Molecular and cytological characterization of repetitive DNA sequences in Brassica

Summary We isolated three different repetitive DNA sequences from B. campestris and determined their nucleotide sequences. In order to analyze organization of these repetitive sequences in Brassica, Southern blot hybridization and in situ hybridization with metaphase chromosomes were performed. The sequence cloned in the plasmid pCS1 represented a middle repetitive sequence present only in B. campestris and not detected in closely related B. Oleracea. This sequence was localized at centromeric regions of six specific chromosomes of B. campestris. The second plasmid, pBT4, contained a part of the 25S ribosomal RNA gene, and its copy number was estimated to be 1,590 and 1,300 per haploid genome for B. campestris and B. oleracea, respectively. In situ hybridization with this sequence showed a clear signal at the NOR region found in the second largest chromosome of B. Campestris. The third plasmid, pBT11, contained a 175-bp insert that belongs to a major family of tandem repeats found in all the Brassica species. This sequence was detected at centromeric regions of all the B. campestris chromosomes. Our study indicates that in situ hybridization with various types of repetitive sequences should give important information on the evolution of repetitive DNA in Brassica species.     

Effects of non-MHC loci on resistance to retrovirus-induced immunodeficiency in mice

Mice of certain strains are highly sensitive to development of a severe immunodeficiency disease following inoculation as adults with LP-BM5 murine leukemia viruses (MuLV) whereas others are extremely resistant. These strain-dependent differences in response to infection have been shown to be genetically determined with resistance to disease being, in general, associated with homozygosity for Fv-1 ⁿand H-2 haplotypes a and d and sensitivity with homozygosity for Fv-1 ^band other H-2 haplotypes including b, s, and q. The Fv-1 ^b, H-2 ^rstrain RIIIS/J (RIIIS) was found to be highly resistant to disease even though B10.RIII(71NS)/J (B10.RIII), also H-2 ^r, was very sensitive, thus excluding a role for H-2 in the resistance of RIIIS. The characteristics of RIIIS resistance were evaluated in studies of infected (B10.RIII×RIIIS) F₁, F₂ and reciprocal backcross mice. Resistance to disease was shown to be semidominant and determined by more than one gene, although a preponderant influence of a single gene was suggested. Studies of segregating populations showed that resistance was not associated with or linked to polymorphisms of the V _\complex or genes in proximity to the Emv-2 locus on chromosome 8. However, there was almost complete concordance between absence of disease in infected mice and inhibition of ecotropic virus spread. These results demonstrate that genes other than Fv-1 or H-2 can profoundly influence the development of retrovirus-induced immunodeficiency and replication of ecotropic viruses.Abbreviations MuLV murine leukemia virus - MCF mink cell focus-inducing MuLV - B6 C57BL/6 - BM5d the defective virus in LP-BM5 MuLV - MAIDS murine acquired immunodeficiency syndrome - RIIIS RIIIS/J - B10.RIII B10.RIII (71NS)/J - MLR mixed lymphocyte reaction - FACS fluorescence activated cell sorter     

Immunological detection of the dystrophin molecule with antibody directed against the synthetic peptide.

We synthesized a peptide designated R8 (amino acid residues 1157-1201) based on the primary structure presumed from the nucleotide sequence of the cDNA clone from the gene for Duchenne muscular dystrophy. Antibody to the synthetic R8 generated by immunization of rabbits was tested on human and mouse skeletal muscle by Western blotting analysis. The antibody reacted with a component of the 400K dystrophin of normal human and mouse skeletal muscles, but not with components of the muscles of Duchenne muscular dystrophy patients and mdx mice. Thus we established that this peptide sequence is in fact missing in the protein product 'dystrophin' encoded by the DMD gene. The antibody may prove useful for the diagnosis of the Duchenne types of muscular dystrophy.     

Monoclonal antibodies that depress a specific subset of multiple components of the glutathione-induced response ofHydra

Summary Reduced glutathione evokes a feeding response, the tentacle-ball formation inHydra japonica. This response consists of at least 5 components (R1–R5). We raised 6 monoclonal antibodies (mAbs), each of which depressed a specific subset of these components, and we also examined the immunocytochemical localization of antigens with these mAbs at light microscopic level. The 2 mAbs that depressed R2 and R4 bound to the cnidocils of the desmoneme and the stenotele nematocytes; the 3 mAbs that depressed R5 bound to the apical surface adjacent to the cnidocils of the nematocytes; and the 2 mAbs that depressed R1 and R3 bound to the apical spot structures of unidentified cells in the ectoderm.Together with the specificity of the action of the mAbs on the behavioral response, the correspondence between the effects on the response and the structures visualized with these mAbs suggests that these structures include components of the receptor-effector system relevant to chemoreception.     

Identification of eleven single-strand initiation sequences (ssi) for priming of DNA replication in the F, R6K, R100 and ColE2 plasmids.

Based on the ability to complement the poor growth of an M13 phage derivative lacking the complementary strand origin, eleven single-strand initiation sequences (ssi) for DNA replication are identified in the F, R6K, R100 and ColE2 plasmids. Six of them were from F, two from near the gamma and alpha origins (ori) of R6K, two from the vicinity of the basic replicon of R100 and one from near the ori of ColE2. They can be classified into two groups based on the morphology of the plaques and the length of nucleotide (nt) sequences required for ssi activity; one group that gives rise to larger and clearer plaques and can be reduced to nearly 100 nt (seven out of eleven), and another that generates smaller and less clear plaques and requires more than 200 nt for full activity (four out of eleven). Sequence homology is detected among some members from both groups. The possible biological roles of the ssi are discussed.     

Dehydroepiandrosterone sulfate (DHEA-S) and 3', 5'-cyclic adenosine monophosphate (cAMP) production in a cultured human adrenocortical carcinoma cell line (SW-13)

Very little has been known of the biochemical function of a human adrenocortical carcinoma cell line, SW-13. In this study, the production of several adrenal steroids and 3', 5'-cyclic adenosine monophosphate (cAMP) were investigated in this cell line. The cells were incubated in L-15 medium containing 0.1% bovine serum albumin with several reagents in an atmosphere of 5% CO2 and 95% air for 2 hours at 37 degrees C. Aldosterone (Ald), corticosterone (B), cortisol (F), dehydroepiandrosterone sulfate (DHEA-S) and cAMP were simultaneously assayed by specific radioimmunoassays in the medium and cells. Significant increases in cAMP production were observed by cholera toxin (10 ng/ml) and forskolin (10 nM), both direct stimulators of adenylate cyclase, in the cAMP concentration without an increase in the steroids. The DHEA-S concentration in the medium was significantly increased by angiotensin-II (10(-7)M), noradrenalin (3 X 10(-5) M), adrenalin (3 X 10(-5) M) or alpha-melanocyte-stimulating hormone (alpha-MSH, 10(-7) M), none of which was associated with cAMP production. Neither adrenocorticotropin (10(-10) M) nor human chorionic gonadotropin (500 mIU/ml) stimulated the release of the steroids or cAMP production. A calcium ionophore, A23187 (10(-7) M), and 12-O-tetradecanoylphorbol-13-acetate (10(-8) M), a direct stimulator of protein kinase C, stimulated the release of DHEA-S, but not those of Ald, B and F. The results suggest that SW-13 retains functioning adenylate cyclase which, however, is not linked with steroidogenesis and that DHEA-S is produced probably by the mechanisms which involve protein kinase C system or calcium ion. This report provides the first demonstration of cAMP and DHEA-S production in SW-13 and suggests that this cell line is potentially useful for investigating the mechanisms of steroidogenesis in the human adrenal cortex.     

Purification of Rts1 RepA protein and binding of the protein to mini-Rts1 DNA.

RepA protein, essential for the replication of plasmid Rts1, was purified, and its binding to mini-Rts1 subregions was examined by a DNase I protection assay. RepA protected the incI and incII iterons, a region immediately upstream of the repA promoter, and a 10-base-pair region located between the most external incII iteron and a GATC box. The protection was less efficient when preheated RepA was used.     

Sulfated glycoproteins synthesized by the corneal epithelium of chick embryo having the same molecular weights as cytokeratin polypeptides

The previous study from this laboratory demonstrated that the corneal epithelium of 19-d-old chick embryo synthesizes two classes of sulfated glycoconjugates consisting of sulfated glycoproteins and proteoglycans (Yonekura, H., Oguri, K., Nakazawa, K., Shimizu, S., Nakanishi, Y., & Okayama, M. (1982) J. Biol. Chem. 257, 11166-11175). The present study demonstrated that when the sulfated glycoproteins labeled metabolically with [35S]sulfate and [3H]glucosamine were analyzed by SDS-PAGE, the 70,000 component (accounting for approximately 30% of the 35S and 35% of the 3H of the total sulfated glycoprotein) co-migrated with five major proteins with apparent molecular weights (Mrs) of 70,000, 66,000, 58,000, 51,000, and 48,000, which together accounted for about 57% of the total tissue protein. All five proteins cross-reacted with an antibody against human sole keratin, indicating that they are cytokeratin polypeptides of the corneal epithelium. Amino acid analysis demonstrated that they had high contents of glycine, serine, glutamic acid, leucine, and aspartic acid. Two-dimensional tryptic peptide maps indicated that they were all different. Analysis of radiolabeled materials released by alkaline borohydride treatment of the sulfated glycoproteins which were synthesized in the presence and absence of tunicamycin and co-purified with the five cytokeratin polypeptides, revealed that they contained both N- and O-glycosidically linked sulfated oligosaccharides. All the results obtained in the present study indicate that the five sulfated glycoproteins are similar, if not identical, to the cytokeratin polypeptides. This is consistent with the result in the accompanying paper that these sulfated glycoproteins are localized intracellularly.     

Increase of (Ca2++Mg2+)-ATPase activity of renal basolateral membrane by parathyroid hormone via cyclic AMP-dependent membrane phosphorylation

Studies were made on the mechanism of the effect of parathyroid hormone (PTH) on the activity of (Ca2++Mg2+)-ATPase, a membrane bound Ca2+-extrusion pump enzyme from the basolateral membranes (BLM) of canine kidney (Km for free Ca2+ = 1.3 X 10(-7) M, Vmax = 200 nmol Pi/mg/min). At 1 X 10(-7) M free Ca2+, both PTH (10(-7)-10(-6) M) and cAMP (10(-6)-10(-4) M) stimulated (Ca2++Mg2+)-ATPase activity dose-dependent and their stimulatory effects were inhibited completely by 5 microM H-8, an inhibitor of cAMP-dependent protein kinase. PTH (10(-7) M) also caused 40% increase in 32P incorporation into the BLM and 5 microM H-8 inhibited this increase too. PTH (10(-7) M) was found to stimulate phosphorylation of a protein of Mr 9000 by cAMP dependent protein kinase and 5 microM H-8 was found to block this stimulation also. From these results, it is proposed that PTH stimulates (Ca2++Mg2+)-ATPase activity by enhancing its affinity for free Ca2+ via cAMP-dependent phosphorylation of a BLM protein of Mr 9000.     

Interferon gamma modulates the ability of autologous non-T cells to stimulate T cells to produce and respond to interleukin 2

The interactions of T-cell receptor with self-Ia antigen on non-T cells induced IL-2 production and IL-2 receptors on the cell surface and thus responsiveness to IL-2 of T cells in autologous mixed-lymphocyte reaction (AMLR). Four-day-cultured autologous non-T cells lost their ability to stimulate T cells to produce and respond to IL-2 with concurrent decrease of HLA-DR and HLA-DQ antigen expressed on the cell surface. Culturing of non-T cells with 500 U/ml of recombinant interferon gamma (IFN-gamma) maintained their stimulating ability which was otherwise lost. Treatment of non-T cells with monoclonal anti-HLA-DR or anti-HLA-DQ antibody before mixture with T cells abrogated their ability to induce IL-2 production and IL-2 responsiveness of T cells. The combined data suggested that Ia antigen expressed on non-T cells is modulated by IFN-gamma, which increases the ability of non-T cells to stimulate autologous T cells to produce and respond to IL-2.