Ribosomal protein S14 is not responsible for the Minute phenotype associated with the M(1)7C locus in Drosophila melanogaster.

Summary A locus associated with a severe Minute effect has been mapped at 7C on the X chromosome of Drosophila melanogaster. Previous work has suggested that this Minute encodes ribosomal proteins S14A and S141B. We have made a chromosomal deficiency that removes the S14 ribosomal protein genes, yet does not display the Minute phenotype. These data suggest that the S14 genes do not actually correspond to the Minute locus.     

Physiological Responses of Phytoflagellates to Dissolved Organic Substrate Additions. 2. Dominant Role of Autotrophic Nutrition in Pyrenomonas salina (Cryptophyceae)

The enhancement of algal growth by organic substrate assimilationis a common laboratory observation, yet few studies have addressedthe interaction of dissolved organic compounds and environmentalfactors for controlling the relative contribution of heterotrophyand autotrophy to the nutrition of these algae. The effectsof light intensity and glycerol addition on the growth, cellvolume, pigmentation, and carbon uptake of the facultative heterotroph,Pyrenomonas salina Santore, were examined. Glycerol additionto cultures growing at a limiting light intensity increasedthe growth rate, increased the average cell volume and cellularstarch content, decreased the cellular phycoerythrin to chlorophyll ratio, and had no effect on the CO₂ fixation rate cell^–1.Glycerol addition to cultures growing at a moderate light intensitythat was saturating for photo-autotrophic growth increased theaverage cell volume and cellular starch content but had no effecton the CO₂ fixation rate cell^–1. The results indicatethat autotrophy was the major process for carbon acquisitionduring the growth of P. salina, but that carbon acquisitionfrom glycerol catabolism also was used to partially supportgrowth of the alga at the limiting light intensity. In addition,glycerol presumably was used to fulfill the energy and/or reductantrequirements of the alga, and to increase the reserve carbohydrate(starch). ¹ Current address and address for correspondences: Horn PointEnvironmental Laboratories, University of Maryland, PO Box 775,Cambridge, Maryland 21613, U.S.A. (Received October 29, 1990; Accepted May 31, 1991)     

Polyphosphoinositol lipids inChlamydomonas eugametos gametes

InChlamydomonas eugametos gametes, phosphatidylinositol 4-phosphate (PtdInsP) and phosphatidylinositol 4,5-bisphosphate (PtdInsP₂) comprised 0.4 and 0.3% of the whole-cell phospholipids. They were concentrated in the plasma membrane around the cell body and were present in low concentrations in the flagellar membrane. When gametes were fed³²PO ₄ ^- , the label was rapidly incorporated into PtdInsP and PtdInsP₂ and only slowly incorporated into structural lipids such as phosphatidylethanolamine and phosphatidylglycerol. Similarly, when a pulse of³²PO ₄ ^- was chased with PO ₄ ^- , the label was rapidly lost from the polyphosphoinositol lipids but not from the structural lipids. The major fatty acids in the polyphosphoinositides were C-22 carbon polyenoic acids (70%). The significance of these results in relationship to intracellular signalling via inositol phosphates and Ca²⁺ is discussed.Abbreviations InsP₃ inositol 1,4,5-trisphosphate - mt^–/mt⁺ mating-type plus or minus - PtdA phosphatidic acid - PtdEtn phosphatidylethanolamine - PtdGro phosphatidylglycerol - PtdIns phosphatidylinositol - PtdInsP phosphatidylinositol 4-phosphate; - PtdInsP₂ phosphatidylinositol 4,5-bisphosphate - TCA trichloroacetic acid We thank Frank Schuring for Fig. 5A and Susan Kenter, Hans Kruisselbrink, Saskia Bijvank and Nelleke Corbett for their enthousiastic assistance.     

A method for determining overall protein fold from NMR distance restraints

Summary We describe a simple method for determining the overall fold of a polypeptide chain from NOE-derived distance restraints. The method uses a reduced representation consisting of two particles per residue, and a force field containing pseudo-bond and pseudo-angle terms, an electrostatic term, but no van der Waals or hard shell repulsive terms. The method is fast and robust, requiring relatively few distance restraints to approximate the correct fold, and the correct mirror image is readily determined. The method is easily implemented using commercially available molecular modeling software.     

Characterization of a heat-stable NADP-dependent isocitrate dehydrogenase from the obligate thermophile Thermoleophilum minutum YS-4

Summary A thermostable NADP-dependent isocitrite dehydrogenase (IDH; EC. 1.1.1.42) was purified from the obligately thermophilic hydrocarbonoclastic bacterium Thermoleophilum minutum YS-4 (ATCC 35265). This was accomplished by affinity chromatography and electroelution from a nondenaturing polyacrylamide gel. The enzyme has an M _r of 60 000 and is composed of two identical subunits of M _r 30 500. The amino acid composition has an Arg/Lys ratio of 4:1 and very high levels of glycine. Under nondenaturing conditions, the enzyme has a distinct difference in electrophoretic mobility relative to IDHs obtained from other genera including the genus Thermus. The secondary strcuture consists of 16% -helix, 20% -sheet, 25% -turn and 37% random coil as determined by circular dichroism spectroscopy. The optimum pH and temperature for activity were 7.2 and 75° C respectively and the apparent K _mvalues for DL-isocitrate adn NADP⁺ were 33 M, and 48 M, respectively. The enzyme requires divalent cations, such as Mn²⁺ or Mg²⁺ for activity. NAD⁺ cannot substitute for NADP⁺. Oxaloacetate plus glyoxylate exert considerable inhibition on IDH activity while other glycolytic and tricarboxylic acid cycle intermediates have a lesser effect. p-Chloromercuribenzoic acid was inhibitory to the IDH although isocitrate and Mn²⁺ offered some protection from this inactivation. The enzyme is thermostable, retaining 84% and 57% of initial activity after incubation for 1 h at 60° and 70° C, respectively. Isocitrate provided protection from thermal inactivation allowing the IDH to maintain 21% activity after 1 h at 80° C. Offprint requests to: J. J. Perry     

Structure of equine corticotropin releasing factor

A 41 amino acid peptide, probably identical in structure to human corticotropin releasing factor, was isolated from 70 equine hypothalami by methanol extraction, immunoaffinity chromatography and single step of reverse phase HPLC. The amino acid sequence was determined by gas phase sequence analysis. Probable carboxyl terminal amidation was demonstrated by similar retention times for equine and human corticotropin releasing factor on reverse phase HPLC at pH 8. The likely structure of equine corticotropin releasing factor is: Ser-Glu-Glu-Pro-Pro- Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Glu-Val-Leu-Glu-Met-Ala-Arg-Ala-Glu-Gln-Leu-Ala-Gln-Gln-Ala-His-Ser-Asn- Arg-Lys-Leu-Met-Glu-Ile-Ile-NH₂. The purified peptide is equipotent with human corticotropin releasing factor in an in vitro bioassay and in a human plasma binding protein assay.     

Crosses among Homokaryons from Commercial and Wild-Collected Strains of the Mushroom Agaricus brunnescens (= A. bisporus)

A new technique for the production of hybrid strains of the cultivated mushroom Agaricus brunnescens is described. Homokaryons were recovered from regenerated protoplasts obtained from several heterokaryotic strains. A total of 16 novel hybrids were produced in 63 attempted crosses between paired homokaryons. Recovery of both homokaryons and hybrids was verified by analysis of restriction fragment length polymorphisms. Three of four hybrids fruited in small-scale tests, further confirming that the isolates were true hybrids. Colony morphology alone was found to be a poor indicator of hybrid status. In two instances, three homokaryons crossed successfully in all combinations, suggesting that there are at least three alleles at the putative mating-type locus. Crosses between homokaryons from commercial and wild-collected isolates indicated that these strains belong to the same biological species.     

Thymosin alpha 1-induced modulation of cellular responses and functional T-cell subsets in mice with experimental autoimmune thyroiditis

The effects of Ta-1, a peptide constituent of thymosin fraction 5, were studied on murine autoimmune thyroiditis using two congenic strains of mice, B10.Br (Br) and B10.D2 (D2), which are sensitive and resistant to experimental autoimmune thyroiditis (EAT) induction, respectively. EAT was induced by either 2 weekly iv injections of mouse thyroglobulin with adjuvant lipopolysaccharide (LPS) or intradermal injection of thyroglobulin mixed with complete Freund's adjuvant (CFA). The criteria for induction and intensity of thyroiditis were the level of lymphoid infiltration in the thyroid gland and the titer of anti-thyroglobulin antibodies. Ta-1 was given in 5 or 10 daily sc injections in doses ranging from 0.0001 to 0.1 microgram/injection. The injections were commenced at varying intervals from the 1st to the 4th week after immunization. T-Cell subsets in the spleens were determined 2 weeks after the first antigen injection and thyroid infiltration was determined 3 weeks later. Treatment with Ta-1 between the two antigen injections increased the level of thyroiditis in resistant mice, but had no effect in sensitive mice. Treatment for the first 2 weeks had similar effects in resistant mice, but also suppressed thyroiditis in the sensitive strain. Later treatments, during the 3rd and 4th weeks after immunization also revealed immunomodulating properties of Ta-1, with a suppressing effect on thyroiditis in sensitive mice and an enhancing effect in the resistant strain. Both effects of Ta-1 were dose dependent. The effects of Ta-1 on the individual phenotypes were also dose dependent. The dose of 0.01 microgram greatly lowered the percentages of Lyt-2+3+ cells in D2 mice and mildly increased the percentages in Br mice, but did not change the Lyt-1+ cell level in either strain. On the other hand, the dose of 0.001 microgram greatly increased the percentage of Lyt-1+ cells in D2 mice and mildly decreased it in the Br strain, but did not alter the Lyt-2+3+ cell subset in either strain. Thus, both doses of Ta-1 modulated Lyt-1+/2+3+ ratios, with each dose affecting a different T-cell subset. The changes in the response to thyroglobulin are apparently exerted through the regulation of the functional T-cell subset balance.