J Med Chem, 1987 Aug, 30(8), 1519 - 21 Lysosomotropic agents . 7 . Broad-spectrum antifungal activity of lysosomotropic detergents; Firestone RA et al.; Lysosomotropic detergents, which kill mammalian cells by disrupting lysosomal membranes, have now been found to be antifungals also . All strains in our assay are susceptible . The mode of action is as yet undetermined, but intracellular vacuoles may be the primary targets. Int J Radiat Biol Relat Stud Phys Chem Med, 1987 Aug, 52(2), 185 - 90 Effect of cellular glutathione content on the induction of DNA double strand breaks by 25 MeV electrons; Frankenberg D et al.; The effect of endogenous glutathione (GSH) on the induction of DNA double strand breaks (dsb) by 25 MeV electrons was investigated using stationary haploid yeast cells defective in gamma-glutamyl-cysteine-synthetase (gsh 1) containing less than 5 per cent of the normal GSH content . In gsh 1 cells the induction of dsb is increased by a factor of 1.5 under oxic and 1.8 under anoxic irradiation conditions: whereas the oxygen enhancement ratio was only slightly decreased (1.9) compared to wild-type cells (2.4). Pediatr Dermatol, 1987 Aug, 4(2), 105 - 7 Leiner's disease associated with diminished third component of complement; Sonea MJ et al.; A 6-week-old female infant experienced recurrent diarrhea, wasting, and generalized seborrheic dermatitis . She manifested defective yeast opsonization and concomitant very low level of the third component of complement (C3) . This is the first report of Leiner's disease associated with diminished C3 . It corroborates evidence to suggest that yeast opsonic activity of normal human serum is dependent on C3 levels. EMBO J, 1987 Aug, 6(8), 2449 - 56 Import of an incompletely folded precursor protein into isolated mitochondria requires an energized inner membrane, but no added ATP; Verner K et al.; We have studied the post-translational import of incomplete precursor chains into isolated yeast mitochondria . The precursor was a fusion protein containing a mitochondrial presequence attached to mouse dihydrofolate reductase . In vitro-synthesis of the precursor was interrupted by the elongation inhibitor cycloheximide and the arrested nascent chains cosedimenting with ribosomes were released by EDTA . These incomplete chains were efficiently imported by isolated yeast mitochondria; their import resembled that of the complete precursor in requiring an energized inner membrane and a mitochondrial presequence . It differed from that of the completed precursor in its resistance to methotrexate (which only binds to correctly folded dihydrofolate reductase) and its independence of added ATP . The incomplete chains were also more sensitive to proteinase K than the completed precursor . We conclude that the incomplete chains were incompletely folded and suggest that the lack of tight folding caused import into mitochondria to become independent of added ATP . This implies that ATP may participate, directly or indirectly, in the unfolding of the precursor for its transport into mitochondria. EMBO J, 1987 Aug, 6(8), 2441 - 8 Transport of proteins to the mitochondrial intermembrane space: the 'sorting' domain of the cytochrome c1 presequence is a stop-transfer sequence specific for the mitochondrial inner membrane; van Loon AP et al.; The presequence of yeast cytochrome c1 (an inner membrane protein protruding into the intermembrane space) contains a matrix-targeting domain and an intramitochondrial sorting domain . This presequence transports attached subunit IV of cytochrome c oxidase into the intermembrane space (van Loon et al . (1987) EMBO J., 6, 2433-2439) . In order to determine how this fusion protein reaches the intermembrane space, we studied the kinetics of its import into isolated mitochondria or mitoplasts and its accumulation in the various submitochondrial compartments . The imported, uncleaved fusion precursor and a cleavage intermediate were bound to the inner membrane and were always exposed to the intermembrane space; they were never found at the matrix side of the inner membrane . In contrast, analogous import experiments with the authentic subunit IV precursor, or the precursor of the iron-sulphur protein of the cytochrome bc1 complex also an inner membrane protein exposed to the intermembrane space), readily showed that these precursors were initially transported across both mitochondrial membranes . We conclude that the intramitochondrial sorting domain within the cytochrome c1 presequence prevents transport of attached proteins across the inner, but not the outer membrane: it is a stop-transfer sequence for the inner membrane . Since the presequence of the iron-sulphur protein lacks such 'stop-transfer' domain, it acts by a different mechanism. J Bioenerg Biomembr, 1987 Aug, 19(4), 341 - 50 Molecular genetics of the VDAC ion channel: structural model and sequence analysis; Forte M et al.; The voltage-dependent anion-selective channel of the outer mitochondrial membrane provides a unique system in which to study the molecular basis of voltage gating of ion flow . We have cloned and sequenced a cDNA coding for this protein in yeast . From the derived amino acid sequence, we have generated a preliminary model for the secondary structure of the protein which suggests that the protein forms a "beta-barrel" type structure . Comparison of the VDAC amino acid sequence with that of the bacterial porins has indicated that the two classes of molecules appear to be unrelated. Nucleic Acids Res, 1987 Jul 24, 15(14), 5787 - 801 Crosslinking of elongation factor Tu to tRNA(Phe) by trans-diamminedichloroplatinum (II) . Characterization of two crosslinking sites in the tRNA; Wikman FP et al.; Trans-diamminedichloroplatinum (II) was used to induce reversible crosslinks between EF-Tu and Phe-tRNA(Phe) within the ternary EF-Tu/GTP/Phe-tRNA(Phe) complex . Up to 40% of the complex was specifically converted into crosslinked species . Two crosslinking sites have been unambiguously identified . The major one encompassing nucleotides 58 to 65 is located in the 3'-part of the T-stem, and the minor one encompassing nucleotides 31 to 42 includes the anticodon loop and part of the 3'-strand of the anticodon stem. Nucleic Acids Res, 1987 Jul 24, 15(14), 5699 - 713 The requirement for the A block promoter element in tRNA gene transcription in vitro depends on the ionic environment; Gabrielsen OS et al.; When yeast cell extracts that faithfully transcribe class III genes are provided with different electrolyte ions, the pattern of transcripts changes . A transcription unit in pBR322, silent with 0.1M potassium chloride, becomes active in the presence of 0.1M potassium acetate . This pseudogene depends on transcription factors B and C and RNA polymerase III like a tRNA gene . The transcribed region contains the only sequence in pBR322 homologous to the modified B block consensus sequence GTTCRDNNC found in normal tRNA genes . The presence of a block A sequence is less evident . When a block A deleted tRNA(GLU) gene was constructed, it behaved similarly: poorly transcribed with 0.1M potassium chloride, well transcribed with 0.1M potassium acetate . In fact, the deletion of the A block promoter element from the tRNA(GLU) gene did not dramatically lower its transcription when tested with potassium acetate, while it had a strong negative effect when tested with potassium chloride . Consequently the requirement for this promoter element is not constant but is a function of the electrolyte composition. Nature, 1987 Jul 23-29, 328(6128), 353 - 5 Genetic evidence that zinc is an essential co-factor in the DNA binding domain of GAL4 protein; Johnston M; The 'cysteine-zinc DNA binding finger' is a recently identified sequence motif that is present in a wide variety of transcriptional regulatory proteins and is thought to be directly involved in DNA binding . It has been proposed that an essential component of this structure is a zinc ion bound between two pairs of cysteine residues . The GAL4-encoded protein of Saccharomyces cerevisiae, which binds to DNA and activates the transcription of several genes, contains this sequence motif . Here I describe a gal4 mutant with an alteration in the cysteine-zinc DNA binding finger whose defect is corrected in vivo by high concentrations of ZnCl2 . The DNA binding activity of the altered protein from this mutant is restored by ZnCl2 in vitro . This is evidence that the GAL4 protein indeed contains zinc ions essential for its DNA binding activity. J Immunol Methods, 1987 Jul 16, 101(1), 119 - 25 The use of fluorescence quenching in flow cytofluorometry to measure the attachment and ingestion phases in phagocytosis in peripheral blood without prior cell separation; Hed J et al.; Flow cytofluorimetry identifies and quantifies cell markers of different leukocyte subpopulations by combining cytofluorimetry with the differences in the light scattering properties of the leukocytes in mixed populations . In the phagocytic assay, reported in this paper, the experimental conditions were selected in such a way that it was possible to analyse the phagocytic function of granulocytes in peripheral blood without time-consuming cell separation . The percentage of phagocytosing granulocytes was not dependent on the concentration of granulocytes at the selected incubation time and particle (yeast-C3b) concentration . Furthermore, it was possible to adapt a previously described fluorescence quenching technique (FQ method) to differentiate between attachment and ingestion . Crystal violet, originally used in the FQ method, could not be used in this assay due to its lysomotropic effect . Trypan blue at a concentration of 0.25 mg/ml or higher at pH 4.5 showed a plateau effect in fluorescence quenching indicating an effect on attached but not ingested particles . This assay offers a simple technique to screen the functional properties of phagocytic cells in peripheral blood. Biochim Biophys Acta, 1987 Jul 7, 913(3), 279 - 84 31P-NMR study of the orotate phosphoribosyltransferase equilibrium with thiopyrophosphate as substrate; Tavares A et al.; 31P-nuclear magnetic resonance spectroscopy was used to directly determine the equilibrium of the reaction catalysed by yeast orotate phosphoribosyltransferase, using orotidine monophosphate and inorganic pyrophosphate as substrates . A Keq value of 0.71 was determined, in good agreement with that of 0.49 calculated by Victor, Greenberg and Sloan (J . Biol . Chem . 254 (1979) 2647-2655), from kinetic data . Substitution of thiopyrophosphate as the substrate shifted the position of the equilibrium 55-fold, to yield a Keq value of 39 . Only the beta S analogue of 5-phosphoribosyl 1-diphosphate appeared to be synthesized in this reaction. J Mol Biol, 1987 Jul 5, 196(1), 11 - 25 Structure of the rat L-type pyruvate kinase gene; Cognet M et al.; The total sequence of a 13,021 base-pair (bp) genomic fragment containing the rat L-type pyruvate kinase (L-PK) gene was determined by "shot gun" sequencing . This fragment includes 8360 bp of the L-PK gene, plus 3193 bp of the 5'-flanking and 1468 bp of the 3'-flanking regions . Like the chicken PK-M1 gene, the rat L-PK gene exhibits a fully conserved exon-intron structure, with 11 exons and 10 introns . In the chicken M1 gene, the coding sequences are well conserved (about 70%), in particular at the level of the exons implicated in the formation of PK active sites, exons that are also partially homologous to the corresponding sequences of the yeast gene . Various types of repetitive sequences exist in the L-PK gene, especially two ID (identifier) sequences located in the second intron and the 11th exon . Elements very similar to the "cyclic AMP-dependent regulatory element" recently described in the phosphoenolpyruvate carboxykinase and somatostatin genes are found in the sequenced fragment, but far upstream (-2338) and downstream (+5788) from the cap site . Various sequences homologous to described regulatory elements (glucocorticoid regulatory elements, enhancers, potential Z-DNA) are also observed 5' and 3' of the cap site . A comparison of the 5'-flanking region of the L-PK gene with the same regions of liver-specific or non-specific, cyclic-AMP-responsive or non-responsive genes was also made . It revealed various potentially interesting features that will be used to guide a further functional study . The cap site was determined by primer extension and nuclease S1 mapping using either mature mRNA or precursor RNA as templates . With both templates the start site of transcription appeared to be microheterogeneous, 19 to 14 bp before the ATG translation initiation codon. Cell, 1987 Jul 3, 50(1), 137 - 42 The carboxy-terminal 30 amino acids of GAL4 are recognized by GAL80; Ma J et al.; In wild-type yeast the action of the transcriptional activator GAL4 is inhibited by GAL80, and galactose relieves this inhibition . We show that deletion mutants of GAL4 lacking 30 amino acids of the carboxyl terminus activate transcription constitutively, whereas other deletion mutants bearing the carboxy-terminal 30 amino acids are inhibited by GAL80 . Moreover, GAL4 fragments bearing these 30 amino acids, when expressed from a strong promoter on multicopy plasmids, free the endogenous GAL4 from inhibition by GAL80 . These and other results suggest that GAL80 recognizes the carboxy-terminal 30 amino acids of GAL4, forming a complex that, though bound to DNA, does not activate transcription. J Cell Biol, 1987 Jul, 105(1), 127 - 35 A single histone acetyltransferase from Tetrahymena macronuclei catalyzes deposition-related acetylation of free histones and transcription-related acetylation of nucleosomal histones; Chicoine LG et al.; A salt-extracted histone acetyltransferase activity from Tetrahymena macronuclei acetylates mostly histone H3 and H4 when free histones are used as substrate . Free histone H4 is acetylated first at position 11 (monoacetylated) or positions 11 and 4 (diacetylated) . This activity strongly resembles in vivo, deposition-related acetylation of newly synthesized histones . When acetylase-free mononucleosomes are used as substrate, all four core histones are acetylated by the same extract, and H4 is acetylated first at position 7 (monoacetylated) or positions 7 and 4 (diacetylated) . In this respect, the activity of the extract is indistinguishable from postsynthetic, transcription-related histone acetylation that occurs in vivo or in isolated nuclei . Heat inactivation curves with both substrates are indistinguishable, and free histones compete with chromatin for limiting amounts of enzyme activity . These results argue strongly that two distinct, biologically important histone acetylations, one deposition related and one transcription related, are carried out by a single acetyltransferase. Anal Biochem, 1987 Jul, 164(1), 181 - 9 Measurement of the susceptibility of paramagnetically labeled cells with paramagnetic solutions; Russell AP et al.; A method of measuring the volumetric magnetic susceptibility, in which magnetically labeled cells or other particles are suspended in a paramagnetic solution of known susceptibility over the poles of a magnet, is presented . If the cells are more magnetic than the solution, they are attracted toward the poles; if they are less magnetic, they are repelled . If they have the same susceptibility as the solution, they do not move . Under this condition, the cells are said to be "isomagnetic" with the surrounding solution . Since the volumetric susceptibility of this solution is known, the susceptibility of the cells is obtained . Using the "isomagnetic" method, the volumetric susceptibilities of test metal powders were determined within +/- 8 X 10(-6) SI units . Yeast, colonic carcinoma, and liver cells, rendered magnetic with erbium chloride, had susceptibilities ranging from 13 to 20 X 10(-6) . Particles of articular cartilage treated with erbium chloride were heterogeneous, with susceptibilities ranging between 50 and 125 X 10(-6), while particles of bone had a susceptibility of 560 to 580 X 10(-6) . Eukaryotic cells labeled with ferritin attained susceptibilities of less than 1 X 10(-6). Radiobiologiia, 1987 Jul-Aug, 27(4), 487 - 92 {Effect of the dose rate on the synergism of combined ionizing radiation and hyperthermia}; Zhurakovskaia GP et al.; In experiments with yeast cells it was shown that the synergistic effect of a combination of ionizing radiation and hyperthermia is a function of dose rate . It was demonstrated that the temperature at which radiation is delivered should be elevated to obtain the maximum synergistic effect with the increasing dose rate. Hepatology, 1987 Jul-Aug, 7(4), 743 - 9 Evidence for the cell-surface localization of antigens cross-reacting with the "mitochondrial antibodies" of primary biliary cirrhosis; Ghadiminejad I et al.; Studies with subfractions of Saccharomyces cerevisiae obtained by differential centrifugation showed only two primary biliary cirrhosis-specific antigens . These antigenic species were shown, using preabsorption studies, to have determinants cross-reactive with their mammalian counterpart . Distribution profiles of marker enzymes and primary biliary cirrhosis antigens between sucrose density gradient subcellular fractions of yeast showed that a relatively high concentration of primary biliary cirrhosis-specific antigens was associated with fractions containing plasma membranes, as well as those containing mitochondria . The possible cell-surface localization of the primary biliary cirrhosis antigens was further investigated using an indirect immunofluorescent technique on a number of different mammalian cells . Rat hepatoma cells, isolated rat hepatocytes and human polymorphonuclear leukocytes and lymphocytes stained positively with primary biliary cirrhosis sera, but not with normal sera or primary biliary cirrhosis sera preabsorbed with beef heart mitochondria . However, blood cells from primary biliary cirrhotic patients gave positive immunofluorescence in all tests, which is compatible with prior binding of the patients' own antimitochondrial antibodies to the surface of the cells. Proc Natl Acad Sci U S A, 1987 Jul, 84(14), 4851 - 5 Amino acid sequence of the beta subunit of bovine lung casein kinase II; Takio K et al.; The amino acid sequence of the 209-residue beta subunit of bovine lung casein kinase II has been determined . Excluding the amino-terminal blocking group, which was not identified, the molecular weight of the polypeptide chain is 24,239 . A marked polarity of the beta subunit is indicated by clusters of negative charges in the amino-terminal region and of positive charges in the carboxyl-terminal region . Whereas the beta subunit shows no homology with any known protein, a segment of the sequence of the larger and microheterogeneous alpha subunit exhibits homology with the catalytic domains of other protein kinases, particularly with the yeast cell-division-control protein CDC28. Biol Chem Hoppe Seyler, 1987 Jul, 368(7), 855 - 61 Studies on immunoassays of peptide factors . V . Synthesis of cholecystokinin-58-{1-11}/iso-1-cytochrome c conjugate; Moroder L et al.; In previous studies on model compounds we have found that the maleimide function is sufficiently stable under the conditions of peptide synthesis to allow its incorporation at preselected positions of a peptide chain in earlier steps of the synthetic route . Taking advantage of this observation the N-terminal undecapeptide of canine cholecystokinin-58 containing at its N-terminus the maleimide group became accessible in high yields as chromatographically homogenous and analytically well characterized compound . Via the incorporated anchor group the undecapeptide was linked selectively at its N-terminus to the cysteine residue 107 of iso-1-cytochrome c to yield a well characterized conjugate of 1:1 stoichiometry for immunization experiments. Biol Chem Hoppe Seyler, 1987 Jul, 368(7), 839 - 48 Studies on immunoassays of peptide factors . III . Gastrin/iso-1-cytochrome C as immunogen for raising anti-gastrin antisera; Moroder L et al.; Iso-1-cytochrome c contains in penultimate position of its sequence a cysteine residue which in analogy to the known tertiary structures of cytochromes c should be exposed on the surface of the protein . Its selective reaction with N alpha-maleoyl-beta-alanyl-human-little-gastrin-I-{2-17} led to a well characterized and homogeneous gastrin conjugate to be used as immunogen in rabbits . The antisera raised by this procedure exhibited a degree of specificity for the hormone gastrin parallel to that of the gastrin receptor . This is clearly documented by comparison of the immune crossreactivities of gastrin-peptides of increasing chain length and of fragments corresponding to various regions of the hormone molecule with their biological activity . The immune response provoked in the animals by the use of an homogeneous immunogen was found to be highly reproducible in terms of specificity of the antigastrin antibodies. J Biol Chem, 1987 Jun 25, 262(18), 8875 - 83 ERp99, an abundant, conserved glycoprotein of the endoplasmic reticulum, is homologous to the 90-kDa heat shock protein (hsp90) and the 94-kDa glucose regulated protein (GRP94); Mazzarella RA et al.; We have isolated an expressible full-length cDNA clone encoding murine ERp99, an abundant, conserved transmembrane glycoprotein of the endoplasmic reticulum membrane . ERp99 is synthesized as a 92,475-kDa precursor containing 802 amino acids . It possesses a signal peptide of 21 amino acids which is cleaved cotranslationally . Analysis of the amino acid sequence deduced from the nucleotide sequence of the cDNA clone led us to propose a model for the orientation of ERp99 in the endoplasmic reticulum membrane . In this model, ERp99 possesses one membrane-spanning, stop transfer segment in the N-terminal region . The protein chain passes through the membrane only once, and approximately 75% of the protein remains on the cytoplasmic side of the ER membrane . Comparison of the ERp99 sequence to the sequence of other proteins revealed that ERp99 has extensive homology with the 90-kDa heat shock protein of Saccharomyces cerevisiae (hsp90) and the 83-kDa heat shock protein of Drosophila melanogaster . In addition, the N terminus of mature ERp99 is identical to that of the 94-kDa glucose regulated protein (GRP94) of mammalian cells. Nucleic Acids Res, 1987 Jun 25, 15(12), 4771 - 87 Human mRNA polyadenylate binding protein: evolutionary conservation of a nucleic acid binding motif; Grange T et al.; We have isolated a full length cDNA (cDNA) coding for the human poly(A) binding protein . The cDNA derived 73 kd basic translation product has the same Mr, isoelectric point and peptidic map as the poly(A) binding protein . DNA sequence analysis reveals a 70,244 dalton protein . The N terminal part, highly homologous to the yeast poly(A) binding protein, is sufficient for poly(A) binding activity . This domain consists of a four-fold repeated unit of approximately 80 amino acids present in other nucleic acid binding proteins . In the C terminal part there is, as in the yeast protein, a sequence of approximately 150 amino acids, rich in proline, alanine and glutamine which together account for 48% of the residues . A 2,9 kb mRNA corresponding to this cDNA has been detected in several vertebrate cell types and in Drosophila melanogaster at every developmental stage including oogenesis. Biochemistry, 1987 Jun 16, 26(12), 3453 - 61 Pyridine coenzyme analogues . Synthesis and characterization of alpha- and beta-nicotinamide arabinoside adenine dinucleotides; Kam BL et al.; The synthesis and characterization of a new pyridine coenzyme analogue containing a nicotinamide arabinonucleotide moiety are reported . The redox potentials are -339 mV for beta-oxidized nicotinamide arabinoside adenine dinucleotide and -319 mV for alpha-oxidized nicotinamide adenine dinucleotide, and the lambda max is 346 and 338 nm for beta- and alpha-reduced nicotinamide arabinoside adenine dinucleotides (araNADH), respectively . Anomerization of the reduced analogues leads to a 5:1 ratio of alpha-araNADH to beta-araNADH at 90 degrees C . These results establish that the relative configuration of the 2'-hydroxyl to the base is the primary determinant for the configuration-dependent changes in lambda max, the redox potential of the pyridine nucleotides, and the preferred anomeric configuration of the reduced coenzymes . Comparison of the 1H and 31P NMR spectral data of the analogues with those for the ribo coenzymes is reported and the conformational analysis discussed . The coenzyme properties of the arabino analogues have been evaluated with yeast and horse liver alcohol dehydrogenases . Both the alpha- and beta-anomers are found to serve as coenzymes, and the stereochemistry of hydride transfer is identical for both anomers. Genetica, 1987 Jun 15, 72(2), 151 - 9 Experimental evidence for the adaptive value of sexual reproduction; Wolf HG et al.; It is generally believed that recombination by sexual reproduction is unfavourable in constant environments but is of adaptive value under changing environmental conditions . To test this theory, experimental populations of yeast (Saccharomyces cerevisiae) were set up and maintained at different levels of environmental heterogeneity . Recombination was estimated by determining sporulation rates . Sporulation rates first increased in populations living in highly variable environments, but after some time began to decrease . The decrease started last and was slowest in populations which were maintained under the same conditions for a sufficiently long time, to allow some adaptation of the gene pool to the respective environment . Patterns of genotypic variability could not be interpreted in such simple terms, but there was a statistically significant correlation between sporulation rate and genotypic variability . This correlation is to be expected because recombination generates genotypic variability . Summing up, recombination by sexual reproduction is advantageous in changing environments if the population can track the changes in the environment by changing its genotypic structure. Eur J Biochem, 1987 Jun 15, 165(3), 543 - 5 Fructofuranose 2-phosphate is the product of dephosphorylation of fructose 2,6-bisphosphate; Purwin C et al.; Using comparative ion-exchange chromatography on Dowex 1X4, the product of dephosphorylation of fructose 2,6-bisphosphate with purified yeast fructose-2,6-bisphosphate 6-phosphohydrolase, was shown to be identical to the furanose form of fructose 2-phosphate prepared by chemical synthesis according to Pontis and Fischer {Biochem . J . 89, 452-459 (1963)} . As expected for the furanose form of fructose 2-phosphate, the enzymatically formed product consumes 1 mol periodate/mol fructose 2-phosphate, whereas the chemically synthesized pyranose form consumes 2 mol periodate/mol . In addition, it is shown that the enzymatic product behaves identically to the furanose, not the pyranose, form of fructose 2-phosphate in hydrolysis of the ester bond at pH 4 and 37 degrees C, as described previously for the chemically synthesized compounds {Pontis and Fischer (1963) vide supra}. Yeast, 1987 Jun, 3(2), 71 - 6 Analysis of DNA double strand breakage and repair using orthogonal field alternation gel electrophoresis; Contopoulou CR et al.; Orthogonal field alternation gel electrophoresis (OFAGE) allows separation of DNA molecules in the size range of 200 kb to 3000 kb . These sizes encompass the chromosome sizes of the genome of Saccharomyces cerevisiae . Using this technique, we have found that yeast cells exposed to X-rays generate a smear of DNA fragments corresponding to the products of random, independent double strand breaks, and that the bands corresponding to unbroken chromosomes decrease in intensity in direct proportion to chromosome size . If exposed wild type cells are permitted time to repair (5 h at 30 degrees C on YEPD), the fragments partially disappear and the chromosome bands reappear, although at less than normal intensity . In certain radiation-sensitive mutants (rad51, rad52 and rad54), the fragment smear appears following X-ray exposure but no repair of broken chromosomes occurs . In fact, loss of the fragments occurs; this could appear as partial repair using other procedures. Biochem Cell Biol, 1987 Jun, 65(6), 536 - 42 Effects of ribosome dissociation on the structure of the ribosome-associated 5.8S RNA; Lo AC et al.; Diethyl pyrocarbonate reactivity and thermal denaturation were used to probe potential ribosomal interactions between tRNA and the small 5.8S and 5S rRNAs . Puromycin, an analogue of the terminal aminoacyl-adenosine portion of aminoacyl-tRNA, was observed to increase the accessibility of the 5.8S rRNA, including the highly conserved GAACp sequences . EDTA which releases both tRNA and the 5S rRNA-protein complex resulted in an even greater accessibility in the 5.8S rRNA . The thermal dissociation of whole ribosomes resulted in the release of all three RNAs, with a striking similarity in the denaturation profiles . These results strongly suggest an interdependence in the ribosome-associated structures of the small rRNAs and provide in situ evidence for the various 5S rRNA, 5.8S rRNA, and tRNA containing ribonucleoprotein complexes previously reconstituted through affinity chromatography. J Cell Physiol, 1987 Jun, 131(3), 458 - 64 Recombinant murine granulocyte-macrophage (GM) colony-stimulating factor supports formation of GM and multipotential blast cell colonies in culture: comparison with the effects of interleukin-3; Koike K et al.; We studied the effects of murine recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) on murine hemopoiesis in methylcellulose culture . The GM-CSF was purified from cultures of Saccharomyces cerevisiae transfected with a cloned murine GM-CSF cDNA . In cultures of spleen cells from normal mice, only granulocyte-macrophage (GM) colonies were supported by GM-CSF . Blast cell colonies were the predominant type in cultures of spleen cells from 5-fluorouracil (5-FU)-treated mice . Dose-response studies revealed that maximal GM and blast cell colony formation is achieved with 100 U/ml GM-CSF . Blast cell colonies revealed variable but high replating efficiencies, and the secondary colonies included multilineage colonies . Serial replating of washed blast cell colonies in cultures with GM-CSF provided evidence for the direct effects of GM-CSF on the proliferation of multipotential blast cells . A combination of GM-CSF and interleukin-3 (IL-3) did not increase the number of blast cell colonies over the level supported by IL-3 . This observation indicates that the progenitors for blast cell colonies that responded to GM-CSF are a subpopulation of multipotential progenitors that are supported by IL-3 . Cytological studies of colonies derived from GM-CSF and/or IL-3 suggest that the eosinophilopoietic ability of murine GM-CSF is less than that of IL-3. DNA, 1987 Jun, 6(3), 189 - 97 A genetically engineered P450 monooxygenase: construction of the functional fused enzyme between rat cytochrome P450c and NADPH-cytochrome P450 reductase; Murakami H et al.; A hybrid cDNA encoding a fused enzyme consisting of rat cytochrome P450c and rat NADPH-cytochrome P450 reductase was constructed by combining the cytochrome P450c cDNA with the cDNA fragment encoding the protease-solubilized moiety of the NADPH-cytochrome P450 reductase . The hybrid cDNA was inserted between the yeast alcohol dehydrogenase I promoter and terminator of the expression vector pAAH5 to yield expression plasmid pAMP19 . Saccharomyces cerevisiae AH22 cells transformed with the expression plasmid pAMP19 produced a 130-kD protein reactive with both anti-cytochrome P450c Ig and antireductase Ig . The yeast cells containing the fused enzyme exhibited about four times higher monooxygenase activity toward 7-ethoxycoumarin than those containing rat cytochrome P450c alone . The fused enzyme was purified from the yeast microsomal fraction by sequential chromatography with DEAE-cellulose and 2',5'-ADP Sepharose 4B columns . The preparation had an apparent molecular weight of 130 kD and the same sequence of the 10 amino-terminal amino acids as that of rat cytochrome P450c . Spectral properties of the fused enzyme indicated the presence of a protoheme, flavin adenine dinucleotide, and flavin mononucleotide in the molecule . The reaction mechanism of the fused enzyme followed first-order kinetics . These results clearly indicate that the fused enzyme is a new self-catalytic P450 monooxygenase . Trypsin treatment of yeast microsomes containing the fused enzyme suggested that the P450 moiety is embedded in the microsomal membrane with the reductase moiety lying on the cytoplasmic side. Mol Cell Biol, 1987 Jun, 7(6), 2221 - 30 The site-specific ribosomal insertion element type II of Bombyx mori (R2Bm) contains the coding sequence for a reverse transcriptase-like enzyme; Burke WD et al.; Two classes of DNA elements interrupt a fraction of the rRNA repeats of Bombyx mori . We have analyzed by genomic blotting and sequence analysis one class of these elements which we have named R2 . These elements occupy approximately 9% of the rDNA units of B . mori and appear to be homologous to the type II rDNA insertions detected in Drosophila melanogaster . Approximately 25 copies of R2 exist within the B . mori genome, of which at least 20 are located at a precise location within otherwise typical rDNA units . Nucleotide sequence analysis has revealed that the 4.2-kilobase-pair R2 element has a single large open reading frame, occupying over 82% of the total length of the element . The central region of this 1,151-amino-acid open reading frame shows homology to the reverse transcriptase enzymes found in retroviruses and certain transposable elements . Amino acid homology of this region is highest to the mobile line 1 elements of mammals, followed by the mitochondrial type II introns of fungi, and the pol gene of retroviruses . Less homology exists with transposable elements of D . melanogaster and Saccharomyces cerevisiae . Two additional regions of sequence homology between L1 and R2 elements were also found outside the reverse transcriptase region . We suggest that the R2 elements are retrotransposons that are site specific in their insertion into the genome . Such mobility would enable these elements to occupy a small fraction of the rDNA units of B . mori despite their continual elimination from the rDNA locus by sequence turnover. J Biol Chem, 1987 May 25, 262(15), 7081 - 6 Purification and characterization of arginase from Neurospora crassa; Borkovich KA et al.; We have purified an enzymatically active form of arginase from a wild-type strain of Neurospora crassa to homogeneity . The enzyme has a subunit molecular weight of 38,300 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . The native protein migrated as a hexamer during gel-filtration chromatography with an apparent molecular weight of 266,000 . The enzyme exhibited hyperbolic kinetics at pH 9.5 with an apparent Km for arginine of 131 mM . Antiserum was prepared against the purified enzyme and used to demonstrate the existence of three cross-reactive proteins in crude extracts of wild-type N . crassa . One of these proteins corresponded to the purified protein, whereas the other two were of molecular weights 41,700 and 26,800, respectively . Using the same antiserum, we found that rat liver, but not rat kidney, contains immunoreactive material . We also detected two proteins in extracts of Saccharomyces cerevisiae that were weakly cross-reactive with the antiserum . These data provide evidence for the existence of multiple forms of arginase in fungi as well as in mammals. J Biol Chem, 1987 May 25, 262(15), 7132 - 4 Substrate specificity of acetyl coenzyme A synthetase; Patel SS et al.; Acetyl coenzyme A synthetase (EC 6.2.1.1) has been examined for its ability to accept various carboxylic acids as substrates in place of acetic acid . The activity of the enzyme with these substrates was monitored using a coupled enzyme assay and high pressure liquid chromatography (HPLC) analysis . Short chain carboxylic acids were found to be active including: propionic, acrylic, fluoroacetic, methacrylic, 3-chloropropionic, 3-bromopropionic, and propiolic . The kinetic parameters, Km and % Vmax of the carboxylic acid substrates, are reported and show that these acids are poorer substrates than acetic acid . Several of the acyl CoAs were synthesized on a preparative scale using enzyme catalysis, purified using preparative HPLC, and characterized using proton NMR spectroscopy . In the course of the NMR identification, a complete and fully resolved spectral assignment for all the protons of coenzyme A was made and is reported . The acyl-CoA analogs should be useful as substrate analogs and as potential affinity labels for enzymes that bind acetyl-CoA. Biochemistry, 1987 May 19, 26(10), 2836 - 48 Kinetics of reduction by free flavin semiquinones of the components of the cytochrome c-cytochrome c peroxidase complex and intracomplex electron transfer; Hazzard JT et al.; The kinetics of reduction by free flavin semiquinones of the individual components of 1:1 complexes of yeast ferric and ferryl cytochrome c peroxidase and the cytochromes c of horse, tuna, and yeast (iso-2) have been studied . Complex formation decreases the rate constant for reduction of ferric peroxidase by 44% . On the basis of a computer model of the complex structure {Poulos, T.L., & Finzel, B.C . (1984) Pept . Protein Rev . 4, 115-171}, this decrease cannot be accounted for by steric effects and suggests a decrease in the dynamic motions of the peroxidase at the peroxide access channel caused by complexation . The orientations of the three cytochromes within the complex are not equivalent . This is shown by differential decreases in the rate constants for reduction by neutral flavin semiquinones upon complexation, which are in the order tuna much greater than horse greater than yeast iso-2 . Further support for differences in orientation is provided by the observation that, with the negatively charged reductant FMNH., the electrostatic environments near the horse and tuna cytochrome c electron-transfer sites within their respective complexes with peroxidase are of opposite sign . For the horse and tuna cytochrome c complexes, we have also observed nonlinear concentration dependencies of the reduction rate constants with FMNH. . This is interpreted in terms of dynamic motion at the protein-protein interface . We have directly measured the physiologically significant intra-complex one electron transfer rate constants from the three ferrous cytochromes c to the peroxide-oxidized species of the peroxidase . At low ionic strength these rate constants are 920, 730, and 150 s-1 for tuna, horse, and yeast cytochromes c, respectively . These results are also consistent with the contention that the orientations of the three cytochromes within the complex with CcP are not the same . The effect on the intracomplex electron-transfer rate constant of the peroxidase amino acid side chain(s) that is (are) oxidized by the reduction of peroxide was determined to be relatively small . Thus, the rate constant for reduction by horse cytochrome c of the peroxidase species in which only the heme iron atom is oxidized was decreased by only 38%, indicating that this oxidized side-chain group is not tightly coupled to the ferryl peroxidase heme iron . Finally, it was found that, in the absence of cytochrome c, neither of the ferryl peroxidase species could be rapidly reduced by flavin semiquinones.(ABSTRACT TRUNCATED AT 400 WORDS) Anal Biochem, 1987 May 15, 163(1), 100 - 6 Variations on the "dilution" method for reconstituting cytochrome c oxidase into membrane vesicles; Ramirez J et al.; A method for the rapid incorporation of cytochrome c oxidase into membranes has been developed . This method essentially consists of obtaining a preparation of the enzyme in which it is isolated and then dissolving it in a medium containing 0.5% of the detergent Tween 20, which gives a final concentration of 0.0125% after reconstitution . These studies revealed an optimal ratio of 1 microgram of enzyme to 5 mg of phospholipids . A similar optimal ratio was found when the amount of protein was varied . The optimum temperature was found to be 30 degrees C . Without a peak value being reached, it was found that the best reconstitution was obtained at pH 7.0-8.0 . When measurements were performed either with a fluorescent cyanine (DiSC3) or by the uptake of tetraphenylphosphonium, it was found that the enzyme, with cytochrome c added to the outside, was capable of generating a membrane potential that was negative inside . Using the same procedure, the enzyme could also be reconstituted into vesicles of yeast plasma membrane . The procedure, then, seems adequate for incorporating cytochrome c oxidase into different kinds of membrane vesicles. Biochem Biophys Res Commun, 1987 May 14, 144(3), 1237 - 42 Glucose tolerance factor stimulates 3-O-methylglucose transport into isolated rat adipocytes; Tokuda M et al.; Glucose tolerance factor partially purified from yeast extract powder stimulated {U-14C}-D-glucose uptake to a level 5.6 times greater than the basal level in the absence of insulin in isolated adipocytes prepared from rats fed with normal laboratory chow . The factor also stimulated 3-O-methylglucose transport 2.2-fold from the basal level in the absence of insulin, but not in the presence of 8 nM insulin . Kinetic analysis revealed that glucose tolerance factor increased 3-O-methylglucose transport by decreasing the Ks value for 3-O-methylglucose with little change in the Vmax. Biochim Biophys Acta, 1987 May 13, 919(1), 21 - 5 Studies on the peroxisomal oxidation of palmitate and lignocerate in rat liver; Wanders RJ et al.; We have investigated the pathways involved in the peroxisomal oxidation of palmitate and lignocerate, measured as the cyanide-insensitive formation of acetyl units, in rat-liver homogenates . The peroxisomal beta-oxidation of both fatty acids is dependent on the presence of ATP, coenzyme A, NAD+ and Mg2+ . However, there is a striking difference in the dependence of the rate of oxidation of the two substrates on the concentration of the individual cofactors, especially ATP . The peroxisomal beta-oxidation of lignocerate was inhibited to a progressively greater extent by increasing concentrations of palmitate and vice versa . Activation of lignoceric acid to lignoceroyl-CoA, however, was not inhibited by increasing concentrations of palmitate, and vice versa . It can be concluded that the peroxisomal palmitate and lignocerate beta-oxidation pathways differ in at least one enzymic reaction (the synthetase), but that the two pathways share at least one common step. Mutat Res, 1987 May, 178(1), 11 - 20 NADPH as rate-limiting factor for microsomal metabolism . An alternative and economic NADPH-generating system for microsomal mono-oxygenase in in vitro genotoxicity studies; Paolini M et al.; The effect of NADPH supply on enzymatic activity and its stability were investigated with respect to the mono-oxygenase activities of 7-ethoxyresorufin O-deethylase (ERD), dinemorphan N-demethylase (DND), aminopyrine N-demethylase (APD), 7-ethoxycoumarin O-deethylase (ECD) and p-nitroanisole O-demethylase (p-NAD) under incubation conditions for the liver microsomal assay (LMA) . Experiments with S9 liver fractions of mouse (induced with Na-phenobarbital and beta-naphthoflavone) and rat (induced with Aroclor 1254) were set out at different pre-incubation times with and without exogenous isocitrate dehydrogenase (IC-DH) in the LMA . Such LMA mixtures contain Mn2+, NADP+, DL-isocitrate (IC) and endogenous IC-DH as NADPH-generating machinery . No changes in mono-oxygenase stability and lipid peroxidation (LP) were observed in the presence of exogenous IC-DH . The metabolizing capability at the considered times was the maximal one, as shown by no stability changes after the direct addition of IC-DH to the enzymatic assays . Exogenous IC-DH in the incubation for LMA did not alter the mitotic crossing-over and the mitotic gene conversion of dimethylnitrosamine (DMNA) and AR2MNFN (a nitroimidazo{2,1-b}thiazole) in the tester D7 strain of Saccharomyces cerevisiae . It was concluded that endogenous IC-DH seems to be sufficient to provide a saturating level of NADPH for mono-oxygenase activities during incubations for LMA without additional external NADPH-generating enzyme activity. Derm Beruf Umwelt, 1987 May-Jun, 35(3), 81 - 91 {Problems of occupationally induced respiratory allergies as exemplified by bakers' asthma}; Thiel H; In the Federal Republic of Germany (FRG) approximately every fourth case of occupational lung disease, registered with application for compensation at the "Gewerbliche Berufsgenossenschaften", in 1984 was suspected to be caused by allergic airway obstruction . Respiratory allergies may arise if two prerequisites exist firstly exposure to sensitizing agents in the work environment and secondly individual disposition to allergic reactions . The large range of sensitizing agents includes organic and inorganic substances derived from animals, plants, fungi, metals or chemicals . Most of them are responsible for Type-I IgE-mediated allergic reactions . Three different patterns of respiratory responses can be found following simulated occupational exposure: immediate, late (non-immediate), and the dual-type of asthmatic reaction, whereby the non-immediate asthmatic response must be strictly differentiated from the genuine Type-III hypersensitivity . Today, bakers' asthma is the most frequent and most costly occupational allergy in the FRG, contributing more than 50% of all registered and more than 75% of all compensated cases . Therefore, flour allergies can be used as a classic model of an occupational allergic disease in order to discuss epidemiological, social and clinical problems, such as prevalence, socioeconomic impact, as well as prevention, early diagnosis and therapeutic measures . Despite many well-known clinical and epidemiological data, respiratory allergies display a lot of unresolved questions calling for further research. Anal Biochem, 1987 May 1, 162(2), 521 - 8 Direct determination of the specific activity of RNA uniformly labeled with 32P; Mueller DM et al.; A simple method for the direct determination of the specific activity of RNA uniformly labeled with 32P is described . The procedure is based on the premise that upon disintegration of 32P to 32S, the phosphodiester bond is broken . Analysis of the rate of decay of the full-length molecule by gel electrophoresis and autoradiography can accurately determine the "intramolecular specific activity" of the RNA . An equation that predicts the relative intensity of the intact RNA molecules remaining as a function of time is presented . These predictions are confirmed using in vitro-synthesized RNA labeled at a known specific activity . This procedure has been used to determine the intramolecular specific activity of RNA labeled in vivo in yeast . It can also be employed to choose the best conditions for experiments utilizing uniformly labeled RNA or single-stranded DNA and requiring the detection of intact molecules. Anal Biochem, 1987 May 1, 162(2), 466 - 75 Primer extension analysis of tRNA gene transcripts synthesized in vitro and in vivo; Dingermann T et al.; The primer elongation method has been adapted to analyze tRNA gene transcripts . The primer used to direct cDNA synthesis from a corresponding tRNA template, in the presence of AMV reverse transcriptase, was a restriction fragment, or a synthetic oligonucleotide, containing exclusively coding nucleotides of a tRNA gene . This method not only allows one to identify the exact 5'-end of mature tRNA, but also 5'-ends of primary transcripts are readily determined . Further, analysis of tRNAs synthesized in vitro, as well as tRNAs produced in vivo in homologous and heterologous organisms can be studied . Purification of the tRNAs questioned, from bulk tRNA, is not necessary. Mol Cell Biol, 1987 May, 7(5), 1602 - 11 Eucaryotic RNA polymerase conditional mutant that rapidly ceases mRNA synthesis; Nonet M et al.; We have isolated a yeast conditional mutant which rapidly ceases synthesis of mRNA when subjected to the nonpermissive temperature . This mutant (rpb1-1) was constructed by replacing the wild-type chromosomal copy of the gene encoding the largest subunit of RNA polymerase II with one mutagenized in vitro . The rapid cessation of mRNA synthesis in vivo and the lack of RNA polymerase II activity in crude extracts indicate that the mutant possesses a functionally defective, rather than an assembly-defective, RNA polymerase II . The shutdown in mRNA synthesis in the rpb1-1 mutant has pleiotropic effects on the synthesis of other RNAs and on the heat shock response . This mutant provides direct evidence that the RPB1 protein has a functional role in mRNA synthesis. Proc Natl Acad Sci U S A, 1987 May, 84(10), 3176 - 80 Reaction of argininosuccinase with bromomesaconic acid: role of an essential lysine in the active site; Lusty CJ et al.; We have undertaken studies on bovine liver argininosuccinase (L-argininosuccinate arginine-lyase, EC 4.3.2.1) with the active site-directed reagent bromo{U-14C}mesaconic acid, an analogue of fumaric acid . Reactivity, measured by enzyme inactivation, followed pseudo-first-order kinetics, and the rate increased with reagent concentration . Argininosuccinate completely protected the enzyme against inactivation, but neither arginine nor fumarate was protective . A plot of the degree of inactivation as a function of alkyl groups incorporated was extrapolated to 4 mol per mol of enzyme, or 1 mol per active site . After large-scale alkylation of the enzyme (and digestion with trypsin), two 14C-labeled tryptic peptides were isolated . These were chemically sequenced by the Edman method . The amino acid sequences proved to be identical with regions of the deduced amino acid sequences or argininosuccinases from human and yeast sources {O'Brien, W . E., McInnes, R., Kalumuck, K . & Adcock, M . (1986) Proc . Natl . Acad . Sci . USA 83, 7211-7215; Beacham, I . R., Schweitzer, B . W., Warrick, H . M . & Carbon, J . (1984) Gene 29, 271-279} . The 14C-labeled tryptic peptide in the active site region had the sequence Gly-Leu-Glu-Xaa-Ala-Gly-Leu-Leu-Thr-Lys; Xaa represents an unknown phenylthiohydantoin derivative detected in cycle 4 . The corresponding amino acid was identified as lysine-51 on the basis of sequence similarity with human and yeast amino acid sequences in this region . The reaction of the enzyme with the alkylating agent and the specific protection against inactivation by argininosuccinate suggest that this lysine residue has an essential role in the binding of argininosuccinate to the enzyme and, consequently, is essential for catalysis. J Bacteriol, 1987 May, 169(5), 2142 - 9 Gene fusion is a possible mechanism underlying the evolution of STA1; Yamashita I et al.; DNA from the STA1 (extracellular glucoamylase) gene of Saccharomyces diastaticus was used as a probe to enable the cloning by colony hybridization of three DNA fragments from Saccharomyces cerevisiae; these were designated S1, S2, and SGA (intracellular, sporulation-specific glucoamylase gene) . To examine the evolutionary relationship among these sequences at the nucleotide level, we sequenced S2, S1, SGA and compared them with STA1 . These data and RNA blot analysis revealed that the following regions of STA1 were highly conserved in S2, S1, and SGA: upstream regulatory sequences responsible for transcription, a signal sequence for protein secretion, a threonine- and serine-rich domain, and a catalytic domain for glucoamylase activity . These results suggest that an ancestral STA gene was generated relatively recently in an evolutionary time scale by the sequential fusions of S2, S1, and SGA, with S1 functioning as a connector for S2 and SGA . We describe a model for the involvement of short nucleotide sequences flanking the junctions in the gene fusions. J Biol Chem, 1987 Apr 15, 262(11), 5360 - 5 Bile acid: CoASH ligases from guinea pig and porcine liver microsomes . Purification and characterization; Vessey DA et al.; A procedure for the purification of the enzyme bile acid:CoA ligase from guinea pig liver microsomes was developed . Activity toward chenodeoxycholate, cholate, deoxycholate, and lithocholate co-purified suggesting that a single enzyme form catalyzes the activation of all four bile acids . Activity toward lithocholate could not be accurately assayed during the earlier stages of purification due to a protein which interfered with the assay . The purified ligase had a specific activity that was 333-fold enriched relative to the microsomal cell fraction . The purification procedure successfully removed several enzymes that could potentially interfere with assay procedures for ligase activity, i.e . ATPase, AMPase, inorganic pyrophosphatase, and bile acid-CoA thiolase . On sodium dodecyl sulfate-polyacrylamide gel electrophoresis the purified ligase gave a single band of approximately 63,000 Mr . A molecular size of 116,000 +/- 4,000 daltons was obtained by radiation inactivation analysis of the ligase in its native microsomal environment, suggesting that the functional unit of the ligase is a dimer . The purified enzyme was extensively delipidated by adsorption to alumina . The delipidated enzyme was extremely unstable but could be partially stabilized by the addition of phospholipid vesicles or detergent . However, such additions did not enhance enzymatic activity . Kinetic analysis revealed that chenodeoxycholate, cholate, deoxycholate, and lithocholate were all relatively good substrates for the purified enzyme . The trihydroxy bile acid cholate was the least efficient substrate due to its relatively low affinity for the enzyme . Bile acid:CoA ligase could also be solubilized from porcine liver microsomes and purified 180-fold by a modification of the above procedure . The final preparation contains three polypeptides as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . The three peptides range in size from 50,000 to 59,000, somewhat smaller than the guinea pig enzyme . The functional size of the porcine enzyme in its native microsomal environment was determined by the technique of radiation inactivation analysis to be 108,000 +/- 5,000 daltons . Thus, the functional form of the porcine enzyme also appears to be a dimer. Biochem J, 1987 Apr 15, 243(2), 507 - 17 Resolution and analysis of 'native' and 'activated' properdin; Farries TC et al.; A rapid and reproducible procedure for the resolution of 'native' and 'activated' forms of properdin (a component of the alternative activation pathway of complement), by gel filtration on the polyvinyl matrix Fractogel TSK HW-55(S), is reported . This fractionation permitted effective screening of samples for conditions that cause activation . Only 'native' properdin was detected in serum, even after activation of the alternative pathway by yeast cell walls . Transformation of 'native' into 'activated' properdin in vitro was produced by freeze-thawing of the protein, but not upon binding to and dissociation from the C3 convertase, C3bBb . Electron microscopy showed that only the 'native' population contained the discrete cyclic structures described previously by Smith, Pangburn, Vogel & Muller-Eberhard {(1984) J . Biol . Chem . 259, 4582-4588} . 'Activated' properdin, which was eluted from the gel-filtration column close to the breakthrough peak, was mainly composed of large amorphous aggregates . We therefore conclude that properdin 'activation' is not a physiological event that occurs in serum on complement activation, but is an artifact of isolation . Fractionation of properdin on Fractogel TSK HW-55(S) has, however, enabled detailed analysis of functional heterogeneity within the 'native' population. Biochim Biophys Acta, 1987 Apr 9, 898(2), 172 - 80 Binding of phospholipids to the phosphatidylinositol transfer protein from bovine brain as studied by steady-state and time-resolved fluorescence spectroscopy; Van Paridon PA et al.; The phosphatidylinositol transfer protein isolated from brain, liver, heart and platelets was found to be present in two subforms which could be distinguished on the basis of the isoelectric points . In this study we have demonstrated that the two subforms isolated from bovine brain are due to the presence of either phosphatidylinositol or phosphatidylcholine in the lipid binding site of the protein . The transfer protein accommodates one phosphatidylinositol molecule in the binding site . The binding site for the sn-2 fatty acyl chain was investigated by incorporating in the transfer protein either phosphatidylinositol or phosphatidylcholine carrying a parinaroyl-chain attached at the sn-2 position . Time-resolved fluorescence spectroscopy revealed that the sn-2 fatty acyl chains for both phospholipids in the lipid-protein complex were completely immobilized (i.e., rotational correlation times of 17.4 ns for phosphatidylcholine and 16.3 ns for phosphatidylinositol) . The similarity in correlation times suggests that the sn-2 fatty acyl chains of both phospholipids are accommodated in the same hydrophobic binding site of the protein. Mol Cell Biol, 1987 Apr, 7(4), 1425 - 35 Developmental regulation of SPO13, a gene required for separation of homologous chromosomes at meiosis I; Wang HT et al.; Previous studies have demonstrated that the SPO13 gene is required for chromosome separation during meiosis I in Saccharomyces cerevisiae . In the presence of the spo13-1 nonsense mutation, MATa/MAT alpha diploid cells complete a number of events typical of meiosis I including premeiotic DNA synthesis, genetic recombination, and spindle formation . Disjunction of homologous chromosomes, however, fails to occur . Instead, cells proceed through a single meiosis II-like division and form two diploid spores . In this paper, we report the cloning of this essential meiotic gene and an analysis of its transcription during vegetative growth and sporulation . Disruptions of SPO13 in haploid and diploid cells show that it is dispensible for mitotic cell division . Diploids homozygous for the disruptions behave similarly to spo13-1 mutants; they sporulate at wild-type levels and produce two-spored asci . The DNA region complementing spo13-1 encodes two overlapping transcripts, which have the same 3' end but different 5' ends . The major transcript is 400 bases shorter than the larger, less abundant one . The shorter RNA is sufficient to complement the spo13-1 mutation . While both transcripts are undetectable or just barely detectable in vegetative cultures, they each undergo a greater than 70-fold induction early during sporulation, reaching a maximum level about the time of the first meiotic division . In synchronously sporulating populations, the transcripts nearly disappear before the completion of ascus formation . Nonsporulating cells homozygous for the mating-type locus show a small increase in abundance (less than 5% of the increase in sporulating cells) of both transcripts in sporulation medium . These results indicate that expression of the SPO13 gene is developmentally regulated and starvation alone, independent of the genotype at MAT, can trigger initial induction. Can J Microbiol, 1987 Apr, 33(4), 290 - 9 Quantum thermodynamics approach to phosphorylation and heterotrophic growth yields; Tran VD; A model of cell growth is presented which is based on the double postulates of quantized loss of energy during phosphorylation and reversible biosynthesis of cell structure . An immediate consequence of the postulates is the identical value for the energy efficiency of the phosphorylation and for that of the whole growth process . Another consequence is the relationship between the energy level of the biomass and the phosphorylation potential as embodied in the equation: EO = gamma'M X EATP, where EO is the heat of transfer of a pair of electrons to oxygen, EATP, the molar heat of hydrolysis of ATP, and gamma'M, the degree of reduction of the biomass, gamma M being constant and equal to 5 . The model predicts five levels of growth yields corresponding to five permissible values for the P/O ratio (r = 0, 1, 2, 3, and 4) . Any growth process would be characterized by a set of two integers N and lambda; N is the maximal P/O ratio prescribed by the energy content of the substrate as compared with that of the biomass, and lambda the number of further downward quantum jumps of the P/O ratio resulting from the adversity of the growth condition (N - lambda = r) . Under full aerobiosis, one has 0 less than or equal to lambda less than or equal to N less than or equal to 3 . When growth is limited only by the energy content of the substrate (lambda = 0), the time-independent dispersion of N, owing to substrate-level phosphorylations and (or) dephosphorylations, leads to effective values which are higher than the nominal ones for the yield per mole of oxygen and the heat of transfer of a pair of electrons . Under adverse conditions (lambda greater than 0), the apparent variations of the yields and the P/O ratio in function of the growth rate are shown to be an effect of the random dispersion of lambda and of the existence of a maximal rate of substrate consumption . Statistical evidence for the macroscopic quantum effect in heterotrophic growth is presented. EMBO J, 1987 Apr, 6(4), 1073 - 7 Both ATP and an energized inner membrane are required to import a purified precursor protein into mitochondria; Eilers M et al.; We have investigated the energy requirement of mitochondrial protein import with a simplified system containing only isolated yeast mitochondria, energy sources and a purified precursor protein . This precursor was a fusion protein composed of 22 residues of the cytochrome oxidase subunit IV pre-sequence fused to mouse dihydrofolate reductase . Import of this protein required not only an energized inner membrane, but also ATP . ATP could be replaced by GTP, but not by CTP, TTP or non-hydrolyzable ATP analogs . Added ATP did not increase the membrane potential of respiring mitochondria; it supported import even if the proton-translocating mitochondrial ATPase and the entry of ATP into the matrix were blocked . We conclude that ATP exerts its effect on mitochondrial protein import outside the inner membrane. Arch Microbiol, 1987 Apr, 147(3), 235 - 9 Inhibition of protein phosphorylation by chloroquine; Kalisz H et al.; The rapid phase of fructose-1,6-bisphosphatase (FBPase) inactivation following glucose addition to starved yeast cells {reported previously} is inhibited on addition of 10 mM chloroquine (CQ) at about pH 8 . This inhibition of inactivation was shown to be due to the prevention of phosphorylation of the enzyme . CQ was also found to inhibit general protein phosphorylation in the yeast cells . Glycolysis, as observed by changes in intracellular glucose-6-phosphate and extracellular glucose and ethanol concentrations, was shown to be significantly inhibited in cells treated with CQ . Similarly, a decrease in ATP concentrations was observed . However, during the early stages of phosphorylation of FBPase, levels of ATP were similar in cells containing CQ as in those without CQ . Thus, decrease in ATP levels is not thought to be significantly responsible for the inhibition of protein phosphorylation . However, the phosphorylating activity of cyclic AMP-dependent protein kinases is inhibited in vitro by relatively low concentrations of CQ . Thus, prevention of protein phosphorylation by CQ is believed to be due to inhibition of protein kinases in yeast cells. Arch Biochem Biophys, 1987 Apr, 254(1), 214 - 21 Substrate specificity of choline kinase; Clary GL et al.; The substrate specificity of choline kinase (ATP:choline phosphotransferase, EC 2.7.1.32) from brewer's yeast has been examined using multiple analogs of choline, most of which have been reported to be a substrate of one or another choline-using system from other sources . In contrast to many such systems, choline kinase from brewer's yeast has been found to have relatively stringent and straight-forward structural requirements for its substrates . It is hypothesized that there are at least four points of interaction of the substrate with the enzyme--one for the hydroxyalkyl side chain and one for each of the three substituents on the quaternary nitrogen . Of the latter, one site seems relatively more sterically hindered than the other two . Short, single or double alkyl substitutions on the quaternary nitrogen are possible without a large loss of substrate capacity of the analog . Thus N,N-dimethyl-N-propylethanolamine had a relative Vmax of 116% and a relative Vmax 96% that of choline and a Km of 68 +/- 15 microM {nearly four times that of choline itself (18 microM)} . However, N-butyl-N,N-dimethylethanolamine and N,N,N-triethylethanolamine were very poor substrates . Analogs with substituents on the quaternary nitrogen of longer chain length were without activity as were aromatic derivatives . None of the bisquaternary compounds of the general structure HOCH2CH2N+(CH3)2-(CH2)n-N+(CH3)2CH2CH2OH (n = 2-10) showed any substrate capacity, as well . Restrictions on the hydroxyethyl side chain were also severe . One additional methylene group in this chain greatly reduced substrate capacity of the analog and two additional ones eliminated it entirely, as did almost any substituent on the beta carbon . A single (but not a double) substituent on the alpha carbon was moderately tolerated, however . Thus alpha-methylcholine and N-methyl-2-hydroxymethylpiperidine were substrates (although the latter one was a poor one) but beta-methylcholine and N-methyl-3-hydroxypiperidine were not . Such information may be of use toward designing cholinergic probes targeting specific enzyme or metabolic functions. Neurochem Res, 1987 Apr, 12(4), 335 - 9 Efficacy of RNase inhibitors during brain polysome isolation; Gauthier D et al.; We have investigated the efficiency of heparin, polyvinyl sulfate and yeast RNA (as competitive RNase inhibitors), liver extract (as crude preparation of liver RNase inhibitors) and DEPC (as irreversible non-competitive inhibitor) for the preparation of rat brain polysomes . Sucrose gradient sedimentation profiles, obtained from PMS, were used to determine the optimal concentration of each inhibitor . Diethylpyrocarbonate, whatever the composition of isolation buffer, was found detrimental for brain polysomes . Most of the other inhibitors where found useless or even harmful . A slight positive effect was observed with heparin 0.75 mg/mL both for total yield and sedimentation pattern . It is concluded that the utilisation of most of the widely used RNase inhibitors is of questionnable effectiveness for brain polysome preparation. J Biol Chem, 1987 Mar 25, 262(9), 4387 - 94 Structure, assembly, and secretion of octameric invertase; Esmon PC et al.; Yeast invertase forms a homo-octamer of core glycosylated subunits during assembly in the lumen of the endoplasmic reticulum . This form has been purified from mutant cells (sec18) in which transport of secreted proteins from the endoplasmic reticulum is blocked . No heterologous protein subunits are found in the purified material . Analysis of invertase derived from wild type cells or from mutant cells blocked at subsequent stages in secretion demonstrates that invertase remains a homo-octamer throughout the pathway even though the extent of subunit glycosylation increases . Purified octameric invertase is dissociated into dimer units that reassociate in the presence of polyethylene glycol . Negatively stained preparations show the dissociated enzyme as individual spheres, whereas octameric invertase appears as four associated spheres . Assembly of the octamer in vitro and in vivo is facilitated by the presence of N-linked carbohydrate . Selective release of dimeric glycosylated invertase from intact yeast cells suggests that oligomerization helps retain the enzyme in the periplasmic space. J Biol Chem, 1987 Mar 25, 262(9), 4355 - 9 Molecular cloning of a cDNA for a human ADP/ATP carrier which is growth-regulated; Battini R et al.; We have identified in a human cDNA library a clone (hp2F1) whose cognate RNA is growth-regulated . The insert has been sequenced and the nucleotide sequence shows a strong homology to the nucleotide sequences of the ADP/ATP carrier cDNA and gene, respectively, isolated from Neurospora crassa and Saccharomyces cerevisiae . The putative amino acid sequence of hp2F1 shows an 87% homology to the amino acid sequence of the ADP/ATP carrier from beef heart mitochondria . We conclude that the insert of hp2F1 contains the full coding sequence of a human ADP/ATP carrier . The steady-state RNA levels of the ADP/ATP carrier are growth-regulated . They increase when quiescent cells are stimulated by serum, platelet-derived growth factor, or epidermal growth factor, but not by platelet-poor plasma or insulin . RNA levels of the ADP/ATP carrier decrease instead when growing HL-60 cells are induced to differentiate by either phorbol esters or retinoic acid. Biochemistry, 1987 Mar 24, 26(6), 1503 - 11 Crystal structure of cytochrome c peroxidase compound I; Edwards SL et al.; We have compared the 2.5-A crystal structure of yeast cytochrome c peroxidase (CCP) with that of its semistable two-equivalent oxidized intermediate, compound I, by difference Fourier and least-squares refinement methods . Both structures were observed at -15 degrees C . The difference Fourier map reveals that formation of compound I causes only small positional adjustments of a few tenths of an angstrom . The map's most pronounced feature is a pair of positive and negative peaks bracketing the heme iron position . Least-squares refinement shows that the iron atom moves about 0.2 A toward the distal side of the heme . No significant difference density is evident near the side chains of Trp-51 or Met-172, each of which has been proposed to be the site of the electron paramagnetic resonance (EPR) active radical in compound I . However, the second most prominent feature of difference density is a negative peak near the side chain of Thr-180, which, according to the results of least-squares refinement, moves by 0.15 A in the direction of Met-230 . These observations, together with the results of mutagenesis experiments {Fishel, L . A., Villafranca, J . E., Mauro, J . M., & Kraut, J . (1987) Biochemistry 26, 351-360; Goodin, D . B., Mauk, A . G., & Smith, M . (1986) Proc . Natl . Acad . Sci . U.S.A . 83, 1295-1299} in which Trp-51 and Met-172 have been replaced without loss of the EPR radical signal in compound I, lead us to consider the possibility that the radical site lies within a cluster composed of the side chains of Met-230, Met-231, and Trp-191.(ABSTRACT TRUNCATED AT 250 WORDS) Cell, 1987 Mar 13, 48(5), 847 - 53 Deletion analysis of GAL4 defines two transcriptional activating segments; Ma J et al.; We describe the activities of a wide array of deletion mutants of GAL4, a yeast transcriptional activator . We identify two short regions of GAL4, each of which activates transcription when fused to the DNA-binding region of the molecule . Very large portions of GAL4 are not required for gene activation. Biochim Biophys Acta, 1987 Mar 13, 918(1), 36 - 9 Activity of long chain acyl-CoA synthetase in isolated alveolar type II cells; Haq RU et al.; Fatty-acyl-CoA synthetase activity was determined in rat alveolar type II cells . Compared to whole-lung homogenate, the enzyme specific activity with palmitic acid was 3.6-fold higher in isolated type II alveolar cells . The enzyme in rat alveolar type II cells did not discriminate among various fatty acids, suggesting that supply of fatty acids rather than specificity might be an important factor for their activation in these cells. Anal Biochem, 1987 Mar, 161(2), 508 - 13 Enzymatic assays for 2-deoxyglucose and 2-deoxyglucose 6-phosphate; Chi MM et al.; Methods for 2-deoxyglucose (2-DG) and 2-deoxyglucose 6-phosphate (DG6P) are described which are based on the fact that DG6P is oxidized by glucose-6-phosphate dehydrogenase (G6PDH), but at a rate 1000-fold slower than for glucose 6-phosphate, whereas hexokinase phosphorylates 2DG and glucose at comparable rates . Therefore, by adding the two enzymes in a suitable order, and in appropriate concentrations, 2DG, glucose, DG6P, and glucose 6-P can all be separately measured . To avoid a side reaction from the use of a high level of G6PDH, when measuring DG6P, glucose is first removed with glucose oxidase plus aldose reductase. Mol Cell Biol, 1987 Mar, 7(3), 1271 - 5 The genome of Trypanosoma cruzi contains a constitutively expressed, tandemly arranged multicopy gene homologous to a major heat shock protein; Dragon EA et al.; cDNA libraries have been constructed in the plasmid vector pUC18 with mRNA isolated from both epimastigotes and trypomastigotes of the Peru strain of Trypanosoma cruzi . Pools of randomly selected clones were analyzed by hybridization-selection-translation . Translation products were immunoprecipitated either with normal human sera or with sera from patients with Chagas' disease (chagasic sera), and the immunoprecipitates were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . With this approach, a cDNA clone (pEC5) was identified which encodes a portion of an 85,000-Mr polypeptide . A genomic clone was subsequently isolated (FG1) by using oligonucleotide probes derived from the DNA sequence of this cDNA clone . A portion of this clone was isolated and sequenced, and the coding region for the protein was identified . Computer analysis of the predicted protein sequence indicates that this protein is closely related to the 83,000-Mr heat shock protein (hsp83) of Drosophila melanogaster, the hsp90 of Saccharomyces cerevisiae, and the hsp90 of chicken . This gene is tandemly organized in the T . cruzi genome as a cluster of 6 to 10 copies. Mol Biol (Mosk), 1987 Mar-Apr, 21(2), 347 - 58 {Nucleotide sequence of a mutant allele and wild type allele SUP1 and comparison of transcripts of SUP1 and SUP2 genes}; Surguchev AP et al.; Nucleotide sequences of the yeast recessive suppressor gene SUP1 and one of its mutant alleles (supl-ts36) are compared . One open reading frame is found in the gene able to code 438 amino acid residues . The reading frame is not interrupted by introns . Mutant allele supl-ts36 contains one nucleotide change at position + 101 (T----C) inducing the exchange of leucine on serine in N-terminal part of the polypeptide product . The homology between the structure of SUP1 gene and two groups of proteins is found (1.tRNA-binding or nucleotide-binding proteins; 2 . mitochondrial proteins coded by mitochondrial genome) . The size of transcript for SUP1 gene is 1600 nucleotides corresponding to the coding region of the gene . For SUP2 gene two stable transcripts are found corresponding to approximately 2500 and approximately 1400 nucleotides . The homology between SUP1 and SUP2 genes is not found . The absence of splicing for both SUP1 and SUP2 genes transcripts is demonstrated in the experiments with RNA2 conditional mutants with impaired splicing. Yeast, 1987 Mar, 3(1), 33 - 42 PHO5 upstream sequences confer phosphate control on the constitutive PHO3 gene; Bajwa W et al.; To identify the sequences involved in the regulation of the yeast acid phosphatase gene (PHO5) we constructed a series of hybrid promoters . Increasing lengths of 5'-flanking sequences of the PHO5 gene were placed in front of the TATA-box of constitutively expressed acid phosphatase gene (PHO3) . The PHO5/PHO3 promoter constructions were used to replace the entire PHO5, PHO3 gene cluster on chromosome II . Depending on the length of PHO5 5'-flanking sequences present the PHO3 gene driven by the hybrid promoter could now be derepressed in response to inorganic phosphate (low Pi) exactly as the PHO5 wild type gene . A critical regulatory element was located between position -402 to -351 (upstream from ATG) and sequences further downstream (from -351 to -300) could increase transcriptional activation . The transcription levels of PHO3 were determined by northern blot analysis, under repressed (high Pi) and derepressed (low Pi) conditions which was paralleled by an increase in extra-cellular acid phosphatase activity . Fully regulated promoter hybrids showed a 40-fold induction of mRNA levels, comparable to wild type PHO5 promoter . Sl-nuclease protection experiments revealed that the PHO5 5'-flanking sequences, placed in front of PHO3, did not change the PHO3 transcription initiation site/s. Nature, 1987 Feb 19-25, 325(6106), 673 - 8 The role of small nuclear ribonucleoprotein particles in pre-mRNA splicing; Maniatis T et al.; A small set of distinctive short RNA molecules are found in the nuclei of all higher eukaryotic cells and yeast, in protein complexes known as 'small nuclear ribonucleoprotein particles', or snRNPs . Recent work has confirmed early suggestions that these particles form part of the machinery by which primary RNA transcripts are processed to their mature, functional form . In particular, snRNPs have been shown to be an integral part of the 'spliceosome', a multi-component complex involved in the removal of intron sequences from the coding regions of messenger RNA precursors. Biochim Biophys Acta, 1987 Feb 14, 917(2), 247 - 57 Biosynthesis of glycerolipids by hepatoma and liver microsomes . I . Fatty acyl-CoA ligase and acyl-CoA:sn-glycerol-3-phosphate acyltransferase; Harvey BE et al.; The intracellular membranes of hepatomas exhibit an altered content and composition of lipid compared to the membranes of normal liver . In order to elucidate the role of lipid biosynthetic enzymes in these membrane differences, we first examined the fatty acyl-CoA ligase and acyl-CoA:sn-glycerol 3-phosphate (Gro-3P) acyltransferase activities and acyl specificities of microsomes from liver, Morris hepatoma 7288C, and hepatoma tissue culture (HTC) cells . Based upon incorporation of fatty acid and Gro-3P, it is concluded that acyl-CoA:sn-Gro-3P acyltransferase activities are markedly elevated (6-30-fold) in the microsomes of Morris Hepatoma 7288C and HTC cells compared to microsomes from liver, whereas the fatty acyl-CoA ligase activity is reduced (30-50-fold) . Therefore, the low phospholipid content of these tumor cells does not appear to result from reduced acyltransferase activity . Though diminished ligase activity may play a role, it appears that activation of fatty acid may not be rate-limiting, even at the low levels of fatty acyl-CoA ligase present in the tumor and HTC cells . Preliminary evidence suggests that another factor that may be responsible for the low tumor phospholipid content is the limited availability of Gro-3P, a lipid precursor . The phospholipid in hepatoma 7288C is also characterized by an elevated ratio of monenoic to dienoic fatty acid . We have found that this change does not reflect an altered specificity of acyl-CoA:sn-Gro-3P acyltransferase. Biochim Biophys Acta, 1987 Feb 14, 917(2), 341 - 3 Early effect of myo-inositol deficiency on phosphatidylinositol metabolism in rat liver; Anderson LL et al.; Young rats (100 g) were fed either a myo-inositol-deficient or supplemented (control) diet for up to 14 days following a 12 h fast . At various times during this period animals were killed, livers were removed, and a microsomal fraction was prepared and assayed for CDPdiacylglycerol inositol transferase activity and for phosphatidylinositol-inositol exchange activity . Within 2 days after beginning the regimen, rats consuming the deficient diet had a 40% lower activity of the transferase than rats consuming the control diet . This difference was maintained throughout the feeding period and developed simultaneously with the accumulation of triacylglycerol in the deficient livers . In contrast, the specific activity of the exchange enzyme was unchanged by feeding the deficient diet. Science, 1987 Feb 13, 235(4790), 766 - 71 Splicing of messenger RNA precursors; Sharp PA; A general mechanism for the splicing of nuclear messenger RNA precursors in eukaryotic cells has been widely accepted . This mechanism, which generates lariat RNAs possessing a branch site, seems related to the RNA-catalyzed reactions of self-splicing introns . The splicing of nuclear messenger RNA precursors involves the formation of a multicomponent complex, the spliceosome . This splicing body contains at least three different small nuclear ribonucleoprotein particles (snRNPs), U2, U5, and U4 + U6 . A complex containing precursor RNA and the U2 snRNP particle is a likely intermediate in the formation of the spliceosome. J Chromatogr, 1987 Feb 13, 388(2), 295 - 305 Effects of organic solvents on the partitioning of enzymes in aqueous two-phase systems; Johansson G et al.; Organic solvents (ethylene glycol, glycerol, dimethyl sulphoxide, dimethylformamide, dioxane, methanol and propanol-2, as well as sucrose and urea) have been included in aqueous two-phase (liquid-liquid) systems comprised of water, dextran and poly(ethylene glycol) . The concentration of the organic solvent was in most cases 20% (w/w) . The influence of these solvents on the phase-forming properties, the volume ratio, the freezing point and the partitioning of a polymer-bound ligand, Procion Red HE-3B poly(ethylene glycol), has been studied . The partition coefficients for alkaline phosphatase decrease with ethylene glycol, glycerol, sucrose and urea (factors of 0.25-0.5), but increase with the other substances (factors of 1.2-1.6) . The temperature effects on the partitioning of alkaline phosphatase from calf intestine as well as of phosphofructokinase from yeast in systems containing ethylene glycol have been studied and compared with partitioning in standard systems, not containing solvents . The possible uses of the above systems for partitioning studies of enzymes are discussed. Science, 1987 Feb 6, 235(4789), 658 - 67 Programmed gene rearrangements altering gene expression; Borst P et al.; Programmed gene rearrangements are used in nature to to alter gene copy number (gene amplification and deletion), to create diversity by reassorting gene segments (as in the formation of mammalian immunoglobulin genes), or to control the expression of a set of genes that code for the same function (such as surface antigens) . Two major mechanisms for expression control are DNA inversion and DNA transposition . In DNA inversion a DNA segment flips around and is rejoined by site-specific recombination, disconnecting or connecting a gene to sequences required for its expression . In DNA transposition a gene moves into an expression site where it displaces its predecessor by gene conversion . Gene rearrangements altering gene expression have mainly been found in some unicellular organisms . They allow a fraction of the organisms to preadapt to sudden changes in environment, that is, to alter properties such as surface antigens in the absence of an inducing stimulus . The antigenic variation that helps the causative agents of African trypanosomiasis, gonorrhea, and relapsing fever to elude host defense is controlled in this way. Nature, 1987 Feb 5-11, 325(6104), 499 - 503 A cytosolic protein contains a cryptic mitochondrial targeting signal; Hurt EC et al.; Cytosolic dihydrofolate reductase from mouse contains a cryptic mitochondrial targeting sequence . If this sequence is attached to the amino terminus of 'passenger' proteins which by themselves cannot enter mitochondria, the resulting fusion proteins are transported into yeast mitochondria. Anal Biochem, 1987 Feb 1, 160(2), 316 - 22 A high-performance liquid chromatographic method for the enantiomeric analysis of the enzymatically synthesized coenzyme A ester of 2-tetradecylglycidic acid; Weaner LE et al.; The enantiomeric composition of an enzymatically synthesized sample of the coenzyme A ester of 2-tetradecylglycidic acid (TDGA-CoA) was determined by the use of high-performance liquid chromatography with a chiral stationary phase . The stationary phase was commercially available and consisted of (R)-N-(3,5-dinitrobenzoyl)phenylglycine covalently bonded to aminopropyl silica gel . Analysis was performed using the phenacyl derivative of 2-tetradecylglycidic acid (TDGA), obtained by mild hydrolysis of the TDGA-CoA followed by reaction of the extracted TDGA with phenacyl chloride . Chromatography showed the enantiomeric purity of TDGA-CoA, synthesized in a rat liver microsomal enzyme mixture over a 2-h period, to be a 15.6:1 ratio of the R:S enantiomers (88% ee) . The result demonstrates the stereoselectivity of the long-chain fatty acid-coenzyme A synthetase for chiral fatty acid epoxide, TDGA. Naturwissenschaften, 1987 Feb, 74(2), 78 - 85 {"Artificial" chromosomes}; Roth GE; The structural elements required for chromosome replication, segregation, and stability are replication origins, centromeres, and telomeres . DNA sequences capable of organizing these three elements have been isolated from yeast chromosomal DNA by means of recombinant DNA techniques and yeast cell transformation . It is now possible to combine these sequences into "artificial" chromosomes for yeast cells to obtain more insight into chromosome structure and function . Evidence is presented that the construction of artificial chromosomes functional in higher eukaryotes will be possible in the near future. Clin Chim Acta, 1987 Jan 30, 162(2), 129 - 39 Biosensor for lactate determination in biological fluids . I . Construction and properties of the biosensor; Racek J et al.; The preparation of a biosensor for lactate determination is described . The biosensor is based on an immobilized suspension of the aerobic yeast Hansenula anomala, containing flavocytochrome b2 in high activity . The conditions for yeast cultivation were optimized to gain a sufficiently high activity of this enzyme converting lactate in the cells . The properties of the biosensor are compared with those of a sensor based on immobilized enzyme flavocytochrome b2 . The yeast lactate biosensor has a sufficient sensitivity and linearity and short time of response . The precision and accuracy of lactate determination as well as the results of comparisons using an enzyme electrode and the spectrophotometric UV-test, enables this biosensor to be used in routine work . Analysis can be performed in blood plasma or whole blood . The stability of the biosensor makes it possible to work for 4 weeks with one yeast cell pellet. J Biol Chem, 1987 Jan 25, 262(3), 1030 - 6 Amino-terminal processing of proteins by N-myristoylation . Substrate specificity of N-myristoyl transferase; Towler DA et al.; Using synthetic octapeptides, we examined the amino-terminal sequence requirements for substrate recognition by myristoyl-CoA:protein N-myristoyl transferase (NMT) . NMT is absolutely specific for peptides with amino-terminal Gly residues . Peptides with Asn, Gln, Ser, Val, or Leu penultimate to the amino-terminal Gly were substrates, whereas peptides with Asp, D-Asn, Phe, or Tyr at this position were not myristoylated . Peptides with aromatic residues at this position competitively inhibited myristoylation of substrates, introducing the possibility of developing specific in vivo inhibitors of NMT . Peptides having sequences which correspond to those of known N-myristoyl proteins, including p60src, appear to be recognized by a single enzyme, and yeast and murine NMT have identical substrate specificities . The catalytic selectivity of NMT for myristoyl transfer accounts for the remarkable acyl chain specificity of this enzyme. J Mol Biol, 1987 Jan 20, 193(2), 413 - 7 The efficiency of folding of some proteins is increased by controlled rates of translation in vivo . A hypothesis; Purvis IJ et al.; We propose that the way in which some proteins fold is affected by the rates at which regions of their polypeptide chains are translated in vivo . Furthermore, we suggest that their gene sequences have evolved to control the rate of translational elongation such that the synthesis of defined portions of their polypeptide chains is separated temporally . We stress that many proteins are capable of folding efficiently into their native conformations without the help of differential translation rates . For these proteins the amino acid sequence does indeed contain all the information needed for the polypeptide chain to fold correctly (even in vitro, after denaturation) . However, other proteins clearly do not fold efficiently into their native conformation in vitro . We argue that the efficiency of folding of these problematic proteins in vivo may be improved by controlled synthesis of the nascent polypeptide. J Biol Chem, 1987 Jan 15, 262(2), 576 - 82 Use of directed mutagenesis to probe the role of tyrosine 198 in the catalytic mechanism of carboxypeptidase A; Gardell SJ et al.; Derivatization of Tyr198 in carboxypeptidase A (CPA) results in lowered catalytic activity toward peptide substrates (Cueni, L., and Riordan, J.F . (1978) Biochemistry 17, 1834-1842) . We have synthesized via directed mutagenesis a rat CPA variant {Phe198} CPA containing a Tyr198-to-Phe substitution in order to test whether the phenolic hydroxyl plays a critical role in catalysis . A double mutant {Phe193, Phe248}CPA in which both Tyr198 and Tyr248 have been replaced by phenylalanine has also been engineered . Enzymatic characterization of {Phe198}CPA indicates that the Tyr198 hydroxyl is not obligatory for the hydrolysis of peptide and ester substrates . Furthermore, parallel studies with {Phe198, Phe248}CPA show that simultaneous removal of both the Tyr198 and Tyr248 hydroxyls does not abolish catalytic activity . Analysis of the acetylated derivatives of {Phe198}CPA, {Phe248}CPA, and {Phe198, Phe248}CPA establishes that Tyr198 and Tyr248 are the active site tyrosines which are modified by N-acetylimidazole . In addition, the perturbations of enzymatic activity which accompany acetylation of native CPA can be largely assigned to derivatization of Tyr248 . The changes in the kinetic constants of substrate hydrolysis due to the Tyr198-to-Phe substitution are manifested as small decreases in the kcat values, but the Km values are essentially unaffected . This exclusive effect on the kcat values suggests that the Tyr198 hydroxyl participates in catalysis by stabilizing the rate-determining transition-state complex. Mol Cell Biol, 1987 Jan, 7(1), 470 - 7 Virus-encoded toxin of Ustilago maydis: two polypeptides are essential for activity; Peery T et al.; Cells of Ustilago maydis containing double-stranded RNA viruses secrete a virus-encoded toxin to which other cells of the same species and related species are sensitive . Mutants affected in the expression of the KP6 toxin were characterized, and all were viral mutants . A temperature-sensitive nonkiller mutant indicated that the toxin consists of two polypeptides, 12.5K and 10K, that are essential for the toxic activity . The temperature-sensitive nonkiller mutant was affected in the expression of the 10K polypeptide, and its toxic activity was restored by the addition of the 10K polypeptide to its secreted inactive toxin . These results led to the reexamination of other mutants that were known to complement in vitro . Each was found to secrete one of the two polypeptides . Here we show for the first time that P6 toxin consists of two polypeptides that do not interact in solution, but both are essential for the toxic effect . Studies on the interaction between the two polypeptides indicated that there are no covalent or hydrogen bonds between the polypeptides . Toxin activity is not affected by the presence of 0.3 M NaCl in the toxin preparations and in the medium, suggesting that no electrostatic forces are involved in this interaction . Also, the two polypeptides do not share common antigenic determinants . The activity of the two polypeptides appears to be dependent on a sequential interaction with the target cell, and it is the 10K polypeptide that initiates the toxic effect . The similarity of the U . maydis virus-encoded toxin to that of Saccharomyces cerevisiae is discussed. Radiobiologiia, 1987 Jan-Feb, 27(1), 46 - 51 {Quantitative description of a change in radiosensitivity}; Nekliudov AG et al.; Modification of cell radiosensitivity was expressed mathematically in terms of known experimental values of oxygen enhancement ratios for different yeast cell strains . It was shown that the parameters used permit to quantitate the dependence of the efficiency of radioprotectors and radiosensitizers upon yeast cell genotype. Mutat Res, 1987 Jan, 187(1), 31 - 5 Aneuploidy induced by nocodazole or ethyl acetate is suppressed by dimethyl sulfoxide; Mayer VW et al.; Nocodazole and ethyl acetate have previously been shown to be potent inducers of aneuploidy in Saccharomyces cerevisiae . The elevation in aneuploidy frequency induced by high doses of these compounds was reduced in a dose-response manner in the presence of increasing concentrations of dimethyl sulfoxide . These results imply that compounds dissolved in dimethyl sulfoxide which either are weak inducers of aneuploidy or are of unknown potency may register as false negatives in routine screening procedures. Curr Genet, 1987, 12(4), 257 - 61 Variable abundance of a mitochondrial DNA fragment in cultured tobacco cells; Grayburn WS et al.; The relative abundance of a cloned 4.5 kilobase (kb) pair mitochondrial DNA sequence in two suspension cultures of tobacco (Nicotiana tabacum cv Turkish samsun and Nicotiana tabacum NT-1) has been examined . This sequence is 70-fold reduced in NT-1 relative to Turkish samsun; the reduction is correlated with an increase in supercoiled mitochondrial DNA . This sequence does not hybridize with mitochondrial DNA from watermelon, maize, or Saccharomyces cerevisiae, nor with several cloned mitochondrial genes and is thus probably not a gene . It may represent most of the plant mitochondrial genome thought to be non-essential for mitochondrial function . The sequence complexity of supercoiled mitochondrial DNA from NT-1 cells is about one-third that found for the entire mitochondrial genome and does not include the cytochrome oxidase subunit II gene. Gene, 1987, 61(3), 385 - 99 Cloning, mapping and molecular analysis of the pyrG (orotidine-5'-phosphate decarboxylase) gene of Aspergillus nidulans; Oakley BR et al.; We have modified the transformation procedures of Ballance et al . {Biochem . Biophys . Res . Commun . 112 (1983) 284-289} to give increased rates of transformation in Aspergillus nidulans . With the modified procedures we have been able to complement pyrG89, a mutation in the orotidine-5'-phosphate decarboxylase gene of A . nidulans, by transformation with a library of wild-type (wt) sequences in pBR329 . We have recovered, by marker rescue from one such transformant, a plasmid (pJR15) that carries an A . nidulans sequence that complements pyrG89 efficiently . In three experiments, this plasmid gave an average of 1985 stable transformants/micrograms of transforming DNA . We have analyzed ten of these genetically and by Southern hybridization . In five transformants a single copy of the transforming plasmid had integrated at the pyrG locus, in one transformant several copies of pJR15 had integrated at this locus, in one transformant several copies of the plasmid had integrated into other sites, and in three transformants, the wt allele had apparently replaced the mutant allele with no integration of pBR329 sequences . Sequence and S1 nuclease protection analysis revealed that pJR15 contains a gene that predicts an amino acid sequence with regions of strong homology to the orotidine-5'-phosphate decarboxylases of Neurospora crassa and Saccharomyces cerevisiae . We conclude that this gene is the wt pyrG allele . Finally, we have compared the 5'- and 3'-noncoding sequences and intron splice sequences to other genes of A . nidulans and have mapped the pyrG locus to a region between the fpaB and galD loci on linkage group I. Biorheology, 1987, 24(5), 473 - 82 Importance of convection to the enhancement of erythrocyte sedimentation rates in inclined tubes; Hocking MB et al.; Erythrocytes were settled from whole blood in standard 200 x 2.5 mm erythrocyte settling rate tubes placed vertically and at various angles from 85 degrees to 15 degrees from the horizontal . In all cases sedimentation rates measured along the slope increased with decreasing angle from the horizontal . Vertical settled distances rapidly increased down to an angle of 70 degrees and then changed very little even down to angles as shallow as 30 degrees . Evidence is presented that convection plays a significant role in the inclined settling of erythrocytes, as has already been demonstrated with clay, or glass bead suspensions in water . Inclined settling enhancements obtained are quite similar to those observed under similar conditions with yeast cells in aqueous glucose. Ann N Y Acad Sci, 1987, 506, 208 - 28 Maximizing productivity in an immobilized cell reactor; Vega JL et al.; A vertical immobilized cell reactor employing Saccharomyces cerevisiae cross-linked to a gelatin support with glutaraldehyde has proven to be an effective system to achieve high cell concentrations and high dilution rates . The reactor is very stable over long periods of time, during which the cell concentration increases continuously without achieving steady state . Therefore, periodic regeneration by gas purging is required to remove excess biomass from the interstitial spaces . The glucose concentrations along the reactor follow an exponential profile when plotted as a function of the true residence time . Such profiles are a function of the initial glucose concentration fed to the system . The overall productivity of the reactor is a function of the flow rate and the inlet glucose concentration . For a 99% conversion, the maximum overall productivity is obtained at a substrate concentration of between 15% and 20% . Theoretical cell profiles were obtained and they indicate that mass transfer is promoted at high substrate concentrations and flow rates . The performance of a variety of packing materials having different shapes and materials of construction were compared in a vertical packed bed immobilized cell reactor . With other parameters being constant, the performance of the reactor is dominated by the quantity of cells present . The packing that has the highest surface area per volume of bed yields the most extensive monolayer and gives faster reactor start-up . Packing materials having high biomass loading rates are desirable at prolonged operating periods when growth beyond the monolayer occurs . Ultimately, the packing with the highest initial porosity would be capable of loading the highest cell volume, provided that sufficient interstitial spaces were provided for cell entrapment . When the immobilized cell reactor is operated in the horizontal position, CO2 holdup is decreased, as is evidenced by an increase in liquid holdup . However, the horizontal and vertical reactors showed almost identical substrate profiles at constant cell densities . In addition, under prolonged operation, the performance of the horizontal reactor decayed after several days, while the vertical reactor remained stable for over 40 days . The improved operation of this type of column in the vertical position is attributed to the necessary promotion of mass transfer with CO2 evolution and better liquid distribution . The addition of fatty acids to the media for an ICR results in limiting growth and increasing productivity . Overgrowth can be minimized, thus allowing longer periods of operation without regeneration.(ABSTRACT TRUNCATED AT 400 WORDS) Drugs Exp Clin Res, 1987, 13(9), 577 - 83 In vivo protective role of antioxidants against genotoxicity of metronidazole and azanidazole; Hrelia P et al.; The mutagenicity of metronidazole and azanidazole has been extensively reported . Previous experiments demonstrated, by means of the intrasanguineous host-mediated assay, that they significantly induced mutagenicity in liver, kidney and lung of mice . The treatment of mice with butylated hydroxyanisole (BHA) or butylated hydroxytoluene (BHT) by two different routes of administration (i.p . injection and oral intubation) significantly reduced liver- and kidney-mediated mutagenicity of azanidazole and metronidazole . No significant differences were observed between the routes of treatment in terms of protective effect on genotoxicity of azanidazole in the considered organs, whereas i.p . administration was the most suppressive on the mutagenicity of metronidazole . Even if BHT was the most effective agent in preventing mutation induction in mice, a detectable toxicity, in terms of increased mutagenicity, was evaluated in the liver . Evidence of lung abnormalities was also seen . The results suggest that the possible adverse effects on biological systems limit the prophylactic use of BHA and BHT in preventing the action of chemical carcinogens in man. Basic Appl Histochem, 1987, 31(3), 275 - 80 Topological properties of DNA domains and interaction with RNA polymerase II; Negri R et al.; We have analyzed the relationship between the alterations of the DNA structure induced by topological constraint and the template properties of promoters in vitro . A cause-effect relationship has been defined in several instances . Experimental protocols have been developed for the study of the topological properties of RNA polymerase II promoters . The goal of these studies is the definition of the intrinsic structural informations of DNA. Postgrad Med J, 1987, 63 Suppl 2, 159 - 60 Vaccination of newborns of HBsAg-positive carrier mothers with a recombinant DNA hepatitis B vaccine; Cadranel S et al.; Thirty neonates born to HBsAg-positive mothers were vaccinated with a 20 microgram dose of the SmithKline Biologicals recombinant DNA yeast-derived vaccine within 24 hours after birth, with similar inoculations repeated 1 and 2 months later . Preliminary results indicate that the recombinant vaccine was well tolerated and immunogenic . A 100% seroconversion rate was achieved 4 months after the first vaccine dose and the anti-HBs geometric mean titres progressively rose from 14 to 31,279 and 361 IU/l at months 1, 2, 4, and 6, respectively . Although a longer period of observation and comparison with a historical control group are needed to evaluate its protective efficacy in this high-risk population, the fact that no vaccinated newborns have been infected is encouraging. Postgrad Med J, 1987, 63 Suppl 2, 155 - 8 Immunogenicity of a recombinant hepatitis B vaccine in haemodialysis patients; Bruguera M et al.; The immunogenicity of a recombinant yeast-derived hepatitis B vaccine was evaluated in a randomized trial involving 80 haemodialysis patients in which three 40 microgram doses were administered according to either a 0, 1, 2 month, or a 0, 1, 6 month vaccination schedule . The vaccine induced an anti-HBs seroconversion in 54% of patients who received the three doses at intervals of one month (Group A) and in 55% of those who were vaccinated at months 0, 1, and 6 (Group B) . The geometric mean titres (GMT) seven months after the first injection were 37.7 IU/l in Group A and 91 IU/l in Group B . The seroconversion rate in men (53.6%) was slightly lower than in women (60%), and the respective GMTs were 33.3 and 78.5 IU/l . An age-dependent effect was noted in the anti-HBs response, but the type of renal disease and length of time on dialysis did not influence the antibody response . A 0, 1, and 2 month vaccination schedule seems preferable for haemodialysis patients as it induces more rapid protection. Postgrad Med J, 1987, 63 Suppl 2, 143 - 6 Active immunization against hepatitis B: immunogenicity of a recombinant DNA vaccine in females, heterosexual and homosexual males; Laukamm-Josten U et al.; Three groups of subjects (58 females, 54 heterosexual males, and 50 homosexual males) received three doses of a recombinant DNA yeast-derived hepatitis B vaccine according to a 0, 1, and 6 month vaccination schedule . Local and general side effects were mild . Seroconversion rates after three injections were not significantly different between the groups . Females showed a significantly higher anti-HBs response than both groups of males, and heterosexual males had higher antibody titres than homosexual males . Among the four homosexual non-responders, three were carriers of the human immunodeficiency virus. Postgrad Med J, 1987, 63 Suppl 2, 139 - 41 Immunogenicity of a recombinant DNA hepatitis B vaccine in neonates; Meheus A et al.; Infants of HBsAg-positive mothers (Group I) as well as those born to women without HBV markers (Group II) were vaccinated with a 10 micrograms dose of a recombinant DNA hepatitis B vaccine within 24 hours after birth according to a 0, 1, and 2 month schedule, with a booster dose planned 12 months later . Vaccination results in 14 (Group I) and 47 (Group II) neonates showed that at two months after the third dose of vaccine, 86% (6/7) and 100% (37/37), respectively, seroconverted, with anti-HBs geometric mean titres of 80 IU/l and 266 IU/l in the respective groups . No adverse reactions to the vaccine were observed . These preliminary results indicate that the recombinant DNA hepatitis B vaccine is safe and highly immunogenic in newborns. Postgrad Med J, 1987, 63 Suppl 2, 129 - 35 Persistence of vaccine-induced antibodies to hepatitis B surface antigen and the need for booster vaccination in adult subjects; Ambrosch F et al.; Protection against hepatitis B virus infection can be achieved by the induction of neutralizing antibodies against the hepatitis B surface antigen (HBsAg) . An antibody concentration of 10 IU/l, as measured by radioimmunoassay, is considered to be protective . HBsAg can be produced either from the plasma of chronic carriers or by DNA recombinant technology in yeast cells . Plasma- and yeast-derived vaccines have been compared in several studies and their immunological properties were found to be similar, including the persistence of antibodies induced by either type of vaccine . Finally, yeast-derived hepatitis B vaccine can boost anti-HBs responses initially elicited by either plasma- or yeast-derived vaccine equally and effectively . In order to maintain protective immunity after hepatitis B vaccination, antibody determination and, if necessary, booster vaccination should be performed, particularly in high-risk persons. Postgrad Med J, 1987, 63 Suppl 2, 125 - 8 Comparative multicentre study of the immunogenicity of different hepatitis B vaccines in healthy volunteers; Goudeau A et al.; In a comparative study of the immunogenicity of different hepatitis B vaccines, 339 healthy seronegative adults at three different centres were randomly allocated to receive either three doses of a yeast-derived vaccine at one of three different dose levels (10, 20, or 40 micrograms; SmithKline Biologicals) or one of two commercial plasma-derived vaccines at standard dose levels (5 micrograms, Institut Pasteur Production or 20 micrograms, Merck Sharp & Dohme) . The subjects were inoculated intramuscularly in the deltoid region according to a 0, 1, and 6 month schedule . No severe or serious adverse reactions attributed to any of the vaccines were observed . One month after the third vaccine dose, seroconversion rates ranged from 95% to 100% in all groups with only 6 subjects failing to seroconvert . Although there were no statistically significant intra-centre differences in antibody levels, the Tours/Chateauroux groups generally attained higher antibody levels than those from Limoges for the same yeast-derived vaccine dose . This unexplained difference between centres was not found for the plasma-derived vaccines . Older subjects responded less well than younger ones and females attained higher antibody levels than did males . The yeast-derived vaccine is comparable to the two commercially available plasma-derived vaccines in terms of reactogenicity and immunogenicity. Postgrad Med J, 1987, 63 Suppl 2, 121 - 3 Reactogenicity and immunogenicity of a recombinant hepatitis B vaccine compared with a plasma-derived vaccine in young adults; Just M et al.; A study of the immunogenicity and reactogenicity of three doses of lot L (10, 20, 40 micrograms), two doses of lot N (10, 20 micrograms) of the SmithKline Biologicals recombinant DNA yeast-derived hepatitis B vaccine and a 20 micrograms dose of the Merck, Sharp & Dohme plasma-derived hepatitis B vaccine was conducted in young adults under randomized, double-blind conditions . Immunization was carried out according to a 0, 1, and 6 month vaccination schedule . Results indicated that the yeast-derived hepatitis B vaccine was well tolerated and highly immunogenic . Reactogenicity to both yeast- and plasma-derived vaccines was mild in severity and low in incidence with no significant differences appearing between the study groups . One month after the third dose, the yeast-derived vaccines induced a high degree of seroconversion ranging between 97.8% and 100% . The response was not lot- or dose-dependent . The administration of the plasma-derived vaccine resulted in an anti-HBs geometric mean titre approximately twice as high as those elicited by the different yeast-derived hepatitis B vaccine doses one month after the third inoculation . However, 11 months following the third dose of vaccine, the anti-HBs titres were similar in all groups . Revaccination of subjects who no longer had detectable anti-HBs one year after the last vaccine dose resulted in an anamnestic response. Postgrad Med J, 1987, 63 Suppl 2, 105 - 7 Clinical development of a new recombinant DNA hepatitis B vaccine; Safary A et al.; The clinical development plan for the new recombinant DNA yeast-derived hepatitis B vaccine manufactured by SmithKline Biologicals is summarized . Initially, the emphasis was on assessing the risk of hypersensitivity to yeast-derived contaminants . This was followed by an evaluation of local and general reactions after vaccination . Next, the optimal dose of vaccine to be administered was ascertained followed by an evaluation of the efficacy of different vaccination schedules . The reactogenicity and immunogenicity of the yeast-derived vaccine was compared with that of two commercially available plasma-derived vaccines . The recombinant vaccine's protective efficacy was assessed in chimpanzees and by comparing attack rates in historical homosexual control groups with those in vaccinated homosexuals . Ongoing studies are investigating the protection of neonates born to HBeAg-positive carrier mothers. Gene, 1987, 51(2-3), 129 - 37 Sequence and genomic structure of ras homologues Dmras85D and Dmras64B of Drosophila melanogaster; Brock HW; The ras homologues of Drosophila melanogaster located at 85D and 64B on the polytene chromosome map were cloned using the Ha-ras gene of Harvey murine sarcoma virus as a probe . The genomic sequences of Dmras85D and Dmras64B were determined and shown to differ from previously published sequences . Dmras85D is much more similar to the Ha-ras, Ki-ras, and N-ras genes than it is to either the Dmras64B gene or to the ras genes of Saccharomyces cerevisiae . Comparison of the Dmras85D genomic sequences with the previously published nucleotide sequence (Neuman-Silberberg et al., Cell 37 (1984) 1027-1033) shows that the positions of the two introns are not conserved relative to the positions of the introns in Dmras64B or in vertebrate ras genes . The Dmras64B and Dmras85D transcripts were analyzed by blot hybridization and shown to be dissimilar . The data suggest that the divergence of the Dmras genes was ancient, and that Dmras85D and Dmras64B have different functions. J Mol Evol, 1987, 25(1), 58 - 64 Ubiquitin genes as a paradigm of concerted evolution of tandem repeats; Sharp PM et al.; Ubiquitin is remarkable for its ubiquitous distribution and its extreme protein sequence conservation . Ubiquitin genes comprise direct repeats of the ubiquitin coding unit with no spacers . The nucleotide sequences of several ubiquitin repeats from each of humans, chicken, Xenopus, Drosophila, barley, and yeast have recently been determined . By analysis of these data we show that ubiquitin is evolving more slowly than any other known protein, and that this (together with its gene organization) contributes to an ideal situation for the occurrence of concerted evolution of tandem repeats . By contrast, there is little evidence of between-cluster concerted evolution . We deduce that in ubiquitin genes, concerted evolution involves both unequal crossover and gene conversion, and that the average time since two repeated units within the polyubiquitin locus most recently shared a common ancestor is approximately 38 million years (Myr) in mammals, but perhaps only 11 Myr in Drosophila . The extreme conservatism of ubiquitin evolution also allows the inference that certain synonymous serine codons differing at the first two positions were probably mutated at single steps. Gene, 1987, 55(1), 135 - 9 Characterization of the Cephalosporium acremonium ribosomal RNA genes; Jarai G et al.; To investigate the organization of the ribosomal RNA genes in Cephalosporium acremonium, we cloned the whole r X DNA repeat in pBR322 and pNEO plasmids . Both the cloned and the genomic r X DNA fragments were characterized by restriction mapping . The r X DNA repeat unit was found to be 8.0 kb long and there was no significant heterogeneity among the individual repeats. Complement, 1987, 4(2), 61 - 74 Specificity of membrane complement receptor type three (CR3) for beta-glucans; Ross GD et al.; The binding of the iC3b receptor (CR3) to unopsonized zymosan was shown to result from CR3 attachment to cell wall beta-glucans . A specificity of neutrophil responses for beta-glucan was first suggested by a comparison of yeast (Saccharomyces cerevisiae) cell wall components for stimulation of a neutrophil superoxide burst . Neutrophils responded poorly to heat-killed yeast, but gave increasingly better responses to cell wall polysaccharides devoid of proteins (zymosan) and nearly pure beta-glucan particles derived from zymosan . Zymosan triggered a burst that was 29% as great as that stimulated by phorbol myristate acetate (PMA), and