Microdissection and microcloning from the proximal region of mouse chromosome 7: isolation of clones genetically linked to the pudgy locus

Microdissection and microcloning have been utilized in order to create a bank of clones from the proximal region of mouse chromosome 7. Several important loci map to this area, including the albino locus (c), pink-eye dilution (p), and the developmental mutant, pudgy (pu). By use of interspecific crosses between Mus musculus domesticus and Mus spretus, we have generated backcross progeny segregating for the mutations chinchilla (cch) and pink-eye dilution (p). Exploiting the evolutionary divergence between the two species, we have analyzed the inheritance of restriction fragment length variants of three microclones and their linkage to the two markers cch and p, respectively. All three clones studied map to the dissected region, and as such also show genetic linkage to the pudgy locus. This bank of chromosome 7-derived microclones should provide molecular start points for the isolation of a variety of developmental loci of unknown gene product, including the pudgy locus.     

Effects of growth state and amines on cytoplasmic and vocuolar pH, phosphate and polyphosphate levels in Saccharomyces cerevisiae: a P-nuclear magnetic resonance study

The vacuoles of logarithmic and stationary stage cells were compared by ³¹P-NMR with regard to pH, orthophosphate (P_i) content and average size of polyphosphate. The vacuoles of stationary cells had lower pH higher P_i content, and polyphosphates of longer average chain lenght, although total polyphosphate content was about the same as in logarithmic cells. The lower vacuolar pH in stationary cells was the major cause of a larger cytoplasmic-vacuolar pH gradient. Addition of NH₄Cl, (NH₄)₂SO₄, methylamine or amantadine at pH 8 to cells in either stage caused an icnrease in both cytoplasmic and vacuolar pH, with little or no change in the cytoplasmic-vacuolar pH gradient. However, the administration of ammonium salts to the cells at pH 8.0 resulted in rapid hydrolysis of the intravacuolar polyphosphate to tripolyphosphate and P_i, with attendant redistribution of P_i between the vacuolar and cytoplasmic compartments.     

Respiration and soluble sugar metabolism in sugar pine embryos

Embroys excised from dormant seeds of sugar pine ( Pinus lambertiana Dougl.) incubated at 25°C (non-dormancy-breaking) or stratified at 5°C (dormancy-breaking) were analyzed to determine temperature effects on the relative activities of respiration and fermentative metabolism, the levels of soluble sugers and the activities of the hydrolytic enzymes, invertase and sucrose synthase, as related to the release of dormancy and germinatio. At 25°C, despite a sharp drop in embryo oxygen uptake after 48 h, a simultaneous decline in acetaldehyde and ethanol concentrations indicated that there was not a shift to fermentative metabolism. The concentrations of soluble sugars showed no treatment effects. Embryo invertase (EC 3.2.1.26) activity changed only slightly at either temperature, while stratification was accompanied by a 4-fold increase in sucrose synthase (EC 2.4.1.13) activity (cleavage direction). Upon transfer of stratified seeds to 25°C, embryo sucrose synthase activity rapidly increased almost 10-fold, with the increase beginning prior to germination, while mvertase activity increased 20-fold, concomitant with germination.     

Determination of effective diffusivity of substrate in biological films and particles

Summary Particle supported biofilms of uniform thickness were generated in an aerobic fluidized-bed reactor with phenol as the carbon source. A method was developed for determining the effective diffusivities of oxygen and phenol using trypan blue, a vital stain as the tracer. The effective diffusivities of oxygen and phenol were found to be 2.72×10^–6 cm²/s and 1.12×10^–6 cm²/s respectively.Nomenclature C_i initial solute concentration in bulk, g/cm³ - C_t solute concentration in bulk at time t, g/cm³ - C bulk solute concentration at equilibrium, g/cm³ - D molecular diffusivity, cm²/s - D effective diffusivity, cm²/s - D_o D_p D_tb molecular diffusivity of oxygen, phenol and trypan blue, cm²/s - D_o, D_p, D_tb effective diffusivity of oxygen, phenol and trypan blue, cm²/s - D_s molecular diffusivity of substrate, cm²/s - D_s effective diffusivity of substrate, cm²/s - K partition coefficient - M_t amount of solute in the particle at time t, g - M amount of solute in the particle at equilibrium, g - r particle radius, cm - r _bp radius of the particle with biofilm, cm - S substrate concentration, g/cm³ - S_b substrate concentration in bulk, g/cm³ - S_i initial substrate concentration, g/cm³ - V₁ solute molar volume, cm³/g mol Greek Symbols bf porosity of the biofilm - tortuosity factor     

Sequence analysis of two exons from the murine dystrophin locus

We have isolated two genomic clones from the murine dystrophin locus, containing single exons encoding protein sequence from the putative actin-binding domain of the amino-terminus and the terminal portion of the triple helical domain. Using interspecific backcross progeny mice, both clones were shown to be X-linked. Sequence analysis indicated that the amino-terminal clone contains a 173 bp exon exhibiting 90% nucleotide sequence identity to human dystrophin exon 6, whilst the C-terminal clone contains a 61 bp exon with 93% nucleotide sequence identity to the human cDNA sequence.

     

Heterochromatin staining pattern of quail-chicken hybrid lymphocytes

Feulgen-Rossenbeck staining of lymphoid cells of quail-chicken hybrids in histologic sections revealed a pattern of heterochromatin arrangement distinguishable from that of either parental type. During interphase, hybrid lymphocytes exhibited combined characteristics of both the parental quail and the parental chicken. Hybrid heterochromatin was arranged in a large central mass as in the quail and in fairly evenly distributed small chromacenters around the periphery of the nucleus similar to the arrangement in the chicken. It is suggested that this pattern of staining can be used as a marker for hybrid cells in studies of genetic interactions.     

The side-chain cleavage of cholesterol sulfate--II. The effect of phospholipids on the oxidation of the sterol sulfate by inner mitochondrial membranes and by a reconstituted cholesterol desmolase system

This study compares the side-chain cleavage of aqueous suspensions of cholesterol sulfate with the side-chain cleavage of cholesterol sulfate which is incorporated into phospholipid vesicles. Three different cholesterol desmolase systems are examined: the membrane-bound cholesterol side-chain cleavage system present in inner mitochondrial membranes isolated from bovine adrenal mitochondria; a soluble, lipid-depleted, reconstituted side-chain cleavage system prepared from cytochrome P-450scc, adrenodoxin and adrenodoxin reductase; a membrane associated side-chain cleavage system prepared by adding phospholipid vesicles, prepared from adrenal mitochondrial, to the reconstituted system. Soluble cholesterol sulfate, in low concentration, is a good substrate for the lipid-depleted reconstituted side chain cleavage system. However, at concentrations above 2 microM, in the absence of phospholipids, the sterol sulfate appears to bind at a non-productive site on cytochrome P-450scc which leads to substrate inhibition. Phospholipids, while inhibiting the binding of cholesterol sulfate to the cytochrome, also appear to prevent non-productive binding of the sterol sulfate to the cytochrome. Thus the addition of phospholipids to the lipid-depleted enzyme system leads to an activation of side-chain cleavage of high concentrations of the sterol sulfate. Soluble cholesterol sulfate is a good substrate for both the native and reconstituted membrane-bound systems and no substrate inhibition is observed when the membrane bound enzyme systems are employed in the assay of side-chain activity. However, the cleavage of cholesterol sulfate, which is incorporated into phospholipid vesicles, by both membrane bound enzyme systems appears to be competitively inhibited by the phospholipids of the vesicles. The results of this study suggest that the regulation of the side-chain cleavage of cholesterol sulfate may be entirely different than the regulation of the side-chain cleavage of cholesterol, if cholesterol sulfate exists intracellularly as a soluble non-complexed substrate. If, on the other hand, cholesterol sulfate is present in the cell in lipid droplets as a complex with phospholipids, its metabolism may be under the same constraints as the side-chain cleavage of cholesterol.     

Proteolysis in quaking mouse brain and spinal cord

Six proteolytic enzymes were assayed for activity in quaking CNS in examining the hypothesis that increased proteolytic activity contributes to the hypomyelination characteristic of this mutant. Cathepsin B-like enzyme, cathepsin D, neutral proteinase, calcium-activated neutral proteinase, prolyl endopeptidase, and diaminopeptidase II were assayed in whole homogenate of brain or spinal cord and each was found to have activity similar to that in normal mice. These results do not support a relationship between proteolysis and the genetic defect and suggest that other factors should be investigated to delineate the pathogenesis of this mutant.