EMBO J, 1991 Nov, 10(11), 3321 - 9 Regulatory phosphorylation of the p34cdc2 protein kinase in vertebrates; Norbury C et al.; The p34cdc2 protein kinase is a conserved regulator of the eukaryotic cell cycle . Here we show that residues Thr14 and Tyr15 of mouse p34cdc2 become phosphorylated as mouse fibroblasts proceed through the cell cycle . We have mutated these residues and measured protein kinase activity of the p34cdc2 variants in a Xenopus egg extract . Phosphorylation of residues 14 and 15, which lie within the presumptive ATP-binding region of p34cdc2, normally restrains the protein kinase until it is specifically dephosphorylated and activated at the G2/M transition . Regulation by dephosphorylation of Tyr15 is conserved from fission yeast to mammals, while an extra level of regulation of mammalian p34cdc2 involves Thr14 dephosphorylation . In the absence of phosphorylation on these two residues, the kinase still requires cyclin B protein for its activation . Inhibition of DNA synthesis inhibits activation of wild-type p34cdc2 in the Xenopus system, but a mutant which cannot be phosphorylated at residues 14 and 15 escapes this inhibition, suggesting that these phosphorylation events form part of the pathway linking completion of DNA replication to initiation of mitosis. Lakartidningen, 1991 Oct 30, 88(44), 3665 - 8 {Lysozyme--an enzyme of both historical and current interest as a therapeutical agent}; Burman LG et al.; Lysozyme, a bacteriolytic protein discovered by Fleming in 1922 and found to be phylogenetically ancient and almost ubiquitous among living organisms, is probably the most studied enzyme in biology and medicine . Evidence of its involvement in resistance to bacterial infection is compelling but remains indirect . Muramyl peptides (fragments of bacterial cell wall peptidoglycan) exert many effects on the immune system and the CNS, and appear to contribute to non-specific resistance to infection, fever, fatigue, and the pathogenesis of bacterial infection . Synthetic muramyl peptide analogues are currently used as adjuvants in vaccine trials in humans . Several pathological conditions are associated with changes in lysozyme concentrations, and egg-white lysozyme treatment has been tried on a small scale . With the cloning of the human lysozyme gene in yeast cells the enzyme can now be produced on a large scale, which will enable its therapeutic applications to be evaluated. J Immunol, 1991 Oct 15, 147(8), 2565 - 73 Leukocyte adhesion molecule-1 (LAM-1, L-selectin) interacts with an inducible endothelial cell ligand to support leukocyte adhesion; Spertini O et al.; The human lymphocyte homing receptor, LAM-1, mediates the adhesion of lymphocytes to specialized high endothelial venules (HEV) of peripheral lymph nodes . We now report that LAM-1 is also a major mediator of leukocyte attachment to activated human endothelium . In a novel adhesion assay, LAM-1 was shown to mediate approximately 50% of the adhesion of both lymphocytes and neutrophils to TNF-activated human umbilical vein endothelial cells at 4 degrees C . The contribution of LAM-1 to leukocyte adhesion was only detectable when the assays were carried out under rotating (nonstatic) conditions, suggesting that LAM-1 is involved in the initial attachment of leukocytes to endothelium . In this assay at 37 degrees C, essentially all lymphocyte attachment to endothelium was mediated by LAM-1, VLA-4/VCAM-1, and the CD11/CD18 complex, whereas neutrophil attachment was mediated by LAM-1, endothelial-leukocyte adhesion molecule-1, and CD11/CD18 . Thus, multiple receptors are necessary to promote optimal leukocyte adhesion to endothelium . LAM-1 also appeared to be involved in optimal neutrophil transendothelial migration using a videomicroscopic in vitro transmigration model system . LAM-1-dependent leukocyte adhesion required the induction and surface expression of a neuraminidase-sensitive molecule that was expressed for at least 24 h on activated endothelium . Expression of the LAM-1 ligand by endothelium was optimally induced by LPS and the proinflammatory cytokines TNF-alpha and IL-1 beta, whereas IFN-gamma and IL-4 induced lower levels of expression . The LAM-1 ligand on HEV and cytokine treated endothelium may be similar carbohydrate-containing molecules, because phosphomannan monoester core complex from yeast Hansenula hostii cell wall blocked binding of lymphocytes to both cell types, and identical epitopes on LAM-1-mediated lymphocyte attachment to HEV and activated endothelium . Thus, LAM-1 and its inducible endothelial ligand constitute a new pair of adhesion molecules that may regulate initial leukocyte/endothelial interactions at sites of inflammation. Nature, 1991 Oct 24, 353(6346), 769 - 72 Hypervariable C-terminal domain of rab proteins acts as a targeting signal; Chavrier P et al.; Mammalian cells express many ras-like low molecular mass GTP-binding proteins (rab proteins) that are highly homologous to the Ypt1 and Sec4 proteins involved in controlling secretion in yeast . Owing to their structural similarity and to their variety, rab proteins have been postulated to act as specific regulators of membrane traffic in exocytosis and endocytosis, and rab5 has been shown to be involved in early endosome fusion in vitro . In agreement with their postulated functions, all rab proteins studied so far have been found in distinct subcompartments along the exocytic or endocytic pathways . To define the region mediating their specific localization, we transiently expressed rab2, rab5 and rab7 hybrid proteins in BHK cells, and determined their intracellular localization by immunofluorescence confocal microscopy and subcellular fractionation . Here we present evidence that the highly variable C-terminal domain contains structural elements necessary for the association of rab proteins with their specific target membranes in the endocytic pathway. Nature, 1991 Oct 24, 353(6346), 726 - 30 Peptide-binding specificity of the molecular chaperone BiP; Flynn GC et al.; Members of the heat-shock protein family (hsp70s) can distinguish folded from unfolded proteins . This property is crucial to the role of hsp70s as molecular chaperones and is attributable to the amino-acid specificity of the peptide-binding site . The specificity for peptide ligands is investigated using a set of peptides of random sequence but defined chain length . The peptide-binding site selects for aliphatic residues and accommodates them in an environment energetically equivalent to the interior of a folded protein. FEBS Lett, 1991 Oct 21, 291(2), 192 - 4 Isolation of mitotic p34cdc2 apoenzyme from human cells; Meikrantz W et al.; A simple procedure was devised for isolating from homogenates of mitotic cells the human homolog to the fission yeast cdc2 gene product . The identity of the purified protein was established with anti-p34cdc2 antibodies and p13suc 1, both specific ligands for p34cdc2 . Active-site labeling with oxidized {alpha 32P}ATP showed the purified molecule to be an ATP-binding protein . Its ability to phosphorylate casein but not histone, and its phosphorylation on tyrosine, detected by anti-phosphotyrosine antibodies, indicates the form of p34cdc2 purified is the inactive or apoenzyme form . Purified quantities of human p34cdc2 should be of considerable value in establishing the mechanism of its activation at mitosis by phosphatases. Cell, 1991 Oct 18, 67(2), 283 - 91 Parallel activation of the NIMA and p34cdc2 cell cycle-regulated protein kinases is required to initiate mitosis in A . nidulans; Osmani AH et al.; We show that in Aspergillus nidulans, p34cdc2 tyrosine dephosphorylation accompanies activation of p34cdc2 as an H1 kinase at mitosis . However, the nimA5 mutation arrests cells in G2 with p34cdc2 tyrosine dephosphorylated and fully active as an H1 kinase . Activation of NIMA is therefore not required for p34cdc2 activation . Furthermore, mutation of nimT, which encodes a protein with 50% similarity to fission yeast cdc25, causes a G2 arrest and prevents tyrosine dephosphorylation of p34cdc2 but does not prevent full activation of the NIMA protein kinase . Mitotic activation of p34cdc2 by tyrosine dephosphorylation is therefore not required for activation of NIMA . These data suggest that activation of either the p34cdc2 protein kinase or the NIMA protein kinase alone is not sufficient to initiate mitosis . Parallel activation of both cell cycle-regulated protein kinases is required to trigger mitosis. Biochim Biophys Acta, 1991 Oct 16, 1095(1), 1 - 4 Binding of cathepsin D to the mannose receptor on rat peritoneal macrophages; Young PR et al.; Adherent cultures of rat peritoneal macrophages secrete lysozyme and the lysosomal marker enzymes beta-glucuronidase, beta-N-acetylglucosaminidase and acid phosphatase; the levels of secreted lysosomal cathepsin D, however, were found to be insignificant . Incubation of the cells at 4 degrees C for 15 min with yeast mannan or with 50 mM mannose, methyl alpha-glucopyranoside, or N-acetylglucosamine caused the concentration of cathepsin D in the culture medium to increase 30-40-fold; mannose-6-phosphate had no effect . 125I-labeled cathepsin D was prepared and the binding constant to the macrophage cell surface was determined to be KD = 27 nM . The data suggest that cathepsin D binds to the mannose receptor of macrophages and that binding to this receptor is not in equilibrium with the bulk medium. Proc Natl Acad Sci U S A, 1991 Oct 15, 88(20), 9051 - 5 Origin of human chromosome 2: an ancestral telomere-telomere fusion; IJdo JW et al.; We have identified two allelic genomic cosmids from human chromosome 2, c8.1 and c29B, each containing two inverted arrays of the vertebrate telomeric repeat in a head-to-head arrangement, 5'(TTAGGG)n-(CCCTAA)m3' . Sequences flanking this telomeric repeat are characteristic of present-day human pretelomeres . BAL-31 nuclease experiments with yeast artificial chromosome clones of human telomeres and fluorescence in situ hybridization reveal that sequences flanking these inverted repeats hybridize both to band 2q13 and to different, but overlapping, subsets of human chromosome ends . We conclude that the locus cloned in cosmids c8.1 and c29B is the relic of an ancient telomere-telomere fusion and marks the point at which two ancestral ape chromosomes fused to give rise to human chromosome 2. Proc Natl Acad Sci U S A, 1991 Oct 15, 88(20), 8977 - 81 Autoregulation of human thymidylate synthase messenger RNA translation by thymidylate synthase; Chu E et al.; Thymidylate synthase (TS; 5,10-methylenetetrahydrofolate:dUMP C-methyltransferase, EC 2.1.1.45) is essential for the de novo synthesis of thymidylate, a precursor of DNA . Previous studies have shown that the cellular level of this protein is regulated at both the transcriptional and posttranscriptional levels . The regulation of human TS mRNA translation was studied in vitro with a rabbit reticulocyte lysate system . The addition of purified human recombinant TS protein to in vitro translation reactions inhibited translation of TS mRNA . This inhibition was specific in that recombinant TS protein had no effect on the in vitro translation of mRNA for human chromogranin A, human folate receptor, preplacental lactogen, or total yeast RNA . The inclusion of dUMP, 5-fluoro-dUMP, or 5,10-methylene-tetrahydrofolate in in vitro translation reactions completely relieved the inhibition of TS mRNA translation by TS protein . Gel retardation assays confirmed a specific interaction between TS protein and its corresponding mRNA but not with unrelated mRNAs, including human placenta, human beta-actin, and yeast tRNA . These studies suggest that translation of TS mRNA is controlled by its own protein end product, TS, in an autoregulatory manner. Proc Natl Acad Sci U S A, 1991 Oct 15, 88(20), 8895 - 9 Inhibitors of DNA topoisomerase II prevent chromatid separation in mammalian cells but do not prevent exit from mitosis; Downes CS et al.; DNA topoisomerase II (EC 5.99.1.3) is necessary for chromosome condensation and disjunction in yeast but not for other functions . In mammalian cells, it has been reported to be necessary for progression toward mitosis but not for transit through mitosis . We have found, on the contrary, that specific inhibition of topoisomerase II (but not of topoisomerase I) interferes with mammalian mitotic progression . Metaphase is prolonged, and anaphase separation of chromatids is completely inhibited, in cells given high concentrations of topoisomerase II inhibitors; nevertheless these cells attempt cleavage, sometimes generating nucleate and anucleate daughters . Lower concentrations of inhibitors interfere with anaphase and produce abnormalities of segregation . DNA topoisomerase II activity is therefore necessary for mammalian chromatid separation, but it is not tightly coupled to the control of other mitotic events. Biochemistry, 1991 Oct 15, 30(41), 9873 - 81 Proton linkage of complex formation between cytochrome c and cytochrome b5: electrostatic consequences of protein-protein interactions; Mauk MR et al.; Two potentiometric methods have been used to study the pH-dependent changes in proton binding that accompany complex formation between cytochrome c and cytochrome b5 . With one method, the number of protons bound or released upon addition of one cytochrome to the other has been measured as a function of pH . The results from these studies are correlated with the complexation-induced difference titration curve calculated from the titration curves of the preformed complex and of the individual proteins . Both methods demonstrate that complex formation at acid pH is accompanied by proton release, that complex formation at basic pH is accompanied by proton uptake, and that the change in proton binding at neutral pH, where stability of complex formation is maximal, is relatively small . Under all conditions studied, the stoichiometry of cytochrome c-cytochrome b5 complex formation is 1:1 with no evidence of higher order complex formation . Although the dependence of complex formation on pH for interaction between different species of cytochrome c and cytochrome b5 are qualitatively similar, they are quantitatively different . In particular, complex formation between yeast iso-1-cytochrome c and lipase-solubilized bovine cytochrome b5 occurs with a stability constant that is 10-fold greater than observed for the other two pairs of proteins under all conditions studied . Interaction between these two proteins is also significantly less dependent on ionic strength than observed for complexes formed by horse heart cytochrome c with either form of cytochrome b5.(ABSTRACT TRUNCATED AT 250 WORDS) Nucleic Acids Res, 1991 Oct 11, 19(19), 5307 - 12 Binding of human glutaminyl-tRNA synthetase to a specific site of its mRNA; Schray B et al.; The human glutaminyl-tRNA synthetase is able to bind to its own mRNA . The enzyme contains two binding regions . One is located in the central section of the enzyme which includes its most hydrophilic portion with ten lysine residues in a block of 20 amino acids . This part of the enzyme binds unspecifically to all RNA sequences tested . A second binding region is located in that part of the enzyme which shows high degrees of sequence similarities with the bacterial and yeast glutaminyl-tRNA synthetases, and which is most likely responsible for the charging of tRNA with glutamine . This second RNA binding region specifically interacts with a site in the 3' noncoding region of the synthetase's mRNA . The binding site in the mRNA is characterized by an extended secondary structure that includes elements of the 'identity set' of nucleotides recognized by the enzyme when interacting with tRNA . We discuss possible physiological implications of the interaction between glutaminyl-tRNA synthetase and its mRNA. Nucleic Acids Res, 1991 Oct 11, 19(19), 5269 - 74 A rat brain mRNA encoding a transcriptional activator homologous to the DNA binding domain of retroviral integrases; Duilio A et al.; We have isolated a rat cDNA, named FE65, hybridizing to an mRNA of about 2,300 nucleotides present in rat brain, undetectable in rat liver and very poorly represented in other tissues . An mRNA of the same size is present in human neuroblastoma cells and is absent from other human cell lines . The FE65 cDNA contains an open reading frame (ORF) coding for a polypeptide of 499 amino acids in which 143 residues can be aligned with the DNA binding domain of the integrases encoded by mammalian immunodeficiency viruses . The remaining part of the FE65 ORF is not homologous with the correspondent regions of the integrases; the first 206 residues of the FE65 ORF show numerous negative charges and a short sequence not dispensable for the function of the transactivating acidic domain of the jun family transcriptional factors . A plasmid which expresses FE65 amino acids 1-232 fused to the yeast GAL4 DNA binding domain was co-transfected with a plasmid containing five GAL4 binding sites upstream of a minimal Adenovirus promoter controlling the expression of the CAT gene . This experiment showed that the fused protein GAL4-FE65 is able to obtain a 30-40 fold increase of the CAT gene expression compared to the expression observed in the presence of the GAL4 DNA binding domain alone . Two types of FE65 mRNA are present in rat brain, differing only for six nucleotides . We demonstrate that this is the consequence of a neuron-specific alternative splicing of a six-nucleotide miniexon, which is also present in the human genome, in an intron/exon context very similar to that of the rat FE65 gene. Biochim Biophys Acta, 1991 Oct 10, 1075(2), 181 - 6 Cooperative stacking and hydrogen bond pairing interactions of fragment peptide in cap binding protein with mRNA cap structure; Ueda H et al.; The stacking and hydrogen bonding abilities of Trp-(Gly)n-Glu (n = 0 approximately 3) for the interaction with 7-methylguanine (m7G) base were examined by fluorescence and 1H-NMR methods, and it was shown that they correlate with the distance between the Trp and Glu residues, and become most significant when both residues are separated from each other by two Gly residues (n = 2) . Based on this insight, the sequence conserved between the human and yeast cap binding proteins (CBPs) was surveyed, and the sequence of Trp-Glu-Asp-Glu (No . 102-105 in human CBP) was selected as a probable site for the binding with mRNA cap structure . Thus, the stacking and hydrogen bonding abilities of Trp-Glu-Asp-Glu with m7G cap structure were examined by comparative experiments using its analogous peptides . The results showed that the fourth Glu residue is important not only for the construction of hydrogen bond pairing with m7G base but also for strengthening the stacking interaction between the Trp indole ring and m7G base . Taking account of the recognition analysis using the mutant CBP proteins by site-directed mutagenesis (Ueda, H., Iyo, H., Doi, M., Inoue, M., Ishida, T., Morioka, H., Tanaka, T., Nishikawa, S . and Uesugi, S . (1991) FEBS Lett . 280, 207-210), this cooperative interaction could be important for the recognition of mRNA cap structure. Biochim Biophys Acta, 1991 Oct 8, 1090(2), 277 - 80 Isolation and characterisation of a bovine cDNA encoding eukaryotic initiation factor 2 alpha; Green SR et al.; Two cDNA clones have been isolated, from a bovine lymphosarcoma library, that encode the alpha-subunit of eukaryotic initiation factor 2 (eIF-2 alpha) . The predicted 315 amino acid sequence showed more than 99% amino acid identity with rat and human eIF-2 alpha . Galactose-regulated expression of a full length bovine eIF-2 alpha cDNA in yeast resulted in the synthesis of a polypeptide of the predicted molecular mass (36 kDa) . Furthermore, the expressed polypeptide cross-reacted with an antibody raised against rabbit eIF-2 alpha confirming the identity of the cDNA. Cell, 1991 Oct 4, 67(1), 197 - 211 cdc25 is a specific tyrosine phosphatase that directly activates p34cdc2; Gautier J et al.; cdc25 controls the activity of the cyclin-p34cdc2 complex by regulating the state of tyrosine phosphorylation of p34cdc2 . Drosophila cdc25 protein from two different expression systems activates inactive cyclin-p34cdc2 and induces M phase in Xenopus oocytes and egg extracts . We find that the cdc25 sequence shows weak but significant homology to a phylogenetically diverse group of protein tyrosine phosphatases . cdc25 itself is a very specific protein tyrosine phosphatase . Bacterially expressed cdc25 directly dephosphorylates bacterially expressed p34cdc2 on Tyr-15 in a minimal system devoid of eukaryotic cell components, but does not dephosphorylate other tyrosine-phosphorylated proteins at appreciable rates . In addition, mutations in the putative catalytic site abolish the in vivo activity of cdc25 and its phosphatase activity in vitro . Therefore, cdc25 is a specific protein phosphatase that dephosphorylates tyrosine and possibly threonine residues on p34cdc2 and regulates MPF activation. Cell, 1991 Oct 4, 67(1), 189 - 96 The cdc25 protein contains an intrinsic phosphatase activity; Dunphy WG et al.; Genetic and biochemical studies have indicated that the cdc25 protein controls the entry into mitosis by triggering tyrosine dephosphorylation of the cdc2 protein kinase . We show that the isolated cdc25 protein can catalyze dephosphorylation of several model phosphatase substrates, including p-nitrophenyl phosphate and two distinct tyrosine-phosphorylated peptides . The cdc25-dependent cleavage reaction closely resembles dephosphorylation by known tyrosine phosphatases: the reaction requires a reducing agent, shows high sensitivity to sodium vanadate, and proceeds efficiently in the presence of metal chelators . Moreover, the phosphatase activity of the cdc25 protein is eliminated by treatment with N-ethylmaleimide or by alteration of a single conserved cysteine residue by site-directed mutagenesis . These observations indicate that the cdc25 protein can function as a tyrosine phosphatase in the absence of any other protein. Nature, 1991 Oct 3, 353(6343), 437 - 40 A product of the prune locus of Drosophila is similar to mammalian GTPase-activating protein; Teng DH et al.; The X-linked prune (pn) eye-colour mutation of Drosophila melanogaster has a highly specific, complementary lethal interaction with the conditional dominant Killer of prune (awdK-pn) mutation . Although awdK-pn flies have no apparent phenotype on their own, pn awdK-pn double mutants die as second or third larval instars . The awd locus encodes a nucleoside diphosphate kinase, an enzyme that catalyses the transfer of high-energy phosphate bonds between nucleoside diphosphates and nucleoside triphosphates, which is essential for the normal development of Drosophila . Analysis of the pn locus has suggested that the complementary DNA, TcD37, encodes a putative pn+ product . Here we report the nucleotide sequence of TcD37 and the similarity of its deduced protein product to the catalytic domain of mammalian GTPase-activating proteins (GAPs); GAPs stimulate the GTPase activity of Ras (ref . 6), which are plasma membrane-bound proteins involved in the regulation of cell proliferation and differentiation . These results suggest that the Drosophila TcD37 protein participates in a biochemical pathway similar to that of Ras and GAPs in mammals and yeast . We propose that the interaction between pn and awd is due to a neomorphic mutation that enhances the ability of AwdK-pn nucleoside diphosphate kinase to induce a regulatory GTPase into a GTP-bound 'on' state, whereas Pn modulates the activity of this GTPase either by switching it to a GDP-bound 'off' state or by interfering with its effector function. Mutat Res, 1991 Oct, 261(2), 85 - 91 Dichloroacetonitrile, a by-product of water chlorination, induces aneuploidy in Drosophila; Osgood C et al.; Nitriles have been shown to be potent inducers of aneuploidy in yeast and Drosophila test systems . Haloacetonitriles are by-products of water chlorination that have been shown to be mutagenic and carcinogenic following topical application . In this report we show that dichloroacetonitrile, but not dibromoacetonitrile, is an effective inducer of aneuploidy in oocytes of Drosophila melanogaster . Following inhalation exposure of ZESTE adult females, dichloroacetonitrile (8.6 ppm) induced highly significant increments in the frequencies of sex chromosome loss and gain . Sodium cyanide was also found to be a highly effective inducer of germline aneuploidy, suggesting that cyanide toxicity may contribute to potency of nitriles as inducers of aneuploidy. Mol Cell Biol, 1991 Oct, 11(10), 4822 - 9 Dominant inhibitory mutations in the Mg(2+)-binding site of RasH prevent its activation by GTP; Farnsworth CL et al.; We have previously demonstrated that substitution of Asn for Ser at position 17 of RasH yields a dominant inhibitory protein whose expression in cells interferes with endogenous Ras function (L . A . Feig, and G . M . Cooper, Mol . Cell . Biol . 8:3235-3243, 1988) . Subsequent structural studies have shown that the hydroxyl group of Ser-17 contributes to the binding of Mg2+ associated with bound nucleotide . In this report, we show that more subtle amino acid substitutions at this site that would be expected to interfere with complexing Mg2+, such as Cys or Ala, also generated dominant inhibitory mutants . In contrast, a Thr substitution that conserves a reactive hydroxyl group maintained normal Ras function . These results argue that the defect responsible for the inhibitory activity is improper coordination of Mg2+ . Preferential affinity for GDP, observed in the original Asn-17 mutant, was found exclusively in inhibitory mutants . However, this binding specificity did not completely block the mutant proteins from binding GTP in vivo since introduction of the autophosphorylation site, Thr-59, in 17N Ras resulted in the phosphorylation of the double mutant in cells . Furthermore, inhibitory mutants failed to activate a model downstream target, yeast adenylate cyclase, even when bound to GTP . Thus, the consequence of improper complexing of Mg2+ was to lock the protein in a constitutively inactive state . A model is presented to explain how these properties could cause the mutant protein to inhibit the activation of endogenous Ras by competing for a guanine nucleotide-releasing factor. Exp Parasitol, 1991 Oct, 73(3), 354 - 61 Angiostrongylus costaricensis: culture of third-stage larvae to young adults in a defined medium; Hata H et al.; The third-stage larvae of Angiostrongylus costaricensis were successfully cultured to young adults in a chemically defined medium . The most suitable medium for the development was Waymouth's medium among eight defined media examined . Twenty-eight days after cultivation in this medium, 77% of the larvae developed to young adults, although these worms gradually died thereafter . When Waymouth's medium was supplemented with mouse red blood cells, these young adult worms continued their development . The mean body lengths of the worms cultivated in Waymouth's medium supplemented with RBCs were significantly larger than those of the worms in the medium without RBCs on Days 14 and 21 after cultivation . Addition of RBCs was essential for their further development . At 28 days after cultivation, the maximum body length of the worms was 2.1 mm for males and 3.3 mm for females . Additions of serum, yeast extract lactalbumin hydrolysate, and growth factors to Waymouth's medium did not provide any additional benefits for worm development. EMBO J, 1991 Oct, 10(10), 2965 - 73 Transcriptional activation by heterodimers of the achaete-scute and daughterless gene products of Drosophila; Cabrera CV et al.; The achaete-scute complex (AS-C) and the daughterless (da) genes encode helix-loop-helix proteins which have been shown to interact in vivo and to be required for neurogenesis . We show in vitro that heterodimers of three AS-C products with DA bind DNA strongly, whereas DA homodimers bind weakly and homo or heterocombinations of AS-C products not at all . Proteins unable to dimerize did not bind DNA . Target sequences for the heterodimers were found in the promoters of the hunchback and the achaete genes . Using sequences of the former we show that the DNA binding results obtained in vitro fully correlate with the ability of different combinations to activate the expression of a reporter gene in yeast . Embryos deficient for the lethal of scute gene fail to activate hunchback in some neural lineages in a pattern consistent with the lack of a member of a multigene family. Plant Mol Biol, 1991 Oct, 17(4), 773 - 85 Substances in nuclear wheat germ extracts which interfere with polymerase III transcriptional activity in vitro; Furter R et al.; Wheat germ nuclear extracts inhibited an active yeast polymerase III (pol III) transcription extract . We isolated two chromatin-associated fractions which harbored biochemically distinguishable inhibitory activities, each contributing about 40-50% to the total inhibitory activity . One fraction, which was released from the chromatin upon treatment with 350 to 900 mM NaCl, was purified to homogeneity and identified as histone H1 . It inhibited the yeast extract by excluding the transcription machinery from the template DNA . It can be partially antagonized by additional nontemplate DNA together with templates that have strong pol III promoters . The other fraction, which was released from the chromatin between 0 and 350 mM NaCl, inhibited transcription by affecting transcription complex formation partially through transcription factor-inhibitor interactions . Furthermore, it affected the rate of transcription reinitiation but not the elongation rate . Ways to move towards an active DNA-dependent pol III plant extract are discussed. Mol Gen Genet, 1991 Oct, 229(2), 273 - 7 The initiation site for recombination cog is at the 3' end of the his-3 gene in Neurospora crassa; Bowring FJ et al.; Recombination at his-3 in Neurospora crassa is thought to be initiated through a site designated cog which lies in the his-3 to ad-3 interval of linkage group I . Fragments of the his-3 gene were used to transform various his-3 mutant alleles to prototrophy in order to link the genetic map to the nucleotide sequence . It was established that cog is at the 3' end of his-3 and is therefore not the his-3 promoter . This suggests that cog may be dissimilar to a number of yeast recombinators which are associated with promoters of transcription. Vrach Delo, 1991 Oct, (10), 57 - 60 {The characteristics of the humoral interaction of lymphocytes with neutrophilic granulocytes of the peripheral blood in patients with chronic lympholeukemia under the influence of Proper-Myl}; Tishchenko LM et al.; A study of 32 patients with chronic lympholeucosis (CL) revealed a reduction of the number of phagocytosing neutrophil granulocytes and a reduction of production of humoral mediators by lymphocytes that stimulated the phagocytic function of granulocytes . It is shown that treatment by usually used cytostatic agents did not essentially effect the investigated values . Inclusion of proper-myl in the treatment course of patients with chronic lympholeucosis furthered increase of the number of phagocytosing neutrophil granulocytes and an increased production of humoral mediator stimulating the phagocytic activity of granulocytes. Protein Eng, 1991 Oct, 4(7), 821 - 9 Mutations in the FAD-binding fold of alcohol oxidase from Hansenula polymorpha; de Hoop M et al.; Alcohol oxidase of methylotrophic yeast is an FAD-containing enzyme . When in its active form, the enzyme is an octamer and located in the peroxisomes . To study the importance of FAD-binding on the activity, octamerization and intracellular localization of the enzyme, alcohol oxidase of Hansenula polymorpha was mutated in its presumed nucleotide-binding domain, which is formed by the N-terminal sequence . Whereas mutations of a glutamic acid residue (E42) reduced the stability of the octamer, it hardly affected enzyme activity and expression . However, replacements of three conserved glycines (G13, G15 and G18) and a conserved glutamic acid (E39) within the fold had severe effects . The mutations not only resulted in loss of enzyme activity but in reduced protein levels as well, probably due to decreased stability of the mutant alcohol oxidase . However, octamerization of the protein still occurred . The existence of inactive octameric proteins provides information about the formation pathway of this octameric flavoprotein. Biologicals, 1991 Oct, 19(4), 347 - 53 Immunogenicity of hepatitis B surface antigen derived from the baculovirus expression vector system: a mouse potency study; Attanasio R et al.; A standard mouse potency test was performed to evaluate the immunogenicity of recombinant hepatitis B surface antigen (HBsAg) produced in the baculovirus/insect cell expression system . Groups of NIH Swiss mice were immunized with serial four-fold amounts of either baculovirus-derived HBsAg adsorbed to aluminum sulfate or a commercially available yeast-derived recombinant HBsAg vaccine preparation . Results from these experiments showed that the effective dose of baculovirus- and yeast-derived HBsAg vaccine preparations necessary to seroconvert 50% of the animals were similar . The duration of the antibody response to HBsAg was studied in mice immunized with the highest doses of the two recombinant vaccine preparations 3 and 6 months after injection . No decrease in the anti-HBs response was observed 6 months after injection . No decrease in the anti-HBs response was observed 6 months after immunization with either of the two vaccine preparations . These results indicate that the baculovirus-derived recombinant HBsAg could serve as an alternative vaccine candidate for hepatitis B virus. J Pharm Sci, 1991 Oct, 80(10), 928 - 30 Kinetics of drug action in disease states . XXXVIII: Effect of body temperature on the convulsant activity of pentylenetetrazol in rats; Walker JS et al.; The purpose of this investigation was to determine the effect of body temperature on the pharmacodynamics (convulsant activity) of pentylenetetrazol (PTZ) . Rats received an iv infusion of PTZ until the onset of maximal seizures, at which time samples of cerebrospinal fluid (CSF), brain, and blood (for serum) were obtained for subsequent determination of PTZ concentrations by HPLC . The PTZ infusion caused a decrease in body temperature of approximately 4 degrees C within 20 min and onset of seizures in approximately 40 min . Compared with animals whose temperature was maintained in the normal range by heating pads, the hypothermic rats required significantly larger doses and higher serum, brain, and CSF concentrations of PTZ to produce seizures . Other rats received an injection of brewer's yeast to produce fever . Then, PTZ was infused 6, 12, or 24 h later when body temperature was elevated by an average of 1.3, 1.1, or 0.4 degrees C, respectively . Compared with control rats, whose temperature was maintained in the normal range by heating pads, moderate hyperthermia had no significant effect on the dose and concentrations of PTZ required to produce maximum seizures . Pentylenetetrazol exemplifies a drug that can produce hypothermia which, in turn, reduces the sensitivity of rats to its pharmacologic action . Unlike the central nervous system (CNS) depressants phenobarbital and ethanol, whose pharmacologic activity in rats is enhanced at elevated body temperature, the activity of the CNS stimulant PTZ is apparently not altered by fever. J Gen Microbiol, 1991 Oct, 137 ( Pt 10), 2469 - 75 A metabolic grid among versiconal hemiacetal acetate, versiconol acetate, versiconol and versiconal during aflatoxin biosynthesis; Yabe K et al.; Dichlorvos treatment of aflatoxigenic Aspergillus parasiticus SYS-4 (NRRL 2999) or a verscolorin A-accumulating mutant, NIAH-9, resulted in accumulation of versiconol acetate (VOAc) and versiconal hemiacetal acetate (VHA), whereas the production of aflatoxins, versicolorin A (VA), and versiconol (VOH) decreased . In feeding experiments using another non-aflatoxigenic mutant, NIAH-26, aflatoxins were newly produced from each of VHA, VOAc, VOH, versicolorin B (VB) and versicolorin C (VC) . In these experiments, aflatoxin production from VHA or VOAc was inhibited by dichlorvos, whereas that from each of VOH, VB and VC was insensitive to dichlorvos . In cell-free experiments using the cytosol fraction of NIAH-26, VHA was converted to VC (or VB) and a substance tentatively identified as versiconal (VHOH) . By further addition of NADH or NADPH to the same reaction mixture, VOAc and VOH were also formed together with VC (VB) and VHOH . VOH was produced from VOAc irrespective of nicotinamide adenine nucleotide . Also, the incubation of VOH in the presence of NAD or NADP led to the formation of VC (VB) . The production of VC (VB) and VHOH from VHA, and that of VOH from VOAc was inhibited by dichlorvos, whereas the production of VOAc from VHA, and that of VC (VB) from VOH, was insensitive to dichlorvos . These results indicate that a metabolic grid catalysed by dehydrogenase and esterase among VHA, VOAc, VOH and VHOH, and a reaction from VHOH to VC (VB) are involved in aflatoxin biosynthesis . These enzyme activities were also detected when yeast extract peptone medium was used, or when A . oryzae SYS-2 was examined. J Rheumatol, 1991 Oct, 18(10), 1606 - 10 Alkaline phosphatase dissolves calcium pyrophosphate dihydrate crystals; Xu Y et al.; We have shown that yeast pyrophosphatase dissolves calcium pyrophosphate dihydrate (CPPD) crystals in solutions . In this investigation we demonstrate that alkaline phosphatase (ALP) effectively dissolves CPPD crystals in vitro . CPPD dissolution by ALP had a pH optimum of 7.4, which is the optimum pH for its pyrophosphatase (PPiase) activity . The CPPD dissolution and PPiase activity by ALP are magnesium dependent, whereas its phosphoester hydrolytic activity is not . Calcium, which inhibited the enzymatic CPPD dissolution and PPiase activity of ALP had no effect on its phosphoester hydrolytic activity . These data indicate that PPiase activity of ALP is responsible for CPPD dissolution and not its phosphoester hydrolytic activity . Matrix molecules such as proteoglycans and chondroitin sulfate had no effect on the enzymatic and nonenzymatic dissolution of CPPD crystals . ALP acted more effectively on CPPD crystals than on soluble pyrophosphate relative to yeast PPiase . Our data suggest that chondrocyte ALP may play an important role in the dissolution of CPPD crystals in cartilage. Antimicrob Agents Chemother, 1991 Oct, 35(10), 2128 - 30 Treatment of murine invasive candidiasis with amphotericin B and cilofungin: evidence for enhanced activity with combination therapy; Sugar AM et al.; The in vivo interactions of cilofungin, an echinocandin antifungal agent, and amphotericin B, a polyene derivative, in a murine model of disseminated candidiasis have been investigated . While single therapy with either drug alone prolonged survival of infected mice, kidney colony counts were not appreciably reduced . In contrast, combination therapy, especially at higher doses of both drugs, resulted in significant prolongation of survival and suppression of growth of yeast cells in the kidneys . Combination therapy of experimental candidiasis with cilofungin and amphotericin B did not result in antagonism; rather, additive or synergistic effects were seen . Future preclinical work with other echinocandin and polyene derivatives should include studies evaluating the in vivo interactions of both classes of compounds. Immunol Lett, 1991 Oct, 30(2), 241 - 8 Genetic regulation of macrophage priming/activation: the Lsh gene story; Blackwell JM et al.; This paper describes functional and genetic studies on the macrophage resistance gene Lsh/Ity/Bcg first described almost two decades ago . Working in vitro with resident peritoneal, liver (Kupffer cells) and bone marrow derived macrophages from congenic B10 (LshS) and B10.L-LshR mice it has been possible to demonstrate that the final effector mechanism for the gene in regulating antileishmanial activity involves production of reactive nitrogen rather than reactive oxygen intermediates . This in turn is dependent upon priming/activation of macrophages for enhanced TNF-alpha release which acts back on the macrophage in an autocrine manner to increase nitric oxide production . The precise point at which Lsh acts to control macrophage priming/activation has not been identified, but studies of early response gene expression show differences in KC mRNA levels at 2 h after LPS stimulation, and in c-fos mRNA as early as 20 min after stimulation with PMA plus ionophore, in peritoneal macrophages from congenic LshS and LshR mice . Data available suggest that both negative and positive signals may be involved in macrophage priming/activation, with LshS macrophages down-regulating their capacity for continued response to the autocrine loop . Work in progress will examine the role of TPA and cAMP response element-binding proteins in regulating gene expression in Lsh congenic mice . A major new initiative has also commenced to clone the Lsh gene by reverse genetics using yeast artificial chromosomes to walk towards Lsh from the closet proximal and distal markers on mouse chromosome 1.(ABSTRACT TRUNCATED AT 250 WORDS) Zentralbl Bakteriol, 1991 Oct, 275(4), 436 - 50 Minimal requirements for growth of Brucella suis and other Brucella species; Plommet M; Minimal nutritional requirements and temperature limits of growth were studied in Brucella suis and, comparatively, in a few other Brucella species . In a saline basic medium including thiosulphate, ammonium sulphate and glucose with addition of 2 or 4 vitamins (nicotinic acid, thiamin and panthotenic acid, biotin), 24 out of 25 B . suis, 4/6 B . melitensis and 1/6 B . abortus strains were able to grow . Some strains, however, needed to be initially induced to grow by other ingredients, CO2, other vitamins, or amino acids, or by a prolonged incubation . In the saline basic medium without ammonium, glutamic acid and/or alanine and arginine, with or without glucose, supported the growth of all the B . suis and B . melitensis strains, except 2 which required a sulphur amino acid . Five out of 6 B . abortus strains did not grow in either medium without addition of one or several aromatic amino acids or, for one strain, aspartic acid, or valine . One strain could also be induced to grow in ammonium medium by other amino acids . In a rich medium with yeast extract, all Brucella species grew at 18 degrees C and 42.5 degrees (except one) while most B . suis (14/17) grew also at 15 degrees C and 44 degrees C, in contrast to other brucellae of which a few strains only grew at these temperatures . In saline ammonium glucose medium, yeast extract at 0.1 g/l provided all the required vitamins and amino acids for all brucellae and at 1 g/l, it even provided enough nitrogen to support growth without ammonium . Such basic saline medium with yeast extract may be advantageously used in routine Brucella culture, instead of the classic undefined peptone mediums . B . suis biovar 1 strains did not differ significantly in their minimal nutritional requirements, precluding the use of these requirements to differentiate the strains, in particular the Chinese vaccine strain S2 from the reference strain 1330 or from other strains from different parts of the world . Finally, B . suis which is endowed with a nearly complete synthetic potential may represent the parental Brucella species from which the melitensis and abortus species may have evolved. Alcohol Clin Exp Res, 1991 Oct, 15(5), 804 - 7 Multi-enzyme catalyzed rapid ethanol lowering in vitro; Whitmire DR et al.; Ethanol was oxidized to acetate by an enzyme system using yeast alcohol dehydrogenase (YADH), yeast aldehyde dehydrogenase (YALDH), and lactic dehydrogenase (LDH) recycling NAD in two model duodenal fluids and in canine duodenal aspirate in vitro . Sufficient enzyme activities were maintained to convert as much as 34% of the original ethanol to acetate with negligible acetaldehyde accumulation. Clin Microbiol Rev, 1991 Oct, 4(4), 411 - 21 Histoplasma variation and adaptive strategies for parasitism: new perspectives on histoplasmosis; Eissenberg LG et al.; This review summarizes the biology of Histoplasma capsulatum in relation to a wide variety of corresponding pathologies in histoplasmosis . Features of these disease syndromes can be explained in part by natural variations within the fungal population and adaptations made by individual organisms to specific environments . H . capsulatum grows as mycelia and conidia in the soil; once inhaled, the organism undergoes a dramatic morphological and physiological conversion to a yeast form . The yeasts proliferate within the phagolysosomes of macrophages, using a variety of specific strategies for intracellular survival . Even avirulent strains or variants are able to avoid being killed by macrophages and instead establish inapparent or persistent infections . The ingested avirulent organisms assume enlarged shapes similar in appearance to those seen in histological sections of tissues from patients with histoplasmosis . Respiratory tract epithelial cells also appear to play a role in persistence: within them yeasts undergo phenotypic switching akin to the phase variation observed in other pathogens . This particular change involves the loss or modification of cell wall alpha-(1,3)-glucan, which is also correlated with the spontaneous appearance of avirulent variants . The repertoire of adaptive responses and natural variations within this species probably evolved from the need to adjust to a wide range of dynamic environments . In combination with the immune status of the host, these characteristics of H . capsulatum appear to influence the epidemiology, extent, and persistence of histoplasmosis. Virus Res, 1991 Oct, 21(2), 141 - 54 Structural relationships between hepatitis B surface antigen in human plasma and dimers from recombinant vaccine: a monoclonal antibody study; Thanh LT et al.; Ten monoclonal antibodies were obtained from mice immunized with a yeast recombinant hepatitis B vaccine . They were selected at an early stage for their ability to bind to native surface antigen particles (HBsAg) in human plasma . All antibodies recognized conformational epitopes which were destroyed completely or almost completely by reduction of disulphide bridges . They were divided into five epitope groups by their competition for binding to recombinant S protein, though epitopes within each group are not identical . Recombinant S protein migrated on SDS-PAGE in the absence of reducing agents as a mixture of monomers and dimers/oligomers . Sucrose gradient analysis suggests that all these forms are co-aggregated into HBsAg-like particles . On Western blots, all ten antibodies either bound only to dimers/oligomers or strongly preferred them over monomers . The results suggest that, of the antibodies produced in response to recombinant vaccine in mice, most of those which bind strongly to 'native' HBsAg particles in human plasma recognize surface structures created by interaction between two subunits. Clin Exp Immunol, 1991 Oct, 86(1), 66 - 70 Analysis of antibodies to RNA in patients with systemic lupus erythematosus and other autoimmune rheumatic diseases; Blanco F et al.; The frequency and clinical associations of anti-RNA antibodies measured by ELISA were assessed in 138 patients with systemic lupus erythematosus (SLE) . Of the sera from these patients 9.4% had anti-RNA antibodies but no distinguishing features, clinical, serological or immunogenetic, between those with or without these antibodies could be identified . However, investigations of patients with other autoimmune rheumatic diseases did not reveal any anti-RNA positivity, which indicates a marked disease specificity for anti-RNA antibodies in SLE . The initial anti-RNA antibody screen used a soluble yeast extract as test antigen . The positive sera were further tested against a range of RNAs from 10 different types of rat tissue . In essence few differences were observed, suggesting that the anti-RNA response is directed against common, highly conserved epitopes. Lab Anim Sci, 1991 Oct, 41(5), 407 - 10 An epizootic of histoplasmosis duboisii (African histoplasmosis) in an American baboon colony; Butler TM et al.; Histoplasmosis duboisii, a chronic granulomatous disease caused by Histoplasma capsulatum var . duboisii, was diagnosed in 21 baboons at a large primate colony in San Antonio, Texas . Diagnosis was based on finding 8 to 15 microns-diameter yeast cells in histologic sections . Therapy with drugs was unsuccessful . Surgical removal of lesions was the primary treatment . Epidemiologic data suggest the incubation period to be at least 9 months . The most likely route of infection was oral and happened during grooming by the baboons. J Mol Evol, 1991 Oct, 33(4), 367 - 78 The evolution of rhodopsins and neurotransmitter receptors; Fryxell KJ et al.; Rhodopsins share a limited number of amino acid identities with a variety of other integral membrane proteins . Most of these proteins have seven putative transmembrane segments and are likely to play a role in transmembrane signaling . We have undertaken a systematic series of comparisons of primary and secondary structure in order to clarify the functional and evolutionary significance of these sequence similarities . On the basis of consistently high similarity scores, we find that the most internally consistent definition of the rhodopsin gene family would include vertebrate rhodopsins, alpha- and beta-adrenergic receptors, M1 and M2 muscarinic acetylcholine receptors, substance K receptors, and insect rhodopsins, while excluding bacteriorhodopsin, the mass human oncogene, vertebrate and insect nicotinic acetylcholine receptors, and the yeast STE2 and STE3 peptide receptors . The rhodopsin gene family is highly diverged at the primary sequence level but has maintained a conserved secondary structure, including a previously unidentified hierarchy of transmembrane segment hydrophobicity . We have developed new computer algorithms for progressive multiple sequence alignment and the analysis of local conservation of protein domains, and we have used these algorithms to examine the phylogeny of the rhodopsin gene family and the changing domains of sequence conservation . The results show striking differences and similarities in the conserved domains in each of the three main branches of the rhodopsin gene family, and indicate that color vision arose independently in the lines of descent leading to modern humans and fruit flies. J Virol Methods, 1991 Oct, 34(3), 333 - 41 Detection of immobilised Murray Valley encephalitis virus RNA using oligonucleotide probes with varying degrees of mismatch; Howard MJ et al.; The design of oligonucleotides used for hybridisation studies often utilises available sequence information of the type strain of a particular virus . If hybridisation studies, using such oligonucleotides, are carried out with field isolates of the same virus, the problem of base pair mismatches and consequent difficulties in detection may arise . This study examined the effect of base pair mismatches on the hybridisation between membrane-bound Murray Valley encephalitis virus (MVE) RNA derived from various strains and deliberately mismatched oligonucleotide probes . Under conditions of very low stringency, probes containing up to 5 mismatches were able to detect MVE RNA, but not yeast RNA . Under washing conditions of increased stringency, hybridisation could be detected between MVE virus RNA and probes with only 3 to 4 mismatches . However, the extent of this interaction was dependent on the number and type of mismatches and their relative sequence position. Exp Mol Pathol, 1991 Oct, 55(2), 119 - 34 The effect of mild hyperthermia on the morphology and function of murine resident peritoneal macrophages; van Bruggen I et al.; During short term culture of murine resident peritoneal macrophages, increasing the temperature from 37 to 39 degrees C resulted in an increased activity of several surface receptors (FcR and receptor for gluteraldehyde-fixed sheep red blood cells), enhanced phagocytosis of yeast particles, improved spreading, and an accelerated reduction of nitroblue tetrazolium . At 41 degrees C, however, significant reduction of several functional properties (endocytosis of colloidal gold and horseradish peroxidase, phagocytosis of yeast particles) and a decrease in the reduction of nitroblue tetrazolium, the incorporation of tritiated uridine, and Fc and C3b surface receptor activity were observed . In addition morphological evidence of apoptosis, observed in a small number of cells cultured at 39 degrees C and in the majority of macrophages maintained at 41 degrees C, was confirmed by DNA electrophoresis . The data indicates that a reduction of several functional activities of macrophages occurs at 41 degrees C and apoptosis may largely account for these effects. Curr Opin Biotechnol, 1991 Oct, 2(5), 735 - 41 Expression cloning systems; Aruffo A; This review will cover the use of expression cloning in Xenopus oocytes, fission yeast, and mammalian cells . Of the systems covered herein, transient expression cloning systems in Xenopus oocytes and mammalian cells have proven to be the most effective and versatile, as demonstrated by the large number of cDNA clones isolated by these two methods in the past year . Of particular interest, are recent advances in the screening methodologies used in conjunction with transient expression in mammalian cells which have permitted the application of this system in the isolation of cDNAs encoding intracellular proteins. Biochem Biophys Res Commun, 1991 Sep 30, 179(3), 1181 - 6 Sequence requirements for proteolytic cleavage of precursors with paired basic amino acids; Oda K et al.; When expressed in COS cells, human prorenin was secreted into the medium without being processed to an active renin . Co-expression of furin, a mammalian homologue of the yeast KEX2 gene product, did not affect proteolytic processing of prorenin . A mutant proreninR-4 constructed by site-directed mutagenesis of Pro (-4) to Arg was not cleaved by an endoprotease in the COS cell . However, proreninR-4 was detectably cleaved to yield the active renin upon co-transfection with furin DNA, indicating that Arg at position -4 is important for recognition and processing by furin in addition to the absolute requirement for paired basic amino acids . Another mutant precursor in which Leu (+1) of proreninR-4 was replaced with Ser was found to be much more efficiently processed than proreninR-4, regardless of co-expression of furin . The results suggest that not only a basic amino acid at position -4 but also Leu at position +1 significantly affect the processing of prorenin catalyzed by the COS cell endoprotease or furin. Cell, 1991 Sep 6, 66(5), 981 - 93 Activation of class II gene transcription by regulatory factors is potentiated by a novel activity; Meisterernst M et al.; A novel activity (USA) stimulated activator-dependent transcription in a reconstituted system in conjunction with natural TFIID, resulting in 10- to 50-fold levels of induction by regulatory factors . USA mediated a modest induction by USF in conjunction with either recombinant human TFIID, intact yeast TFIID, or the evolutionarily conserved C-terminal portion of yeast TFIID . Upon further purification, USA was resolved into two components that had opposite effects on core promoter activity and that in combination potentiated activator function . Gel mobility shift experiments indicated physical interactions between the inhibitory activity and TFIID, suggesting that the additional components (cofactors) associate with the preinitiation complex both to reduce promoter activity in the absence and to increase promoter activity in the presence of transcriptional activators. J Biol Chem, 1991 Sep 5, 266(25), 16954 - 9 Functional expression of furin demonstrating its intracellular localization and endoprotease activity for processing of proalbumin and complement pro-C3; Misumi Y et al.; We have cloned a rat cDNA encoding furin which is structurally related to yeast Kex2 protease . Products of 88 and 94 kDa were obtained by in vitro transcription/translation of the cDNA in the absence and presence of microsomes . When the cDNA was transfected into COS-1 cells, furin was expressed as a major glycosylated form of 94 kDa, accompanied by a minor proteolytic form of 86 kDa, and found to be localized in the Golgi complex . Proalbumin and complement pro-C3 are intracellularly processed into their mature forms by cleavage at the dibasic residues Arg-Arg, a common cleavage signal found in many pro-type precursors . In cells transfected with the cDNA of C3 or albumin alone, only about half of each proform expressed was processed by an endogenous activity of the cells . When furin was coexpressed, the proforms of both C3 and albumin were completely processed into their mature forms . In addition, co-expression of rat alpha 1-protease inhibitor mutant (Met352----Arg) resulted in inhibition of the endogenous and exogenous processing activities, as observed for the naturally occurring mutant Pittsburgh which has been identified as a specific inhibitor for the processing enzyme . Taken together, these results indicate that furin is an endoprotease localized to the Golgi complex and capable of processing proalbumin and pro-C3 into the mature forms. Biochemistry, 1991 Sep 3, 30(35), 8661 - 5 Subsite interactions of ribonuclease T1: viscosity effects indicate that the rate-limiting step of GpN transesterification depends on the nature of N; Steyaert J et al.; We report on the effect of the viscogenic agents glycerol and ficoll on the RNase T1 catalyzed turnover of GpA, GpC, GpU, and Torula yeast RNA . For wild-type enzyme, we find that the kcat/Km values for the transesterification of GpC and GpA as well as for the cleavage of RNA are inversely proportional to the relative viscosity of glycerol-containing buffers; no such effect is observed for the conversion of GpU to cGMP and U . The second-order rate constants for His40Ala and Glu46Ala RNase T1, two mutants with a drastically reduced kcat/km ratio, are independent of the microviscosity, indicating that glycerol does not affect the intrinsic kinetic parameters . Consistent with the notion that molecular diffusion rates are unaffected by polymeric viscogens, addition of ficoll has no effect on the kcat/Km for GpC transesterification by wild-type enzyme . The data indicate that the second-order rate constants for GpC, GpA, and Torula yeast RNA are at least partly limited by the diffusion-controlled association rate of substrate and active site; RNase T1 obeys Briggs-Haldane kinetics for these substrates (Km greater than Ks) . Calculations suggest that the equilibrium dissociation constants (Ks) for the various GpN-wild-type enzyme complexes are virtually independent of N whereas the measured kcat values follow the order GpC greater than GpA greater than GpU . This is also revealed by the steady-state kinetic parameters of Tyr38Phe and His40Ala RNase T1, two mutants that follow simple Michaelis-Menten kinetics because of a dramatically reduced kcat value (i.e., Km = Ks).(ABSTRACT TRUNCATED AT 250 WORDS) Ophthalmology, 1991 Sep, 98(9), 1356 - 9 Ocular histoplasmosis with retinitis in a patient with acquired immune deficiency syndrome; Specht CS et al.; Disseminated histoplasmosis is one of the life-threatening opportunistic infections associated with acquired immune deficiency syndrome (AIDS) . A 29-year-old man with AIDS and disseminated histoplasmosis complained of a hazy spot in the vision of his left eye . Results of examination showed distinct creamy white intraretinal and subretinal infiltrates in both eyes . The patient died within a month from pulmonary infection with Histoplasma capsulatum and cytomegalovirus . Examination with light microscopy showed that the right and left eyes contained histoplasma yeast cells in lesions of retinitis, optic neuritis, and uveitis . These lesions contained variable numbers of lymphocytes and histiocytes . Electron microscopy of the histoplasma in tissue showed characteristic features . This case illustrates the funduscopic appearance and histopathology of histoplasmic retinitis, an uncommon although important ophthalmologic complication of AIDS. Acta Cytol, 1991 Sep-Oct, 35(5), 557 - 9 Fine needle aspiration diagnosis of Penicillium marneffei infection; Ma KF et al.; A disseminated infection with Penicillium marneffei, a rare human pathogen that may infect both healthy and immunocompromised patients, was diagnosed by fine needle aspiration cytology in a patient infected with the human immunodeficiency virus . The presence of yeast-form organisms with an eccentric or central dot and occasional septate and elongated forms highly suggested the diagnosis, which was confirmed on culture . Establishment of the diagnosis is important because this infection is potentially curable. Oncogene, 1991 Sep, 6(9), 1555 - 9 Differential expression of two types of the neurofibromatosis type 1 (NF1) gene transcripts related to neuronal differentiation; Nishi T et al.; A 360 residue region encoded by the neurofibromatosis type 1 (NF1) gene shows significant homology to the catalytic domains of both mammalian GTPase-activating proteins (GAP) and yeast IRA proteins . This GAP-related domain of the NF1 gene (NF1-GRD), like the GAP and IRA protein, has been reported to mediate hydrolysis of Ras-bound GTP to GDP, resulting in inactivation of Ras protein . In the present study, we identified two different types of NF1-GRD cDNA . One (type I) is identical to the previously reported sequence, and the other (type II) contained an additional 63 bp insertion that encodes for a region of 21 amino acids in the center of the NF1-GRD molecule . Alternative splicing is the most likely mechanism by which these two types of transcripts arise . Our observations reveal that the type I transcript is predominantly expressed in undifferentiated cells, whereas the type II transcript predominates in differentiated cells . Furthermore, the expression pattern of type I and type II NF1-GRD mRNA immediately changed in SH-SY5Y neuroblastoma cells when neuronal differentiation programs were induced by retinoic acid treatment . We propose that the differential expression of type I and type II NF1-GRD transcripts might be an 'on/off' switch that regulates the catalytic activity of the NF1 gene product, which plays an important role in the regulation of neuronal differentiation. Mol Gen Genet, 1991 Sep, 228(3), 424 - 32 Identification of the genes coding for the second-largest subunits of RNA polymerases I and III of Drosophila melanogaster; Seifarth W et al.; We have isolated cDNA and genomic clones of Drosophila melanogaster by cross-hybridization with a 658 bp fragment of the yeast gene coding for the second-largest subunit of RNA polymerase III (RET1) . Determination of the sequence by comparison of genomic and cDNA regions reveals an ORF of 3405 nucleotides which is interrupted in the genomic sequence by an intron of 48 bp . The deduced polypeptide consists of 1135 amino acids with a calculated molecular weight of 128 kDa . The protein sequence shows the same conserved regions of homology as those observed for all the second-largest subunits of RNA polymerases cloned so far . The gene (DmRP128) obviously codes for a second-largest subunit of an RNA polymerase which is different from DmRP140 and DmRP135 . We have purified three distinct RNA polymerase activities from D . melanogaster . By using specific RNA polymerase inhibitors in enzyme assays and by comparing their subunit composition we were able to distinguish between RNA polymerase I, II, and III . RNA polymerase preparations of D . melanogaster were blotted and the second-largest subunits were identified with antibodies raised against polypeptides expressed from DmRP128 and DmRP135 . Anti-DmRP135 antibodies react strongly with the second-largest subunit of RNA polymerase I but do not react with the respective subunits of RNA polymerase II and III . The second-largest subunit of RNA polymerase III is only recognized by anti-DmRP128 . Previously, we have claimed that DmRP135 codes for the second-largest subunit of RNA polymerase III.(ABSTRACT TRUNCATED AT 250 WORDS) Am J Hum Genet, 1991 Sep, 49(3), 656 - 61 Isolation of a human DNA sequence which spans the fragile X; Kremer EJ et al.; To identify the sequences involved in the expression of the fragile X and to characterize the molecular basis of the genetic lesion, we have constructed yeast artificial chromosomes (YACs) containing human DNA and have screened them with cloned DNA probes which map close to the fragile site at Xq27.3 . We have isolated and partly characterized a YAC containing approximately 270 kb of human DNA from an X chromosome which expresses the fragile X . This sequence in a yeast artificial ring chromosome, XTY26, hybridizes to the two closest DNA markers, VK16 and Do33, which flank the fragile site . The human DNA sequence in XTY26 also spans the fragile site on chromosome in situ hybridization . When a restriction map of XTY26, derived by using infrequently cutting restriction enzymes, is compared with similar YAC maps derived from non-fragile-X patients, no large-scale differences are observed . This YAC, XTY26, may enable (a) the fragile site to be fully characterized at the molecular level and (b) the pathogenetic basis of the fragile-X syndrome to be determined. Proc Natl Acad Sci U S A, 1991 Sep 1, 88(17), 7674 - 8 Evidence for a factor required for transcriptional stimulation by the chimeric acidic activator GAL-VP16 in HeLa cell extracts; White JH et al.; We provide biochemical evidence for the existence of a transcriptional intermediary factor (TIF) in HeLa whole-cell extracts (WCE) that is distinct from the basic transcription factors and that is required for transcriptional stimulation by the chimeric acidic activator GAL-VP16 . We have fractionated HeLa WCE by heparin-agarose chromatography . Of transcriptionally active fractions eluting in a step between 0.24 and 0.6 M KCl, the initial fractions are refractory to GAL-VP16 stimulation, whereas subsequent fractions are strongly stimulated by the activator . Aliquots of GAL-VP16-responsive fractions efficiently complement refractory fractions for transcriptional stimulation . Aliquots of responsive fractions are also far more efficient than those of refractory fractions in overcoming transcriptional inhibition that is brought about by high concentrations of GAL-VP16 . Experiments performed with heat-treated WCE support the idea that HeLa cells contain a TIF that is essential for GAL-VP16 stimulation, but that is not required for basal transcription . Addition of recombinant yeast or human transcription factor TFIID (rTFIIDY and rTFIIDH, respectively) to a WCE heated at 48 degrees C for 15 min restores basal transcription, but in neither case is the reconstituted system activated by GAL-VP16 . However, a 45 degrees C heat-treated WCE reconstituted with either rTFIIDH or rTFIIDY is stimulated by GAL-VP16, suggesting that a HeLa TIF can be selectively inactivated by heating at 48 degrees C, but not at 45 degrees C . Interestingly, a TFIID fraction partially purified from HeLa cell extracts, but not rTFIIDH, efficiently relieves transcriptional inhibition by GAL-VP16, suggesting that there may be an association between TIF(s) and TFIID and, moreover, that TIF(s) may be the direct target of the acidic domain of GAL-VP16 . In summary, our results support the existence of a TIF that is not essential for basal transcription but that is required to mediate the stimulatory activity of the acidic activator GAL-VP16. Mol Cell Biol, 1991 Sep, 11(9), 4389 - 97 Double-strand gap repair in a mammalian gene targeting reaction; Valancius V et al.; To better understand the mechanism of homologous recombination in mammalian cells that facilitates gene targeting, we have analyzed the recombination reaction that inserts a plasmid into a homologous chromosomal locus in mouse embryonic stem cells . A partially deleted HPRT gene was targeted with various plasmids capable of correcting the mutation at this locus, and HPRT+ recombinants were directly selected in HAT medium . The structures of the recombinant loci were then determined by genomic Southern blot hybridizations . We demonstrate that plasmid gaps of 200, 600, and 2,500 bp are efficiently repaired during the integrative recombination reaction . Targeting plasmids that carry a double-strand break or gap in the region of DNA homologous to the target locus produce 33- to 140-fold more hypoxanthine-aminopterin-thymidine-resistant recombinants than did these same plasmids introduced in their uncut (supercoiled) forms . Our data suggest that double-strand gaps and breaks may be enlarged prior to the repair reaction since sequence heterologies carried by the incoming plasmids located close to them are often lost . These results extend the known similarities between mammalian and yeast recombination mechanisms and suggest several features of the insertional (O-type) gene targeting reaction that should be considered when one is designing mammalian gene targeting experiments. EMBO J, 1991 Sep, 10(9), 2635 - 44 Different effects of intron nucleotide composition and secondary structure on pre-mRNA splicing in monocot and dicot plants; Goodall GJ et al.; We have found previously that the sequences important for recognition of pre-mRNA introns in dicot plants differ from those in the introns of vertebrates and yeast . Neither a conserved branch point nor a polypyrimidine tract, found in yeast and vertebrate introns respectively, are required . Instead, AU-rich sequences, a characteristic feature of dicot plant introns, are essential . Here we show that splicing in protoplasts of maize, a monocot, differs significantly from splicing in a dicot, Nicotiana plumbaginifolia . As in the case of dicots, a conserved branch point and a polypyrimidine tract are not required for intron processing in maize . However, unlike in dicots, AU-rich sequences are not essential, although their presence facilitates splicing if the splice site sequences are not optimal . The lack of an absolute requirement for AU-rich stretches in monocot introns in reflected in the occurrence of GC-rich introns in monocots but not in dicots . We also show that maize protoplasts are able to process a mammalian intron and short introns containing stem--loops, neither of which are spliced in N.plumbaginifolia protoplasts . The ability of maize, but not of N.plumbaginifolia to process stem--loop-containing or GC-rich introns suggests that one of the functions of AU-rich sequences during splicing of dicot plant pre-mRNAs may be to minimize secondary structure within the intron. Plant J, 1991 Sep, 1(2), 141 - 8 Identification of a novel S-phase-specific gene during the cell cycle in synchronous cultures of Catharanthus roseus cells; Ito M et al.; The cell-cycle specific cDNAs were isolated from a cDNA library prepared from cells in the S phase in the synchronous cultures of Catharanthus roseus . One of the isolated genes, which we refer to as cyc07, was analyzed in detail . The full-length cDNA of cyc07 contains an open reading frame of 735 nucleotides, encoding a protein of 245 amino acids with a molecular weight of 28,356 Da . The protein predicted from the nucleotide sequence is highly basic, as are mammalian histones . cyc07 mRNA was detected specifically in cells at the S phase in synchronous cultures . The induction and accumulation of mRNA in the S phase were suppressed when DNA synthesis was inhibited by aphidicolin . In the intact plant, cyc07 mRNA was found preferentially in root tips that contained meristematic tissue . A databank search revealed that a sequence homologous to the nucleotide sequence of cyc07 cDNA is present in the downstream region of the SIR3 gene in the yeast genome . The amino acid sequence predicted from the corresponding region of the yeast genome exhibited significant homology with that of cyc07 protein . These similarities between cyc07 and the corresponding region in yeast suggest that the homologous sequence in yeast is a novel gene that is functionally homologous to cyc07 . Our results presented here suggest the possibility that cyc07 may play a role in the proliferation of higher plant cells, in particular in the entry into or progression of the S phase of the cell cycle. Clin Exp Dermatol, 1991 Sep, 16(5), 331 - 8 An immunological study in patients with seborrhoeic dermatitis; Bergbrant IM et al.; The humoral and cellular immune-status was studied in 30 patients with seborrhoeic dermatitis . Increased frequencies of natural killer cells were found in 46% of patients . Furthermore, subnormal mitogen stimulation responses were demonstrated in 13 patients, whereas two individuals were found to have very high numbers of activated T lymphocytes in peripheral blood . Higher-than-normal total serum IgG and IgA was observed in 14 and 11 patients, respectively . For nine of 12 patients with skin lesions, dermal perivascular cell infiltrates were seen . The majority of the infiltrating cells reacted with anti-CD4 antibodies . HLA-DR-expressing keratinocytes were found in two biopsies . The study suggests that patients with seborrhoeic dermatitis may have depressed T-cell function . This could have a bearing on their susceptibility to the Pityrosporum ovale-associated dermatitis . The very high frequencies of activated T cells observed in the peripheral blood of two otherwise healthy seborrhoeic individuals suggests that intermittent systemic immune activation may occur . Seborrhoeic dermatitis is a common skin disease . It can be diagnosed by its characteristic red to yellow-brown lesions covered with greasy scales distributed in areas with a high number of sebaceous glands, such as the scalp, face and upper trunk . There is an association between seborrhoeic dermatitis and the lipophilic yeast Pityrosporum ovale but its exact aetiological role is not known . The yeast is a member of the normal cutaneous flora but also the aetiological agent of pityriasis versicolor and Pityrosporum folliculitis . P . ovale can activate complement via the direct and alternative pathways . This may play some part in the induction of inflammation.(ABSTRACT TRUNCATED AT 250 WORDS) Arh Hig Rada Toksikol, 1991 Sep, 42(3), 267 - 79 {Immunologic changes and pulmonary ventilatory function in animal feed processing workers}; Zuskin E et al.; Respiratory symptoms and immunological reactions were examined in 35 animal food workers . The most frequent positive skin prick reactions occurred to fish flour (82.9%), followed by carotene (77.1%), cornflour (65.7%), four-leaf clover (62.9%), sunflower (54.3%), chicken meat (31.4%), soy (28.6%) and yeast (22.7%) . The IgE serum level was increased in 40% of the animal food workers and in 2.6% of the controls . A significantly higher prevalence of chronic respiratory symptoms was found in animal food workers than in controls . However, there was no significant difference in prevalence of chronic respiratory symptoms between workers with positive and those with negative skin tests to house dust and fish flour or between those with increased and those with normal IgE levels (except for dyspnoea) . There were significant acute across-shift reductions in ventilatory capacity, particularly for FEF25 . The workers with positive skin tests to fish flour demonstrated significantly larger acute FEF25 reductions than those with negative skin tests . An extract of animal food caused constriction of isolated guinea pig tracheal smooth muscle in vitro . It appears that animal food dust in addition to immunological response may produce a direct irritative effect on the airways of exposed workers. Chem Pharm Bull (Tokyo), 1991 Sep, 39(9), 2301 - 7 Synthesis and antifungal activities of a series of (1,2-disubstituted vinyl)imidazoles; Ogawa M et al.; A series of vinylimidazoles containing a hetero atom such as sulfur or oxygen at a beta-position of the vinyl group was prepared and the antifungal activities were tested . It was found that sulfur-substituted derivatives such as (E)-1-{2-(methylthio)-1-{2-(pentyloxy)phenyl}ethenyl}-1H-imidazole (5a-5) and (E)-1-{1-{2-(hexyloxy)phenyl}-2-(methylthio)ethenyl}-1H-imidazole (5a-6) showed excellent antifungal activities against dermatophytes and yeast cells . The stereochemistry of the hydrochloride salt of 5a-5 was determined by X-ray crystallography . The structure-activity relationships were discussed. Indian J Exp Biol, 1991 Sep, 29(9), 875 - 6 Effect of selected chemicals on mosquito larval orientation behaviour using a new apparatus; Vartak PH et al.; An apparatus was designed and fabricated for studying the effects of aromatic chemicals on the IV instar larvae of Aedes aegypti (L.) . The test chemicals (essential oils and monoterpenoids) were incorporated in agar along with yeast and dog biscuits which formed an attractant food source (bait) . A fairly rapid decrease in the migration of larvae towards the bait cum chemical source was observed during the first hour after which a steady state was observed . Minimum migration (9.67%) was seen with terpeneol anhydrous and maximum (15.0%) with beta-citronellal as compared to control (65.67%), at the end of a 4 hr exposure period . Food particles were also detected on microscopic examination. Mol Biochem Parasitol, 1991 Sep, 48(1), 47 - 58 Induction and localization of Plasmodium falciparum stress proteins related to the heat shock protein 70 family; Kumar N et al.; Induction of heat shock-related stress proteins Pfhsp and Pfgrp, similar in sequence to hsp70 (heat shock protein) and grp78 (glucose-regulated protein), respectively, was studied in culture-derived parasite Plasmodium falciparum . Elevation in temperature from 26 degrees C to 37 degrees C and higher caused significant induction of Pfhsp with a moderate effect on the synthesis of Pfgrp also . Synthesis of Pfgrp, however, was not induced by partial glucose deprivation . On the contrary, lack of glucose in the medium resulted in cessation of protein synthesis in the parasites . Other known inducers of grp synthesis in mammalian cells, i.e., calcium ionophore A23187 and inhibitors of glycosylation (tunicamycin, 2-deoxy glucose) were also without any apparent effect on the synthesis of Pfgrp . Heat shock-induced responses were transient in nature: removal of stress caused repression of these responses . The effect of glucose deprivation was only partially reversible with better recovery if parasites were subjected to glucose starvation at 26 degrees C than at 37 degrees C . Northern blot analysis and in vitro translation of mRNA revealed a parallel increase in the levels of mRNA for Pfhsp upon heat shock . Immuno-gold electron microscopy with cultured parasites revealed nuclear location of Pfhsp and primarily cytoplasmic (probably endoplasmic reticulum) location of Pfgrp . These findings suggest that SDEL (carboxy terminal sequence of Pfgrp) might play a similar role in the cellular localization of Pfgrp as does the sequence KDEL in mammalian cells and HDEL in yeast. Blood Rev, 1991 Sep, 5(3), 177 - 92 Cell cycle regulation; Bybee A et al.; Over the past few years there has been a resurgance of interest in the cell cycle . The excitement has mainly been due to the fact that researchers all over the world who had been working on seemingly different processes in yeast, fruit flies, frogs and man have found that many of the processes and individual proteins have been highly conserved . In essence, what regulates the cell cycle in yeast also works in man . This review is biased towards cell cycle regulation in mammalian cells but we have also covered what is known about similar mechanisms in other organisms to provide a broader perspective to the field . We outline what is currently known about the cell cycle and the key points at which cell proliferation is controlled . We summarise recent work on cell cycle control genes and antioncogenes and the post-transcriptional regulation of the proteins for which they code . Finally we cover the relationship between the cell cycle and differentiation. J Clin Microbiol, 1991 Sep, 29(9), 2007 - 12 Application of an indirect immunofluorescence test for detection of Mycoplasma pneumoniae in respiratory exudates; Hirai Y et al.; We prepared polyclonal antibody specific to Mycoplasma pneumoniae and examined the conditions influencing the ability of an indirect immunofluorescence test to detect the specific antigen in respiratory exudates . The antibody did not cross-react with normal human serum or with respiratory exudates from 10 healthy persons . Cross-reactivity of the antibody with species of mycoplasmas other than M . genitalium was fully diminished when absorbed with horse serum and yeast extract, components of the culture medium . Though the absorbed antibody cross-reacted with M . genitalium, the titer was significantly lower than when tested against M . pneumoniae . Two types of antigen-specific fluorescence were observed in clinical specimens: one is large or small fluorescent granular aggregates found in mucus, and the other is fine fluorescent particles diffused on the entire surface of small epithelial cells . Throat smears from 49 patients with serologically confirmed M . pneumoniae infections were examined by our indirect immunofluorescence method . Positive results were obtained in 42 cases, many of which were positive before a rise in serum antibody titer could be demonstrated, indicating that the method is useful for a preliminary diagnosis at an early stage of the infection. Vopr Med Khim, 1991 Sep-Oct, 37(5), 73 - 6 {The effect of selenium on enzymes metabolizing xenobiotics in rat liver}; Kravchenko LV et al.; Activity of enzymes involved in metabolism of xenobiotics was not altered in liver tissue of rats kept on a ration enriched with selenium at a dose of 2.5 mg/kg . Both organic form of selenium (yeast meal, selenomethionine) and inorganic derivatives (sodium selenite) at a dose of 5 mg/kg of ration caused distinct activation of epoxide hydrolase, UDP-glucuronosyl transferase and glutathione transferase within 6 weeks after the experiment beginning, while content of cytochrome P-450, glutathione-SH and glutathione peroxidase activity were not significantly altered . Within 9 weeks the enzymatic activity remained at the higher rate only in rats kept on the ration with sodium selenite . Relationship between toxic effects of selenium high doses and alterations in activity of enzymes involved in metabolism of xenobiotics is discussed. J Dairy Sci, 1991 Sep, 74(9), 2965 - 75 Isolation and phagocytic properties of neutrophils and other phagocytes from nonmastitic bovine milk; Sandgren CH et al.; A technique for the separation of neutrophils from macrophages-epithelial cells in samples of nonmastitic bovine milk with low cell counts has been developed . The procedure is based on centrifugation in a discontinuous metrizamide gradient and is rapid, taking less than 40 min . The recovery of the neutrophils is about 30% and their viability about 90% . The isolated neutrophils showed an appreciable unstimulated luminol- and lucigenin-dependent chemiluminescence, which was due to NADPH oxidase rather than to xanthine oxidase . The neutrophils had a higher rate of ingestion of C3-opsonized particles than macrophages-epithelial cells, whereas no significant differences in phagocytosis of IgG-opsonized yeast or unopsonized yeast were detected between the two cell populations . The macrophages-epithelial cells produced no luminol-dependent chemiluminescence and induced considerably lower activity in the lucigenin-dependent system than neutrophils, indicating that these cells contain no myeloperoxidase . Analyses of the activity of the neutrophils in response to C3-opsonized yeast particles showed that the luminol-dependent chemiluminescence of cells isolated from residual milk increased significantly over the lactation period . Moreover, a tendency to a higher phagocytosis and chemiluminescence of neutrophils isolated from residual milk than from stripping milk was indicated. Curr Genet, 1991 Sep, 20(4), 319 - 29 The first analysed archegoniate mitochondrial gene (COX3) exhibits extraordinary features; Marienfeld JR et al.; The first mitochondrial-encoded gene of an archegoniate has been identified, cloned and sequenced . The cytochrome oxidase III gene (cox3) of the moss Physcomitrella patens consists of a 618 bp open reading frame with high homology (around 72%) to known cox3 sequences of higher plants . Nevertheless, it is a quarter shorter than these . The cox3 gene of P . patens contains no introns and reveals a G + C-content of 41.3% . The region containing the cox3 gene exists as a single copy in the mitochondrial genome as shown by restriction mapping . In the 5' flanking sequence a putative ribosome binding site and a putative secondary structure were found . Two main transcripts of 2.4 kb and 2.6 kb were detected indicating a complex mitochondrial transcription pattern possibly due to co-transcription . Additional open reading frames were found downstream from, as well as upstream of, the cox3 gene . In Western blots a polyclonal cox3 antibody from yeast detected one single band with an apparent molecular weight of 22 kDa. Biochem Cell Biol, 1991 Sep, 69(9), 586 - 607 Cytochrome c oxidase: structure, function, and membrane topology of the polypeptide subunits; Cooper CE et al.; Mitochondrial cytochrome c oxidase and its bacterial homologs catalyze electron transfer and proton translocation reactions across membranes . The eukaryotic enzyme complex consists of a large number of polypeptide subunits . Three of the subunits (I, II, and III) are mitochondrially encoded while the remaining 6 (yeast) to 10 (bovine) are nuclear encoded . Antibody and chemical-labelling experiments suggest that subunits I-III and most (but not all) of the nuclear-encoded subunits span the inner mitochondrial membrane . Subunits I and II are the catalytic core of the enzyme . Subunit I contains haem a, haem a3 and CuB, while subunit II contains CuA and the cytochrome c binding site . Subunit III and most of the nuclear subunits are essential for the assembly of a functional catalytic enzyme . Some nuclear subunits are present as isozymes, although little functional difference has yet been detected between enzyme complexes composed of different isozymes . Therefore, any additional role attributed to the nuclear-encoded subunits beyond that of enzyme assembly must be tentative . We suggest that enough evidence exists to support the idea that modification of the larger nuclear subunits (IV, V, and possibly VI) can effect enzyme turnover in vitro . Whether this is a physiological control mechanism remains to be seen. Nature, 1991 Aug 29, 352(6338), 821 - 4 Genetic evidence for base pairing between U2 and U6 snRNA in mammalian mRNA splicing; Datta B et al.; Removal of introns from eukaryotic nuclear messenger RNA precursors is catalysed by a large ribonucleoprotein complex called the spliceosome, which consists of four small nuclear ribonucleoprotein particles (U1, U2, U5, and U4/U6 snRNPs) and auxiliary protein factors . We have begun a genetic analysis of mammalian U2 snRNA by making second-site mutations in a suppressor U2 snRNA . Here we find that several mutations in the 5' end of U2 (nucleotides 3-8) are deleterious and that one of these can be rescued by compensatory base changes in the 3' end of U6 (nucleotides 92-95) . The results demonstrate genetically that the base-pairing interaction between U2 (nucleotides 3-11) and U6 snRNA (nucleotides 87-95), originally proposed on the basis of psoralen photocrosslinking experiments, can influence the efficiency of mRNA splicing in mammals . The U2/U6 interaction in yeast, however, is fairly tolerant to mutation (D.J . Field and J.D . Friesen, personal communication), emphasizing the potential for facultative RNA interactions within the spliceosome. Nature, 1991 Aug 29, 352(6338), 803 - 7 Nuclear association of a T-cell transcription factor blocked by FK-506 and cyclosporin A; Flanagan WM et al.; Cyclosporin A and FK506 inhibit T- and B-cell activation and other processes essential to an effective immune response . In T lymphocytes these drugs disrupt an unknown step in the transmission of signals from the T-cell antigen receptor to cytokine genes that coordinate the immune response . The putative intracellular receptors for FK506 and cyclosporin are cis-trans prolyl isomerases . Binding of the drug inhibits isomerase activity, but studies with other prolyl isomerase inhibitors and analysis of cyclosporin-resistant mutants in yeast suggest that the effects of the drug result from the formation of an inhibitory complex between the drug and isomerase, and not from inhibition of isomerase activity . A transcription factor, NF-AT, which is essential for early T-cell gene activation, seems to be a specific target of cyclosporin A and FK506 action because transcription directed by this protein is blocked in T cells treated with these drugs, with little or no effect on other transcription factors such as AP-1 and NF-kappa B . Here we demonstrate that NF-AT is formed when a signal from the antigen receptor induces a pre-existing cytoplasmic subunit to translocate to the nucleus and combine with a newly synthesized nuclear subunit of NF-AT . FK506 and cyclosporin A block translocation of the cytoplasmic component without affecting synthesis of the nuclear subunit. Biochim Biophys Acta, 1991 Aug 27, 1090(1), 115 - 8 An adult male specific gene in Drosophila containing the repetitive element opa; Grabowski DT et al.; A cDNA has been isolated for an adult male specific gene in Drosophila that contains the repetitive element opa . We have named this gene Dromsopa for Drosophila male specific opa containing gene . The 0.6 kb mRNA for this gene is only found in the abdominal region of adult male Drosophila and in no other tissue or at other developmental stages . The Dromsopa opa repeat codes for the usual stretch of poly(glutamine) interrupted by histidine residues . The opa repetitive element was originally found in the Drosophila Notch gene (Kidd, S . et al . (1983) Cell 34, 431-433 and Wharton, K.A . (1985) Cell 40, 55-62) and has, more recently, been found in genes under developmental or tissue specific control from yeast to humans . The gene was cloned using a genomic fragment during a chromosomal walk upstream of the AP3 gene located at chromosomal location 79CD on the left arm of the third chromosome (Kelley, M.R . et al . (1989) Mol . Cell . Biol . 9, 965-973) . The Dromsopa gene has no other identity with genes currently in the databases, once the opa repeat is excluded . The possibility that the Dromsopa gene is a male specific regulatory factor is under investigation as is its precise location within the abdomen, such as in germ line tissue. Biochim Biophys Acta, 1991 Aug 27, 1090(1), 102 - 8 Molecular cloning of cDNA for antimycin A-inducible mRNA and its role in cyanide-resistant respiration in Hansenula anomala; Sakajo S et al.; A cDNA for mRNA induced by antimycin A in Hansenula anomala was cloned . The mRNA for the cDNA was expressed in the yeast under the conditions expressing the cyanide-resistant respiration activity . The nucleotide sequence revealed a long open reading frame of 342 codons encoding a protein with a molecular weight of 40,282 in the cDNA . An antibody recognizing the protein encoded by the open reading frame was produced . Immunoblotting of H . anomala proteins with this antibody showed that a 36 kDa protein localized in mitochondria was a mature form of the protein encoded by the cDNA . It is suggested that the cloned cDNA encodes a protein involved in the cyanide-resistant respiratory pathway. Biochim Biophys Acta, 1991 Aug 27, 1090(1), 125 - 8 Nucleotide sequence of a cDNA coding for the mitochondrial precursor protein of cytochrome c oxidase subunit IV from the slime mold Dictyostelium discoideum; Rizzuto R et al.; Subunit-specific polyclonal antibodies were used to isolate cDNA clones encoding subunit IV of Dictyostelium discoideum cytochrome c oxidase . DNA sequence analysis reveals an open reading frame of 149 amino acids . As shown by sequencing of the protein N-terminus, the subunit is synthesized with a 24 residue cleavable presequence which leads to a mature polypeptide of 14305 Da . The slime mold subunit exhibits a low but significant degree of similarity with subunit Va of human and subunit VI of yeast cytochrome c oxidase. J Biol Chem, 1991 Aug 25, 266(24), 15992 - 8 Missense mutation serine106----proline causes 17 alpha-hydroxylase deficiency; Lin D et al.; Steroid 17 alpha-hydroxylase deficiency is caused by defects in cytochrome P450c17, the single enzyme that has 17-alpha hydroxylase and 17,20-lyase activities . We describe a rapid and efficient polymerase chain reaction tactic for identifying these genetic lesions and identify Ser106----Pro as the cause of 17 alpha-hydroxylase deficiency in two unrelated homozygous patients from Guam . We used site-directed mutagenesis of the normal P450c17 cDNA to construct the Pro106 mutant, and expressed both the normal and mutant sequences in monkey COS-1 cells and in yeast . Expression of the normal sequence permitted the cells to convert pregnenolone to 17-OH pregnenolone, progesterone to 17-OH progesterone, and 17-OH pregnenolone to dehydroepiandrosterone, showing the normal sequence conferred both 17 alpha-hydroxylase and 17,20-lyase activities . Expression of the mutant sequence generated P450c17 mRNA, but conferred none of these activities, proving that the Ser106----Pro mutation abolished the 17 alpha-hydroxylase and 17,20-lyase activities . An HhaI restriction site created by the mutation should permit screening of large populations. J Mol Biol, 1991 Aug 20, 220(4), 903 - 14 Use of high coverage reference libraries of Drosophila melanogaster for relational data analysis . A step towards mapping and sequencing of the genome; Hoheisel JD et al.; Three differently made, primary Drosophila cosmid libraries of 16-fold genome coverage have been generated . Also, a jumping library has been created by a new method that takes advantage of methylation differences between genomic DNA and vector . Thirdly, two cDNA libraries have been picked . All these libraries have been arrayed on high-density in situ filters, each containing 9216 clones . As a reference system, such filters are distributed and identified clones are provided . Single-copy probes have identified on average 1.4 cosmids per genome equivalent . Together with cytogenetically mapped yeast artificial chromosomes, the libraries are also being used for physically mapping the genome, mainly by oligonucleotide fingerprinting and pool hybridizations . cDNA clones are further examined by a partial sequencing analysis by oligomer hybridization. J Mol Biol, 1991 Aug 20, 220(4), 843 - 53 Unidirectional replication as visualized by two-dimensional agarose gel electrophoresis; Martin-Parras L et al.; Two-dimensional (2D) agarose gel electrophoresis is progressively replacing electron microscopy as the technique of choice to map the initiation and termination sites for DNA replication . Two different versions were originally developed to analyze the replication of the yeast 2 microns plasmid . Neutral/Neutral (N/N) 2D agarose gel electrophoresis has subsequently been used to study the replication of other eukaryotic plasmids, viruses and chromosomal DNAs . In some cases, however, the results do not conform to the expected 2D gel patterns . In order to better understand this technique, we employed it to study the replication of the colE1-like plasmid, pBR322 . This was the first time replicative intermediates from a unidirectionally replicated plasmid have been analyzed by means of N/N 2D agarose gel electrophoresis . The patterns obtained were significantly different from those obtained in the case of bidirectional replication . We showed that identification of a complete are corresponding to molecules containing an internal bubble is not sufficient to distinguish a symmetrically located bidirectional origin from an asymmetrically located unidirectional origin . We also showed that unidirectionally replicated fragments containing a stalled fork can produce a pattern with an inflection point . Finally, replication appeared to initiate at only some of the potential origins in each multimer of pBR322 DNA. Biochim Biophys Acta, 1991 Aug 20, 1085(1), 126 - 30 Lipid hydrogenation induces elevated 18:1-CoA desaturase activity in Candida lipolytica microsomes; Horvath I et al.; Microsomal membranes prepared from the mesophilic yeast Candida lipolytica grown at 10 degrees C were hydrogenated by the homogeneous Pd-catalyst, palladium di (sodium alizarine sulfonate) (Pd(QS)2) . After hydrogenation to various levels, the microsomes were washed free of the Pd-complex and transferred to a reaction mixture (containing NADH, MgCl2, ATP, CoA and {14C}18:1-CoA) for assay of 18:1-CoA desaturase activity . Microviscosity alterations were also followed by measuring changes in DPH fluorescence polarization . Rapid catalytic hydrogenation of unsaturated fatty acids of the lipids occurred within 20-120 s, resulting in large increases in 16:0, 18:0 and 18:1 acids and decreases in 18:2 acid . In the range 7-20% 18:0 content, a pronounced increase in desaturase activity was observed, with a maximum of greater than 2-fold at a 18:0 content of 12%, followed by a decrease to the initial activity at 33% 18:0 content . These changes were well-correlated with changes in microviscosity, maximal desaturase activity occurring in the DPH fluorescence anisotropy range of 0.23-0.24; above and below this range, desaturase activities were close to the initial control values . It is suggested that the hydrogenation-induced increase in the formation of 18:2 from 18:1-CoA (proceeding partly through direct desaturation of PC) may be due to changes in conformation of the membrane-bound desaturase enzyme complex as a result of controlled rigidification of the surrounding lipids . The operation of such a self-regulating control mechanism would be consistent with a previously proposed model for microsomal desaturase action. Proc Natl Acad Sci U S A, 1991 Aug 15, 88(16), 7006 - 10 Functional reintroduction of human telomeres into mammalian cells; Farr C et al.; Telomeric sequences of eukaryotes consist of short tandem repeats organized in arrays of variable length in which the guanine-rich strand runs 5'----3' toward the chromosomal end . The terminal repeats in yeast are the only elements necessary for telomere function in this organism . To test whether mammalian terminal repeats can function after reintroduction into a mammalian cell, a repeat-containing terminal fragment from a human chromosome was electroporated into a hamster-human hybrid cell line . In 6 of 27 independent transformants analyzed, the introduced sequences were found at the ends of chromosomes, based on all available criteria . Terminal restriction-fragment heterogeneity and the survival of these chromosomes demonstrate that these telomeres are functional . Cytogenetic evidence from one of these cell lines suggests that chromosome breakage with healing at the integration site is the mechanism responsible for the terminal location. J Biol Chem, 1991 Aug 15, 266(23), 15202 - 12 Structural requirements for transformation of substrates by the (S)-adenosyl-L-methionine:delta 24(25)-sterol methyl transferase; Nes WD et al.; The membrane-bound enzyme of microsomes obtained from sunflower embryos that catalyzes the bi-substrate transfer reaction whereby the methyl group of (S)-adenosyl-L-methionine is transferred to C-24 of the sterol side chain has been investigated . Optimal incubation conditions for assay of the microsomal (S)-adenosyl-L-methionine:sterol delta 24-methyl transferase (SMT) have been established for the first time . The microsomal preparation was found to catalyze the formation of a delta 24(28)-sterol and to be free of contaminating methyl transferase enzymes, e.g . those which form delta 23-24 methyl sterols (cyclosadol) and delta 25-24 beta-methyl sterols (cyclolaudenol) and other sterolic enzymes which might transform the acceptor molecule to metabolites which could compete in the assay with the test substrate . From a series of incubations with 27 sterol and sterol-like (triterpenoids) substrates of which 23 compounds possessed a 24,25-double bond, we observed a marked dependence on precise structural features and three-dimensional shape of the acceptor molecule in its ability to be transformed by the SMT . In contrast to the yeast SMT where cycloartenol fails to bind to the SMT and zymosterol is the best substrate for methylation, the sunflower SMT studied here utilizes cycloartenol preferentially to zymosterol and the other substrates . Of the chemical groups which distinguishes cycloartenol, a free 3 beta-OH,9 beta,19-cyclopropyl group, trimethylated saturated nucleus, and delta 24-double bond, only the nucleophilic centers at C-3 and C-24 were obligatory for substrate binding and methylation . Of the bent or flat conformations which cycloartenol may orient in the enzyme-substrate complex, our results indicate a selection for acceptor molecules which possess the shape that closely resembles the crystal state and solution orientation of cycloartenol which is now known to be flat rather than bent (Nes, W . D., Benson, M., Lundin, R . E., and Le, P . H . (1988) Proc . Natl . Acad . Sci . U.S.A . 85, 5759-5763). J Biol Chem, 1991 Aug 15, 266(23), 15060 - 7 Isolation and structural characterization of the Chlamydomonas reinhardtii gene for cytochrome c6 . Analysis of the kinetics and metal specificity of its copper-responsive expression; Hill KL et al.; We have isolated a 5-kilobase pair fragment of genomic DNA containing the entire coding region for the Chlamydomonas reinhardtii gene encoding the copper-repressible Cyt c6 . A region comprising 2.6 kilobase pairs contains the entire transcribed region plus 852 nucleotides upstream of the Cyt c6 transcription start site and 495 nucleotides downstream of the conserved C . reinhardtii polyadenylation signal . Comparison of the genomic sequence with the cDNA sequence (Merchant, S., and Bogorad, L . (1987) J . Biol . Chem . 262, 9062-9067) revealed that the coding region is interrupted by two introns, each of which is flanked by C . reinhardtii consensus intron/exon boundaries . Primer extension and S1 nuclease protection analyses identified the 5' border of the Cyt c6 mRNA at approximately 79 base pairs upstream from the initiator methionine . Analysis of the 5' upstream region reveals no significant similarity to sequences found in upstream regions of other copper-regulated genes . Time-course studies indicate that 1) the mature Cyt c6 mRNA has a half-life of approximately 45-60 min and is completely lost within 4 h, and 2) the primary, unspliced transcript has a half-life of approximately 10 min and is completely lost within 30 min after the addition of copper ions to copper-depleted cells . These results indicate that the response to copper occurs very rapidly upon elevation of extracellular copper levels . Although this gene is unresponsive to silver ions in vivo, in contrast to the yeast copper-responsive CUP1 gene (Furst, P., Hu, S., Hackett, R., and Hamer, D . (1988) Cell 55, 705-717), it does respond to mercury ions, albeit with less sensitivity . Mercury ions cannot, however, substitute for copper in allowing the accumulation of plastocyanin in vivo. Genomics, 1991 Aug, 10(4), 921 - 6 Linkage mapping and fluorescence in situ hybridization of TCTE1 on human chromosome 6p: analysis of dinucleotide polymorphisms on native gels; Kwiatkowski TJ Jr et al.; Highly informative dinucleotide repeat polymorphisms were identified at the T-complex-associated-testes-expressed-1 (TCTE1) locus on human chromosome 6p . Electrophoresis of single-stranded DNA on native gels facilitated the analysis of the dinucleotide polymorphisms . Linkage mapping positions this marker midway between the centromere and HLA with recombination fractions as follows: D6Z1-0.21-TCTE1-0.24-HLA . Two-color fluorescence in situ hybridization places TCTE1 proximal to CRIL171 (D6S19) . Together, linkage and in situ hybridization indicate that the order of the loci is D6Z1-D6S4-D6S90-TCTE1-D6S19-D6S29-HL A-telomere . A sequence tagged site (STS) was established, and three yeast artificial chromosome (YAC) clones were identified for the TCTE1 locus. Nucleic Acids Res, 1991 Aug 11, 19(15), 4075 - 81 Transcript mapping reveals different expression strategies for the bicistronic RNAs of the geminivirus wheat dwarf virus; Dekker EL et al.; We have characterised the major transcripts of the Czech isolate of wheat dwarf virus (WDV-CJI) which show that WDV uses two different mechanisms for expressing overlapping open reading frames (ORFs) . Mapping of the virion sense RNAs identified a single polyadenylated transcript of 1.1kb spanning the overlapping ORFs V1 and V2 which encode cell-cell spread functions and the coat protein respectively . This finding distinguishes WDV from other monocot-infecting geminiviruses studied so far which were shown to encode two 3' co-terminal transcripts capable of expressing either the V1 or V2 ORF . A survey of codon usage at the junction between the V1 and V2 ORF has led us to propose that translational frame shifting analogous to that in the yeast Ty element may occur . Analysis of polymerase chain reaction (PCR) amplified complementary sense cDNA clones has revealed the presence of mature spliced and unspliced RNAs which could encode products of an intron mediated C1:C2 ORF fusion or the C1 ORF product alone . Mapping of the 5' and 3' extremities of the major WDV encoded transcripts has allowed us to identify putative transcription regulatory sequences and the presence of multiple overlapping transcripts may suggest temporal regulation of transcription. Cell, 1991 Aug 9, 66(3), 451 - 64 The discs-large tumor suppressor gene of Drosophila encodes a guanylate kinase homolog localized at septate junctions; Woods DF et al.; Mutations of the lethal(1)discs large-1 (dlg) tumor suppressor gene of Drosophila cause neoplastic overgrowth of the imaginal discs . Sequencing of a near full-length cDNA predicts a protein containing a domain homologous to yeast guanylate kinase and a region homologous to SH3, a putative regulatory motif in nonreceptor protein tyrosine kinases and other signal transduction proteins . Immunofluorescence analysis using antibodies directed against fusion peptides shows that the dlg gene product is localized in an apical belt of the lateral cell membrane, at the position of the septate junction . The results suggest that a signal transduction process involving guanine nucleotides occurs at the septate junction and is necessary for cell proliferation control in Drosophila epithelia. J Biol Chem, 1991 Aug 5, 266(22), 14754 - 62 Human lysosomal protective protein has cathepsin A-like activity distinct from its protective function; Galjart NJ et al.; The protective protein was first discovered because of its deficiency in the metabolic storage disorder galactosialidosis . It associates with lysosomal beta-galactosidase and neuraminidase, toward which it exerts a protective function necessary for their stability and activity . Human and mouse protective proteins are homologous to yeast and plant serine carboxypeptidases . Here, we provide evidence that this protein has enzymatic activity similar to that of lysosomal cathepsin A: 1) overexpression of human and mouse protective proteins in COS-1 cells induces a 3-4-fold increase of cathepsin A-like activity; 2) this activity is reduced to approximately 1% in three galactosialidosis patients with different clinical phenotypes; 3) monospecific antibodies raised against human protective protein precipitate virtually all cathepsin A-like activity in normal human fibroblast extracts . Mutagenesis of the serine and histidine active site residues abolishes the enzymatic activity of the respective mutant protective proteins . These mutants, however, behave as the wild-type protein with regard to intracellular routing, processing, and secretion . In contrast, modification of the very conserved Cys60 residue interferes with the correct folding of the precursor polypeptide and, hence, its intracellular transport and processing . The secreted active site mutant precursors, endocytosed by galactosialidosis fibroblasts, restore beta-galactosidase and neuraminidase activities as effectively as wild-type protective protein . These findings indicate that the catalytic activity and protective function of the protective protein are distinct. Mol Cell Biol, 1991 Aug, 11(8), 4053 - 64 Dominant inhibitory Ras mutants selectively inhibit the activity of either cellular or oncogenic Ras; Stacey DW et al.; Two dominant inhibitory Ras mutant proteins were analyzed by microinjection . One, {Asn-17}Ras, had a substitution in the putative Mg(2+)-binding site of Ha-Ras . The other, RAST, had a mutation in a yeast RAS protein that impaired its GTPase activity and increased its affinity for GAP . RAST also had a mutation that blocked its localization to the plasma membrane . In NIH 3T3 cells {Asn-17}Ras inhibited the function of normal Ras much more efficiently than that of oncogenic Ras . In contrast, RAST interfered with the transforming activity of oncogenic Ras more efficiently than that of normal Ras . These conclusions were based on two separate types of analysis . The inhibitory Ras mutant proteins were first microinjected into cells stably transformed either by oncogenic Ras or by high levels of expression of cellular Ras . Results obtained in stably transformed cells were then verified by coinjection of the inhibitory Ras mutant proteins together with transforming concentrations of either oncogenic or normal Ras protein . Whereas RAST was active in soluble form . {Asn-17}Ras required membrane localization for activity . Furthermore, mutations in the GAP/effector-binding domain reduced or eliminated the inhibitory activity of RAST but had no detectable effect on {Asn-17}Ras . These results are consistent with the possibility that {Asn-17}Ras functions by blocking the activation of endogenous Ras proteins, while RAST functions by blocking the ability of activated Ras to stimulate a downstream target within the cells . The properties of RAST suggest that interference with the GAP/effector-binding function of RAS represents a strategy for the preferential inactivation of oncogenic Ras in cells. Mol Cell Biol, 1991 Aug, 11(8), 3850 - 9 Mapping of replication initiation sites in mammalian genomes by two-dimensional gel analysis: stabilization and enrichment of replication intermediates by isolation on the nuclear matrix; Dijkwel PA et al.; Two complementary two-dimensional gel electrophoretic techniques have recently been developed that allow initiation sites to be mapped with relative precision in eukaryotic genomes at least as complex as those of yeast and Drosophila melanogaster . We reported the first application of these mapping methods to a mammalian genome in a study on the amplified dihydrofolate reductase (DHFR) domain of the methotrexate-resistant CHO cell line CHOC 400 (J.P . Vaughn, P.A . Dijkwel, and J.L . Hamlin, Cell 61:1075-1087, 1990) . Our results suggested that in this 240-kb domain, initiation of nascent DNA strands occurs at many sites within a 30- to 35-kb zone mapping immediately downstream from the DHFR gene . In the course of these studies, it was necessary to develop methods to stabilize replication intermediates against branch migration and shear . This report describes these stabilization methods in detail and presents a new enrichment protocol that extends the neutral/neutra