Interaction of human rad51 recombination protein with single-stranded DNA binding protein, RPA.

Replication protein A (RPA), a heterotrimeric single-stranded DNA binding protein, is required for recombination, and stimulates homologous pairing and DNA strand exchange promoted in vitro by human recombination protein HsRad51. Co-immunoprecipitation revealed that purified RPA interacts physically with HsRad51, as well as with HsDmc1, the homolog that is expressed specifically in meiosis. The interaction with HsRad51 was mediated by the 70 kDa subunit of RPA, and according to experiments with deletion mutants, this interaction required amino acid residues 169-326. In exponentially growing mammalian cells, 22% of nuclei showed foci of RPA protein and 1-2% showed foci of Rad51. After gamma-irradiation, the percentage of cells with RPA foci increased to approximately 50%, and those with Rad51 foci to 30%. All of the cells with foci of Rad51 had foci of RPA, and in those cells the two proteins co-localized in a high fraction of foci. The interactions of human RPA with Rad51, replication proteins and DNA are suited to the linking of recombination to replication.     

ClassAsteretea tripolium on the territory of the former USSR and Mongolia

Higher syntaxa of the classAsteretea tripolium are reviewed. This class covers plant communities of the Euro-Asian continent on low- and medium-salinized soils with medium moisture conditions where perennial herbaceous plants (hemicryptophytes) of non-succulent character prevail. On the territory of the former USSR and Mongolia, the classAsteretea tripolium is represented by the following orders:Glauco-Puccinellietalia Beeftink etWesthoff inBeeftink 1962,Cirsietalia esculenti Mirkin etV. Golub exV. Golub ord. novus,Scorzonero-Juncetalia gerardii Vicherek 1973,Halerpestetalia Mirkin et al. exV. Golub ord. novus,Artemisio santonicae-Limonietalia gmelinii V. Golub etV. Solomakha 1988,Suaedetalia corniculatae V. Golub ord. novus. Superspecies and aggregations of species are used for the diagnosis of higher syntaxa.     

Transposon-mediated insertion of R factor into bacterial chromosome

Summary Insertion of transposon Tn1 into the E. coli JC411 chromosome results in a sharp increase of plasmid RP4 integration frequency. This effect is absent in JC1553 recA cells. The RP4 integration with the chromosome is probably accomplished via recA-dependent recombination between transposon Tn1 inserted into the chromosome and the same transposon in the RP4 plasmid.     

In vitro sensitization of human lymphoid cells to antigens on cultured melanoma cells. I. Culture conditions resulting in augmented cell-mediated cytotoxicity

Culture conditions have been established that result in the sensitization of normal human peripheral blood lymphoid cells on allogeneic melanoma monolayers. Optimal culture conditions require 2 to 8 × 10⁶ mitomycin C treated stimulator melanoma cells to sensitize 5 to 10 × 10⁶ responder lymphoid cells. Neither rocking nor refeeding of the culture is necessary for the sensitization procedure. Stimulator cells grown in either fetal calf serum or human serum will serve as effective stimulator or target cells. Peak cytotoxic activity was detected at 44 hr in a microcytotoxicity assay, although some cytotoxic activity was detectable at 24 hr.     

Leukemia in AKR mice: III. Size distribution of suppressor T-cells in AKR leukemia and neonatal mice

Suppression of in vitro antibody forming potential of normal cells by leukemic cells of AKR and normal neonatal mice have many similarities. In both cases the suppression is by cell contact rather than by the elaboration of soluble suppressive factors and the suppression is sensitive to both x-irradiation and mitomycin C treatment. When the size distribution of suppressing cells in thymus and spleen were compared by velocity sedimentation, both leukemic and neonatal suppressing cells had similar size distribution in each organ. Both large and small cells in the thymus suppress but only large cells (sedimentation velocity >3.5 mm/hr) in the spleen are able to suppress. Leukemic cells in lymph node have a splenic size distribution, viz., only large cells suppress. Both large and small cells of a subcutaneously growing long passage AKR lymphoma are able to suppress. While large cells contain the bulk of cells actively incorporating tritiated thymidine and thus probably in cycle, small but significant amounts of incorporation in small suppressing cells is also seen.     

The effect of age and 1,25-dihydroxyvitamin D3 treatment on the intestinal calcium-binding protein of suckling rats.

The intestinal level of the vitamin D-dependent duodenal calcium-binding protein was assayed by an equilibrated column technique in rat embryos, neonates, and pups. Calcium-binding protein was undetectable in unborn, newborn, and 1- to 2-day-old rats i.e., the level was lower than in severely vitamin D-deficient animals. Calcium-binding protein was detected after the animals were 5-days old and thereafter rose monotonically as a function of body weight. Treatment with 1,25-dihydroxyvitamin D₃ failed to raise the calcium-binding protein levels of newborn or 1-day-old rats, but doubled the level in 11- or 12-day-old pups. Plasma calcium was raised in all treated animals. The failure to detect calcium-binding protein in vitamin D-replete suckling animals provides evidence for a dissociation between calcium absorption and calcium binding protein.     

Review on testicular development, structure, function, and regulation in common marmoset

BACKGROUND: The common marmoset (Callithrix jacchus) is a New World primate that has been used increasingly in toxicological evaluations including testing for testicular toxicity of pharmaceutical and environmental chemicals. Information on structural and functional characteristics of the testis in common marmosets ("marmoset" in this review) is critical for designing experiments, interpreting data collected, and determining relevance to humans in risk assessment. METHODS: This study provides a comprehensive review on testicular development, structure, function, and regulation in common marmosets. RESULTS: There is little information regarding testicular formation and development during gestation. Based on the overall pattern of embryonic development in marmosets, it is postulated that gonadal formation and testicular differentiation most likely takes place during gestational Week 6-12. After birth, the neonatal period of the first 2-3 weeks and the pubertal period from Months 6-12 are critical for establishment of spermatogenesis in the adult. In the adult, a nine-stage model has been used to describe the organization of seminiferous epithelium and multiple stages per tubular cross-section have been observed. Seminiferous epithelium is organized in a wave or partial-wave manner. There are on average two stages per cross-section of seminiferous tubules in adult marmoset testis. Sertoli cells in the marmoset have a uniform morphology. Marmoset spermatogenesis has a high efficiency. The prime determinant of germ cell production is proliferation and survival of spermatogonia. Sertoli cell proliferation during the neonatal period is regulated by follicle-stimulating hormone (FSH), but chorionic gonadotropin (CG), instead of luteinizing hormone (LH), is the only gonadotropin with luteinizing function in marmoset. The receptor gene for CG in marmoset is unique in that it does not have exon 10. Marmosets have a "generalized steroid hormone resistance," i.e., relatively high levels of steroid hormones in circulation and relatively low response to exogenous steroids. Blockage of FSH, CG, and testosterone production during the first 3 months after birth does not cause permanent damage to the male reproductive system. Initiation of spermatogenesis in the marmoset requires unique factors that are probably not present in other mammals. Normal male marmosets respond to estradiol injection positively (increased LH or CG levels), a pattern seen in normal females or castrated males, but not usually in normal males of other mammalian species. CONCLUSIONS: It seems that the endocrine system including the testis in marmosets has some unique features that have not been observed in rodents, Old World primates, and humans, but detailed comparison in these features among these species will be presented in another review. Based on the data available, marmoset seems to be an interesting model for comparative studies. However, interpretation of experimental findings on the testicular effects in marmosets should be made with serious caution. Depending on potential mode of testicular actions of the chemical under investigation, marmoset may have very limited value in predicting potential testicular or steroid hormone-related endocrine effects of test chemicals in humans.     

Activation of human meiosis-specific recombinase Dmc1 by Ca2+

Rad51 and its meiotic homolog Dmc1 are key proteins of homologous recombination in eukaryotes. These proteins form nucleoprotein complexes on single-stranded DNA that promote a search for homology and that perform DNA strand exchange, the two essential steps of genetic recombination. Previously, we demonstrated that Ca2+ greatly stimulates the DNA strand exchange activity of human (h) Rad51 protein (Bugreev, D. V., and Mazin, A. V. (2004) Proc. Natl. Acad. Sci. U. S. A. 101, 9988-9993). Here, we show that the DNA strand exchange activity of hDmc1 protein is also stimulated by Ca2+. However, the mechanism of stimulation of hDmc1 protein appears to be different from that of hRad51 protein. In the case of hRad51 protein, Ca2+ acts primarily by inhibiting its ATPase activity, thereby preventing self-conversion into an inactive ADP-bound complex. In contrast, we demonstrate that hDmc1 protein does not self-convert into a stable ADP-bound complex. The results indicate that activation of hDmc1 is mediated through conformational changes induced by free Ca2+ ion binding to a protein site that is distinct from the Mg2+.ATP-binding center. These conformational changes are manifested by formation of more stable filamentous hDmc1.single-stranded DNA complexes. Our results demonstrate a universal role of Ca2+ in stimulation of mammalian DNA strand exchange proteins and reveal diversity in the mechanisms of this stimulation.     

Inter- and intrasubunit interactions during the formation of RNA polymerase assembly intermediate

We used yeast two-hybrid and in vitro co-immobilization assays to study the interaction between the Escherichia coli RNA polymerase (RNAP) alpha and beta subunits during the formation of alpha(2)beta, a physiological RNAP assembly intermediate. We show that a 430-amino acid-long fragment containing beta conserved segments F, G, H, and a short part of segment I forms a minimal domain capable of specific interaction with alpha. The alpha-interacting domain is held together by protein-protein interactions between beta segments F and I. Residues in catalytically important beta segments H and I directly participate in alpha binding; substitutions of strictly conserved segment H Asp(1084) and segment I Gly(1215) abolish alpha(2)beta formation in vitro and are lethal in vivo. The importance of these beta amino acids in alpha binding is fully supported by the structural model of the Thermus aquaticus RNAP core enzyme. We also demonstrate that determinants of RNAP assembly are conserved, and that a homologue of beta Asp(1084) in A135, the beta-like subunit of yeast RNAP I, is responsible for interaction with AC40, the largest alpha-like subunit. However, the A135-AC40 interaction is weak compared with the E. coli alpha-beta interaction, and A135 mutation that abolishes the interaction is phenotypically silent. The results suggest that in eukaryotes additional RNAP subunits orchestrate the enzyme assembly by stabilizing weak, but specific interactions of core subunits.