Roles of H1 domains in determining higher order chromatin structure and H1 location

Peptides derived from calf thymus H1 and rat liver H1, comprising only the globular and COOH-terminal domains of the intact molecule and therefore lacking NH2-terminal domains, have been shown by reconstitution to be as effective as the complete H1 molecule in inducing higher-order-chromatin structure. As the globular domain of H1 alone cannot induce chromatin folding, our results demonstrate that this function is primarily controlled by the COOH-terminal domain of the molecule. Surprisingly, these peptides do not locate correctly with respect to the nucleosome. This is demonstrated by their failure to confer upon reconstitutes the ability to protect DNA fragments of chromatosome length when digested with micrococcal nuclease. The precise placement of the H1 molecule (globular domain) with respect to the nucleosome is shown to be influenced by the "tail" domains of both H1 and the core histones.     

The relative cytotoxicity and mutagenicity of cyclobutane pyrimidine dimers and (6-4) photoproducts in Escherichia coli cells

In order to calculate the relative cytotoxicity and mutagenicity of (5-6) cyclobutane pyrimidine dimers and (6-4) photoproducts, we have measured survival and mutation induction in UV-irradiated excision-deficient E. coli uvrA cells, with or without complete photoreactivation of the (5-6) dimers. Radioimmunoassays with specificity for (5-6) dimers or (6-4) photoproducts have shown that maximum photoreactivation eliminates all of the (5-6) dimers produced up to 10 Jm-2 254-nm light, while it has no effect on (6-4) photoproducts. These results were confirmed by measuring the frequency of T4 endonuclease V-sensitive sites. Based on the best fit equations for survival and mutation induction, we have found that the calculated cytotoxicity of (6-4) photoproducts is similar to that of (5-6) dimers; however, the former is much more mutagenic than the latter.     

Molecular cloning and expression of flagellar radial spoke and dynein genes of Chlamydomonas

Several flagellar dynein ATPase and radial spokehead genes have been isolated from a Chlamydomonas genomic expression library in lambda gt11. The library was probed with polyclonal and monoclonal antibodies raised against purified flagellar polypeptides, and recombinant phage giving positive signals were cloned. In vitro translation of mRNAs hybrid-selected by the cloned sequences from whole cell RNA provided confirmation of identity for three of the four clones. Evidence supporting the identification of the fourth, which encodes a dynein heavy chain, was provided by antibody selection; the fusion protein produced by this clone selected heavy chain-specific antibodies from a complex polyclonal antiserum recognizing many dynein determinants. One of the radial spoke sequences isolated here is of particular interest because it encodes the wild-type allele of a locus which was defined previously by temperature-sensitive paralyzed flagella mutation pf-26ts (Huang, B., G. Piperno, Z. Ramanis, and D. J. L. Luck, 1981, J. Cell Biol., 88:80-88). The cloned sequence was used to hybrid-select mRNA from mutant pf-26ts cells, and when translated in vitro, the selected mRNA produced a mutant spokehead polypeptide with an altered electrophoretic mobility. This confirms that the pf-26ts mutation alters the primary structure of a radial spokehead polypeptide. To quantify spokehead and dynein mRNAs during flagellar regeneration, all of the cloned sequences were used as hybridization probes in RNA dot experiments. Levels increased rapidly and coordinately after deflagellation, peaked 3-10-fold above nondeflagellated controls, and then returned to control values within 2 h. This accumulation pattern was similar to that of flagellar alpha-tubulin mRNA.     

Stimulator requirements for primed alloreactive T cells: macrophages and dendritic cells activate T cells across all genetic disparities

The cellular requirements for stimulating primed alloreactive T cells have been investigated. In vitro-primed secondary alloreactive cells, long-term lines, and Ly 1+2- noncytolytic clones which reacted with allo-H-2K, D, or Mls (M locus) antigens were tested. The data indicated that a specialized antigen-presenting cell such as a macrophage or a dendritic cell was required for stimulating primed alloreactive cells across all the genetic disparities tested. B and T lymphocytes were ineffective stimulators. The stimulator requirement for secondary and Ly 1+2- clone responses was heterogeneous, since both macrophages and dendritic cells were effective stimulators. Thus, the allostimulator requirement for inducing proliferation and mediator secretion by the primed T-cell populations closely paralleled the requirement for stimulating unprimed populations. The only exception found was the peritoneal washout population, which did not stimulate a primary response but did stimulate secondary responses. The failure of peritoneal macrophages to stimulate a primary response was shown to be due to an inhibitory pathway which did not occur when the responding population was alloantigen primed.     

Inhibition of proliferation without affecting the generation of cytotoxicity in the human mixed lymphocyte reaction

Phosphoramide mustard (PM) is considered to be the major tumoricidal metabolite of cyclophosphamide in vivo. The effects of this metabolite in vitro on several immune functions of human lymphocytes have been investigated. Very low concentrations (10(-7) to 10(-9) M) of PM added to lymphocyte cultures inhibited proliferation of the lymphocytes in response to mitogens and alloantigens. At these concentrations, inhibition of proliferation appeared to be due to a direct action of PM on the proliferative cells. Thus, concanavalin A-stimulated lymphocytes still acquired IL-2 receptors (Tac antigen) normally in the presence of PM (10(-6) to 10(-9) M). Only exceedingly high concentrations of PM (10(-5) M or greater) prevented the acquisition of Tac antigen. Similarly, the inhibition of proliferation was probably not related to endogenous IL-2 levels: addition of exogenous IL-2 to PM-containing cultures did not result in any restoration of proliferation. Further evidence that PM directly affected proliferative cells was that low concentrations of PM inhibited the proliferation of T cells continuously growing in IL-2. The exposure time to PM necessary for inhibition was essentially identical to those for lymphoproliferative responses to mitogens and alloantigens. Paradoxically, however, the generation of cytotoxic lymphocytes in mixed lymphocyte reactions (MLRs) and mixed lymphocyte tumor cell cultures (MLTCs) was very resistant to PM. In parallel MLRs and MLTCs the cytotoxic responses were resistant to approximately 1000-fold more PM than were the proliferative responses. Only at 10(-5) M PM were these inhibited. These data suggest that clonal expansion of cytotoxic lymphocytes or their precursors by proliferation is not an absolute requirement for the generation of cytolytic activity.     

Endogenous stimulant of prostaglandin endoperoxide synthase activity in human amniotic fluid

In this study we describe the discovery and characterization of a substance in human amniotic fluid that stimulates prostaglandin biosynthesis by a microsome-enriched preparation of bovine seminal vesicles. The stimulatory activity is not retained substantially upon anisotropic ultrafiltration through a filter with a molecular weight exclusion limit of 500. Stimulation of prostaglandin biosynthesis by this substance is time- and concentration-dependent; maximal stimulation of approx. 200% being observed within 20 min of commencing incubation with 1 ml-equivalent of stimulant fraction. Stimulatory activity is demonstrable both in the presence of reduced glutathione (1.3 mM) and L-tryptophan (20 mM), either separately or combined, and in the presence of exogenous arachidonic acid (5-120 microM). In the absence of added cofactors, the stimulatory substance increases the rates of biosynthesis of prostaglandin E2 and prostaglandin F2 alpha to equal extents. The amount of stimulatory substance added to incubations is correlated positively with increased oxygen consumption during incubations. The stimulatory substance is stable to heating at 100 degrees C for 10 min but is inactivated substantially (to less than 20% of original activity) by treatment with pronase. It is concluded that human amniotic fluid contains a substance of relatively low molecular weight, which is proteinaceous in character, that stimulates prostaglandin endoperoxide synthase activity.     

Interactions in Syntrophic Associations of Endospore-Forming, Butyrate-Degrading Bacteria and H2-Consuming Bacteria

Butyrate is an important intermediate in the anaerobic degradation of organic matter. In sulfate-depleted environments butyrate is oxidized to acetate and hydrogen by obligate proton reducers, in syntrophic association with hydrogen-consuming methanogens. This paper describes two enrichments of endospore-forming bacteria degrading butyrate in consortia with methanogens. The isolates are readily established in coculture with H₂-consuming, sulfate-reducing bacteria by pasteurizing the culture. The two original enrichments differed in that one grew to an optically dense culture while the second grew in clumps. Examination by scanning electron microscopy showed that clumping resulted from the production of large amounts of extracellular polymer. Several H₂-consuming methanogens were identified in the enrichments. Some of them grew closely associated to the butyrate degraders. This attachment to the hydrogen producer may permit some methanogens to compete for the growth substrate against other bacteria having higher substrate affinity.     

Synthesis of cytoskeletal and contractile proteins by cultured IMR-90 fibroblasts

Models of the assembly of cytoskeletal and contractile proteins of eukaryotic cells require quantitative information about the rates of synthesis of individual component proteins. We applied the dual isotope technique of Clark and Zak (1981, J. Biol. Chem., 256:4863-4870) to measure the synthesis rates of cytoskeletal and contractile proteins in stationary and growing cultures of IMR-90 fibroblasts. Fibroblast proteins were labeled to equilibrium with [14C]leucine over several days, at the end of which there was a 4-h pulse with [3H]leucine. Fractional synthesis rates (percent per hour) were calculated from the 3H/14C ratio of cell protein extracts or protein purified by one- or two-dimensional polyacrylamide gel electrophoresis and the 3H/14C ratio of medium-free leucine. The average fractional synthesis rate for total, SDS- or urea-soluble; Triton-soluble; and cytoskeletal protein extracts in stationary cells each was approximately 4.0%/h. The range of values for the synthesis of individual proteins from total cell extracts or cytoskeletal extracts sliced from one-dimensional gels was similar, though this range was greater than that for major proteins of Triton-soluble protein extracts. Three specific cytoskeletal proteins--actin, vimentin, and tubulin--were synthesized at similar rates that were significantly slower than the average fractional synthesis rate for total protein. Myosin, on the other hand, was synthesized faster than average. Synthesis rates were the same for beta-and gamma-actin and polymerized (cytoskeletal extract) vs. Triton-soluble actin. The same was true for alpha- and beta-tubulin and two different forms of vimentin. Synthesis rates were uniformly higher in growing cells, though the same pattern of differential rates was observed as for stationary cells. Synthesis rates in growing cells were higher than the rate necessary to maintain the growth rate, even for those cytoskeletal proteins being synthesized slowly. Therefore, there appears to be some turnover of these cytoskeletal elements even during growth. We conclude that proteins in cytoskeletal extracts may have nonuniform rates of synthesis, but at least one important subclass of cytoskeletal proteins that comprise filament subunits have the same synthesis rates.