number of injected progenitors needed to produce repopulation with the number of new precursor cells eventually produced suggests that the relevant progenitors are able to undergo 10,000-fold expansion in 12 to 20 wk. Numerical expansion in the periphery from thymic-processed cells could well be a major source of new lymphocytes in adult mice.     

Phenotypic heterogeneity of antisyngeneic tumor killer cells (ASTK) generated in allogeneic mixed lymphocyte reactions

By using monoclonal antibodies to Thy-1, Lyt-2, and Qa-5 differentiation antigens, we demonstrated a heterogeneity of cytotoxic cells developed in allogeneic mixed lymphocyte responses that lyse tumor cells syngeneic with the responder cells. There are minimally two Thy-1+ populations, one of which is Lyt-2+ and the other Lyt-2-. There is probably also a Thy-1- population. Most of the Lyt-2- tumor killer cells are Qa-5+, and most of the Lyt-2+ tumor killer cells are Qa-5-.     

Interstrand duplexes in Friend erythroleukemia nuclear RNA. The interaction of non-polyadenylated nuclear RNA with polyadenylated nuclear RNA and with small nuclear RNAs

Intermolecular duplexes among large nuclear RNAs, and between small nuclear RNA and heterogeneous nuclear RNA, were studied after isolation by a procedure that yielded protein-free RNA without the use of phenol or high salt. The bulk of the pulse-labeled RNA had a sedimentation coefficient greater than 45 S. After heating in 50% (v/v) formamide, it sedimented between the 18 S and 28 S regions of the sucrose gradient. Proof of the existence of interstrand duplexes prior to deproteinization was obtained by the introduction of interstrand cross-links using 4'-aminomethyl-4,5',8-trimethylpsoralen and u.v. irradiation. Thermal denaturation did not reduce the sedimentation coefficient of pulse-labeled RNA obtained from nuclei treated with this reagent and u.v. irradiated. Interstrand duplexes were observed among the non-polyadenylated RNA species as well as between polyadenylated and non-polyadenylated RNAs. beta-Globin mRNA but not beta-globin pre-mRNA also contained interstrand duplex regions. In this study, we were able to identify two distinct classes of polyadenylated nuclear RNA, which were differentiated with respect to whether or not they were associated with other RNA molecules. The first class was composed of poly(A)+ molecules that were free of interactions with other RNAs. beta-Globin pre-mRNA belongs to this class. The second class included poly(A)+ molecules that contained interstrand duplexes. beta-Globin mRNA is involved in this kind of interaction. In addition, hybrids between small nuclear RNAs and heterogeneous nuclear RNA were isolated. These hybrids were formed with all the U-rich species, 4.5 S, 4.5 SI and a novel species designated W. Approximately equal numbers of hybrids were formed by species U1a, U1b, U2, U6 and W; however, species U4 and U5 were significantly under-represented. Most of these hybrids were found to be associated stably with non-polyadenylated RNA. These observations demonstrated for the first time that small nuclear RNA-heterogeneous nuclear RNA hybrids can be isolated without crosslinking, and that proteins are not necessary to stabilize the complexes. However, not all molecules of a given small nuclear RNA species are involved in the formation of these hybrids. The distribution of a given small nuclear RNA species between the free and bound state does not reflect the stability of the complex in vitro but rather the abundance of complementary sequences in the heterogeneous nuclear RNA.(ABSTRACT TRUNCATED AT 400 WORDS)     

Epitopes on foot-and-mouth disease virus outer capsid protein VP1 involved in neutralization and cell attachment.

Foot-and-mouth disease virus structural protein VP1 elicits neutralizing and protective antibody and is probably the viral attachment protein which interacts with cellular receptor sites on cultured cells. To study the relationships between epitopes on the molecule related to neutralization and cell attachment, we tested monoclonal antibodies prepared against type A12 virus, isolated A12 VP1, and a CNBr-generated A12 VP1 fragment for neutralization and effect on viral absorption. The antibodies selected for analysis neutralized viral infectivity with varying efficiencies. One group of antibodies caused a high degree of viral aggregation and inhibited the adsorption of virus to cells by 50 to 70%. A second group of antibodies caused little or no viral aggregation but inhibited the adsorption of virus to cells by 80 to 90%. One antibody, which is specific for the intact virion, caused little viral aggregation and had no effect on the binding of virus to specific cellular receptor sites. Thus, at least three antigenic areas on the surface of foot-and-mouth disease virus which were involved in neutralization were demonstrated. One of the antigenic sites appears to have been responsible for interaction with the cellular receptor sites on the surface of susceptible cells.