Isolation and regional localization of a large collection (2,000) of single-copy DNA fragments on human chromosome 3 for mapping and cloning tumor suppressor genes

Summary A collection of 2,000 lambda phage-carrying human single-copy inserts (> 700 bp) were isolated from two chromosome-3 flow-sorted libraries. The single-copy DNA fragments were first sorted into 3p and 3q locations and about 700 3p fragments were regionally mapped using a deletion mapping panel comprised of two humanhamster and two-human-mouse cell hybrids, each containing a chromosome 3 with different deletions in the short arm. The hybrids were extensively mapped with a set of standard 3p markers physically localized or ordered by linkage. The deletion mapping panel divided the short arm into five distinct subregions (A-E). The 3p fragments were distributed on 3p regions as follows: region A, 26%; B, 31%; C, 4%; D, 4% and E, 35%. We screened 300 single-copy DNA fragments from the distal part of 3p (regions A and B) with ten restriction endonucleases for their ability to detect restriction fragment length polymorphisms (RFLPs). Of these fragments 110 (36%) were found to detect useful RFLPs: 35% detected polymorphisms with frequency of heterozygosity of 40% or higher, and 25% with frequency of 30% or higher. All polymorphisms originated from single loci and most of them were of the base pair substitution type. These RFLP markers make it possible to construct a fine linkage map that will span the distal part of chromosome 3p and encompasses the von Hippel-Lindau disease locus. The large number of single-copy fragments (2,000) spaced every 100–150 kb on chromosome 3 will make a significant contribution to mapping and sequencing the entire chromosome 3. The 300 conserved chromosome 3 probes will increase the existing knowledge of man-mouse homologies.     

Inhibition of a Low Km GTPase Activity in Rat Striatum by Calmodulin

In rat striatum, the activation of adenylate cyclase by the endogenous Ca2+-binding protein, calmodulin, is additive with that of GTP but is not additive with that of the nonhydrolyzable GTP analog, guanosine-5'-(beta, gamma-imido)triphosphate (GppNHp). One possible mechanism for this difference could be an effect of calmodulin on GTPase activity which has been demonstrated to "turn-off" adenylate cyclase activity. We examined the effects of Ca2+ and calmodulin on GTPase activity in EGTA-washed rat striatal particulate fractions depleted of Ca2+ and calmodulin. Calmodulin inhibited GTP hydrolysis at concentrations of 10(-9)-10(-6) M but had no effect on the hydrolysis of 10(-5) and 10(-6) M GTP, suggesting that calmodulin inhibited a low Km GTPase activity. The inhibition of GTPase activity by calmodulin was Ca2+-dependent and was maximal at 0.12 microM free Ca2+. Maximal inhibition by calmodulin was 40% in the presence of 10(-7) M GTP. The IC50 for calmodulin was 100 nM. In five tissues tested, calmodulin inhibited GTP hydrolysis only in those tissues where it could also activate adenylate cyclase. Calmodulin could affect the activation of adenylate cyclase by GTP in the presence of 3,4-dihydroxyphenylethylamine (DA, dopamine). Calmodulin decreased by nearly 10-fold the concentration of GTP required to provide maximal stimulation of adenylate cyclase activity by DA in the striatal membranes. The characteristics of the effect of calmodulin on GTPase activity with respect to Ca2+ and calmodulin dependence and tissue specificity parallel those of the activation of adenylate cyclase by calmodulin, suggesting that the two activities are closely related.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lactation-associated redistribution of the glial fibrillary acidic protein within the supraoptic nucleus

Summary Three clones of myeloproliferative virus (MPV)-transformed rat fibroblasts (NRK) with different growth properties and morphology were transplanted to athymic nude mice. Presence of carbohydrate-binding proteins was inferred by fluorescence microscopy using fluorescent, glycosylated markers. Salt and detergent extracts of tumors from this model system were fractionated under identical conditions on different sets of Sepharose columns, to which lactose, asialofetuin, melibiose, mannan and fucose had been covalently linked. Successive elution by chelating reagent and specific sugar resulted in isolation of the different Ca²⁺-dependent and Ca²⁺-independent endogenous carbohydrate-binding proteins that were assayable as agglutinins. In comparison, the different tumors displayed a pattern with qualitative and quantitative alterations. Since protein-carbohydrate interaction mediated by carbohydrate-binding proteins (lectins) is of importance for cognitive processes, it is remarkable that the pattern of membrane glycoproteins, isolated by affinity chromatography on resins with immobilized plant lectins, had also been found to reveal certain individual properties for receptors specific for peanut agglutinin (PNA) and Ulex europaeus agglutinin (UEA). These demonstrated differences within the system of protein-carbohydrate interaction suggest that endogenous lectins and their ligands have potential significance as markers defining a certain phenotype within this tumor model system.Dedicated to Prof. Dr. W. Lamprecht on the occasion of his 60th birthday     

Selenium uptake by Butyrivibrio fibrisolvens and Bacteroides ruminicola

Abstract The uptake and incorporation of ⁷⁵[Se]selenite by Butyrivibrio fibrisolvens and Bacteroides ruminicola were by constitutive systems. Rates of uptake were higher in chemostat culture than in batch culture and there may be some inducible component. Uptake of [⁷⁵Se]selenite was distinct from sulphate or selenate transport, since sulphate and selenate did not inhibit selenite uptake, nor could sulphate or selenate uptake be demonstrated in these organisms. Selenite uptake in B. fibrisolvens had and apparent K _m of 1.74 mM and a V _max of 109 ng Se · min⁻¹· (mg protein)⁻¹. An apparent K _m of 1.76 mM and V _max of 1.5 μg Se · min⁻¹· (mg protein)⁻¹ was obtained for B. ruminicola . [⁷⁵Se]Selenite uptake by both organisms was partially sensitive to inhibition by 2,4-DNP. Uptake by B. fibrisolvens was also partially inhibited by azide and arsenate and in B. ruminicola it was partially inhibited by fluoride. CCCP, CPZ, DCCD or quinine did not inhibit uptake in either B. fibrisolvens or B. ruminicola . Selenite transport by both organisms was sensitive to IAA and NEM and was strongly inhibited by sulphite and nitrite. [⁷⁵Se]Selenite was converted to selenocystine, selenohomocystine and selenomethionine by B. fibrisolvens. B. ruminicola did not incorporate [⁷⁵Se]selenite into organic compounds, but did reduce it to red elemental selenium.     

Abscisic acid induction of cloned cotton late embryogenesis-abundant (Lea) mRNAs

Summary Earlier studies found that cotton (Gossypium hirsutum L.) cotyledons contain several mRNAs which are more abundant during late embryogenesis than in mid-embryogenesis or early germination. They are here termed Late embryogenesis-abundant mRNAs, encoded by Lea loci. Complementary DNA clones for 18 such mRNA sequences, defined at a hybridization criterion of T_m-15°C, were identified in a mature embryo cDNA library by differential cDNA hybridization. At a lower hybridization criterion, some sequence homology was found within several of these cloned Lea mRNA sequences. Each Lea mRNA sequence comprises 0.04–1.3% of mature embryo poly(A)⁺ mRNA, a level ten-fold to several hundred-fold higher than in young embryo or 24 h seedling poly(A)⁺ mRNA. Of 18 Lea mRNA sequences examined in cultured young embryos, the level of at least 13 are specifically increased by exogenous abscisic acid (ABA), several to a level near that in normal mature embryos. However, the abundance of several of the sequences does not appear to be significantly modulated by ABA. The LEA polypeptides encoded by 10 Lea mRNA sequences were identified by hybrid-arrested translation. They include most of the late embryogenesis-abundant, ABA-inducible, polypeptides previously identified. Preliminary results suggest that many of the individual Lea mRNA sequences are transcribed from 1–3 genes in each of cotton's two subgenomes.