Epidemiol Mikrobiol Imunol, 1994 May, 43(2), 75 - 8 {Comparison of susceptibility to Legionnaires' disease in small laboratory animals}; Spalekova M et al.; The authors tested under laboratory conditions the susceptibility of five species of small laboratory animals (guinea pigs, rats, syrian and chinese hamsters and mice) to legionella infection after intraperitoneal inoculation with 10(8) cells of nine strains of legionellae of the Legionella pneumophila species . The suitability of the animal model was evaluated on the basis of clinical manifestations of the infection, pathological macroscopic changes and detection of legionellae from animal organs on CYE (Charcoal-Yeast Extract) media . The results of the analysis of experimental animal infection revealed that guinea pigs are the most suitable experimental animals for investigations and the diagnosis of legionellosis. Plant J, 1994 May, 5(5), 735 - 44 Analysis of clones carrying repeated DNA sequences in two YAC libraries of Arabidopsis thaliana DNA; Schmidt R et al.; YAC clones carrying repeated DNA sequences from the Arabidopsis thaliana genome have been characterized in two widely used Arabidopsis YAC libraries, the EG library and the EW library . Ribosomal, chloroplast and the paracentromeric repeat sequences are differentially represented in the two libraries . The coordinates of YAC clones hybridizing to these sequences are given . A high proportion of EG YAC clones were classified as containing chimaeric inserts because individual clones carried unique sequences and repetitive sequences originating from different locations in the genome . None of the EW YAC clones analysed were chimaeric in this way . YAC clones carrying tandemly repeated sequences, such as the paracentromeric or rDNA sequences, exhibited a high degree of instability . These observations need to be taken into account when using these libraries in the development of a physical map of the Arabidopsis genome and in chromosome walking experiments. Plant Mol Biol, 1994 May, 25(2), 271 - 81 Molecular features, processing and import of the Rieske iron-sulfur protein from potato mitochondria; Emmermann M et al.; The mitochondrial iron-sulfur protein (also termed Rieske iron-sulfur protein) of cytochrome c reductase was purified from potato tubers and identified with heterologous antibodies . The sequences of the N-terminus of this 25 kDa protein and of an internal peptide were determined to design oligonucleotide mixtures for screening a cDNA library . One class of cDNA clones containing an open reading frame of 265 amino acids was isolated . The encoded protein contains the peptide sequences of the 25 kDa protein and shares about 50% sequence identity with the Rieske iron-sulfur proteins from fungi and around 43% with those from mammals . In vitro transcription and translation of the cDNA reveals that the iron-sulfur protein is made as a larger precursor of 30 kDa which is processed by the cytochrome c reductase/processing peptidase complex from potato . The processing product obtained after in vitro processing has the same size as the mature protein imported into isolated mitochondria . The presequence, which targets the protein to the organelle, is 53 amino acids long and has molecular features different from those found in presequences of fungal iron-sulfur proteins, which are processed in two steps . Our results indicate that, unlike in yeast and Neurospora, the presequence of the iron-sulfur protein from potato is removed by a single processing enzyme in one step. Plant Mol Biol, 1994 May, 25(2), 217 - 27 Differential accumulation of S-adenosylmethionine synthetase transcripts in response to salt stress; Espartero J et al.; NaCl stress causes the accumulation of several mRNAs in tomato seedlings . An upregulated cDNA clone, SAM1, was found to encode a S-adenosyl-L-methionine synthetase enzyme (AdoMet synthetase) . Expression of the cDNA SAM1 in a yeast mutant lacking functional SAM genes resulted in high AdoMet synthetase activity and AdoMet accumulation . We show that tomato plants contain at least four SAM isogenes . Clones corresponding to isogenes SAM2 and SAM3 have also been isolated and sequenced . They encode predicted polypeptides 95% and 92% identical, respectively, to the SAM1-encoded AdoMet Synthetase . RNA hybridization analysis showed a differential response of SAM genes to salt and other stress treatments . SAM1 and SAM3 mRNAs accumulated in the root in response to NaCl, mannitol or ABA treatments . SAM1 mRNA accumulated also in leaf tissue . These increases of mRNA level were apparent as soon as 8 h after the initiation of the salt treatment and were maintained for at least 3 days . A possible role for AdoMet synthetases in the adaptation to salt stress is discussed. Biophys Chem, 1994 May, 50(1-2), 169 - 81 Effects of DNA topology in the interaction with histone octamers and DNA topoisomerase I; Negri R et al.; Several simple proteins and complex protein systems exist which do not recognize a defined sequence but--rather--a specific DNA conformation . We describe experiments and principles for two of these systems: nucleosomes and eukaryotic DNA topoisomerase I . Evidences are summarized that describe the effects of negative DNA supercoiling on nucleosome formation and the influence of DNA intrinsic curvature on their localization . The function of the DNA rotational information in nucleosome positioning and in the selection of multiple alternative positions on the same helical phase are described . This function suggests a novel genetic regulatory mechanism, based on nucleosome mobility and on the correlation between in vitro and in vivo positions . We observe that the same rules that determine the in vitro localization apply to the in vivo nucleosome positioning, as determined by a technique that relies on the use of nystatin and on the import of active enzymes in living yeast cells . The sensitivity of DNA topoisomerase I to the topological condition of the DNA substrate is reviewed and discussed taking into account recent experiments that describe the effect of the DNA tridimensional context on the reaction . These topics are discussed in the following order: (i) Proteins that look for a consensus DNA conformation; (ii) Nucleosomes; (iii) Negative supercoiling and nucleosomes; (iv) DNA curvature/bending and nucleosomes; (v) Multiple positioning; (vi) Multiple nucleosomes offer a contribution to the solution of the linking number paradox; (vii) Rotational versus translational information; (viii) A regulatory mechanism; (ix) DNA topoisomerase I; (x) DNA topoisomerase I and DNA supercoiling: a regulation by topological feedback; (xi) DNA topoisomerase I and DNA curvature; (xii) The in-and-out problem in the accessibility of DNA information; (xiii) The integrating function of the free energy of supercoiling. Eur J Cancer B Oral Oncol, 1994 May, 30B(3), 186 - 90 Occurrence of corrugated white patch lesions on lateral border of tongue in lymphoma patients during cytostatic treatment; Laine PO; The question of whether or not there was an association between immunosuppression and occurrence of corrugated white patch lesions on the lateral border of the tongue was studied in 79 patients being treated for non-Hodgkin lymphoma or Hodgkin's disease . The mouths of 55 patients (mean age 47.8 years, 34 males, 21 females) were examined during periods of chemotherapy . All patients were HIV-seronegative . White non-removable lesions on the lateral margins of the tongue were noted in 27 patients (42.8%) 74 days after commencement of chemotherapy and 10 days after termination of medication . In 12 cases (44.4%) the lesions were bilateral . Epstein-Barr virus (EBV) DNA was found by gene amplification using polymerase chain reaction (PCR) in one of the two biopsy samples taken . No white lesion on the lateral border of tongue had been seen in any patient before treatment, nor were any evident 1 year after treatment . Leucocyte counts were significantly (P = 0.001) lower when the lesion was present than when it was not detected . Before chemotherapy, 70.4% of patients with lesions and 47.6% of patients without lesions had positive salivary yeast cultures . Yeasts could be cultured from the saliva of 80.5% of patients when the lesions were present . In 2 patients clinical oral candidiasis was diagnosed at the time of the lesion . The study revealed a correlation between the occurrence of corrugated white, non-removable lesions of the lateral borders of the tongue, high salivary yeast counts and leucocytopenia.(ABSTRACT TRUNCATED AT 250 WORDS) Genomics, 1994 May 1, 21(1), 144 - 9 Gene order is conserved within the human chromosome 21 linkage group on mouse chromosome 10; Irving NG et al.; One hundred progeny from each of two intersubspecific mouse backcrosses were used to construct a comparative genetic map of a region of mouse chromosome 10 (MMU10) that is homologous to the distal tip of the long arm of human chromosome 21 (HSA21) . The analysis included five genes and three simple sequence repeat markers, two of which flanked the HSA21-homologous cluster on either side . Analysis of 200 backcross progeny detected at least one crossover between each pair of adjacent genes and demonstrated that the proximal to distal orientation of the cluster was reversed between human and mouse . The order was determined to be Fyn-1-D10Mit20-S100b-Col6a1-Itgb2-Pfkl+ ++/D10Mit7-D10Mit11 . Comparative mapping supports the order of corresponding markers on HSA21 determined using pulsed-field gel electrophoresis and radiation hybrid line data . However, sequence tagged site content mapping of human yeast artificial chromosomes (YACs) yielded conflicting data on the relative positions of human COL6A1 and S100B on HSA21 . This discrepancy was resolved here by demonstrating that several key YACs used in the human contig analysis were mistyped for S100B . The murine map reported here provides a scaffold for construction of physical maps and yeast artificial chromosome contigs that will be useful in the development of mouse models for the study of Down syndrome. Hum Mol Genet, 1994 May, 3(5), 735 - 40 BLOCK-based PCR markers to find gene family members in human and comparative genome analysis; D'Esposito M et al.; Degenerate primer pairs that include consensus sequences of evolutionary conserved portions of protein families (BLOCKs or ancient conserved regions) can be used to screen by polymerase chain reaction (PCR) for cognate cDNAs and YACs through much of phylogeny . Nine such primer pairs were developed, and five with sites on human chromosomes 7 or X were shown to identify YACs from chromosome-specific locations, including a candidate for a new zinc finger gene in Xq28 . When linked to contig-based genomic maps, such BLOCK-based PCR assays may provide a route to recover the members and study the development of families containing up to 40% of genes, in genomes as diverse as humans, nematodes, and yeast. Rev Inst Med Trop Sao Paulo, 1994 May-Jun, 36(3), 225 - 30 {Comparison between CSF samples from AIDS and non AIDS patients with neurocryptococcosis}; dos Reis-Filho JB et al.; Neurocryptococcosis was a rare nervous system infection . With the rising number of patients with AIDS it became a very frequent disease . This infection is supposed to infect patients with some kind of immunodeficiency and the CSF alterations often simulate tuberculous meningitis . The purpose of this research was to compare the CSF changes in AIDS and non-AIDS patients with meningoencephalitis caused by Cr . neoformans . There were analysed 41 CSF samples from non-AIDS patients with neurocryptococcosis and 23 CSF samples from AIDS patients with neurocryptococcosis . The results of this research allowed to conclude that the inflammatory changes in the CSF from AIDS patients showed a lower intensity compared to those non-AIDS patients . These results showed as well, that the CSF samples from non-AIDS patients always revealed some changes besides the yeast cells . In some samples of AIDS patients, however the unique change was the presence of the yeast . It was demonstrated also, that the presence of Cr . neoformans in CSF, not accompanied by any other change, may suggest that is a patient with AIDS . In non-AIDS patients CSF alterations often simulates tuberculous meningitis . However these alterations were rare in AIDS patients . The yeast cells were more numerous in CSF samples from AIDS patients than in those from non-AIDS patients. Hum Mol Genet, 1994 May, 3(5), 779 - 85 A 2.8 Mb YAC contig in 11q12-q13 localizes candidate genes for atopy: Fc epsilon RI beta and CD20; Stafford AN et al.; An important locus for Atopy (familial asthma, hay fever and eczema) has been localized to the 11q12-q13 region with the minimum recombination fraction around the CD20 gene . We have constructed a 2.8 megabase (Mb) Yeast Artificial Chromosome (YAC) contig of the candidate region using 15 STSs . A total of seven genes have been mapped within this interval in the order cen-OSBP-TCN1-GIF-Fc epsilon RI beta-CD20-CD5-PGA-q(ter) and can be covered by a minimum of eight YAC clones . Contig integrity was assayed with fluorescence in-situ hybridization (FISH) and the mapping of YAC ends on somatic cell and radiation hybrid panels . A long range restriction map of the contig has been constructed to establish the order of and distance between loci . Two promising candidates for the atopy locus, the beta subunit of the high affinity immunoglobulin E receptor (Fc epsilon RI beta) and CD20, a molecule involved in B cell differentiation, have been placed within the contig. Anal Biochem, 1994 May 1, 218(2), 436 - 43 Electrochemically active DNA probes: detection of target DNA sequences at femtomole level by high-performance liquid chromatography with electrochemical detection; Takenaka S et al.; Electrochemically active DNA probes were prepared by linking a ferrocene unit with 5'-aminohexyl-terminated oligonucleotides . The DNA sequences of probes 5a, 5b, and 5c were 5'-T12-3', 5'-T20-3', and 5'-TGCAG TTCCG GTGGC TGATC-3', respectively . Probe 5a could form a complex selectively with a single-strand poly(A) and a double-strand DNA fragment containing an A13 sequence and these complexes could be detected at femtomole levels by an electrochemical detector (ECD) on HPLC . The observed ECD response was proportional to the amount of the complex over the range 20-100 fmol . Probe 5c was capable of detecting femtomole levels of a restriction DNA fragment having oncogene v-myc . Moreover, probe 5b was able to detect picogram levels of mRNA taken from rat brain or yeast total cellular RNA . This proves that the electrochemically active DNA probes are useful in analyzing traces of DNA and RNA carrying the complementary sequence. Genes Chromosomes Cancer, 1994 May, 10(1), 26 - 9 Deletion of a common region on the long arm of chromosome 6 in acute lymphoblastic leukaemia; Menasce LP et al.; We have characterised a region of deletion on the long arm of chromosome 6 (6q) in six cases of acute lymphoblastic leukaemia, by fluorescence in situ hybridisation, using a series of YAC clones which map to 6q . Conventional cytogenetic analysis of four of these cases had been interpreted as showing terminal deletions of 6q . We demonstrated by FISH that in all cases the deletions were interstitial . D6S246 (6q16.3) was the only marker which was missing in all six cases, indicating a common region of deletion between the markers M6P1 at 6q14-15 and FYN at 6q21 . Our results suggest the presence of a tumour suppressor gene within this interval. Cell Tissue Res, 1994 May, 276(2), 213 - 21 Cytokeratin 18 is an M-cell marker in porcine Peyer's patches; Gebert A et al.; The intermediate filaments of the dome epithelium of porcine Peyer's patches were studied by immunohistochemistry . The labelling patterns of monospecific antibodies directed against cytokeratins 8, 18 and 19 differed considerably . About 40% of the dome epithelial cells were intensely labelled by three different anti-cytokeratin 18 antibodies, indicating that large amounts of cytokeratin 18 are present in these cells . In order to verify that these cytokeratin-18-immunoreactive cells were M-cells, uptake studies using fluorescein-labelled yeast particles were performed . Numerous yeast particles were found exclusively in dome epithelial cells that were highly positive for cytokeratin 18, thus representing M-cells . In contrast, the content of cytokeratin 19 in M-cells was lower than that in neighbouring enterocytes . The labelling intensity of cytokeratin 8 did not differ between M-cells and enterocytes . In addition, the absence of vimentin and desmin from the dome epithelium of porcine Peyer's patches was demonstrated . The results show (1) that porcine M-cells differ from enterocytes in the composition of their cytoskeleton, (2) that cytokeratin 18 is a useful marker for detecting porcine M-cells and (3) that this marker directly correlates with M-cell function. Biochem Biophys Res Commun, 1994 Apr 29, 200(2), 966 - 72 Strand scission in DNA induced by S-nitrosothiol with hydrogen peroxide; Park JW et al.; Strand breaks can be produced in pBluescript plasmid DNA by S-nitrosothiol and H2O2 in the presence of a metal chelator, diethylenetriaminepentaacetic acid . S-Nitrosothiols with a wide range of stability were found to be active as a DNA cleaver in the presence of H2O2 . Strand breaks were temperature dependent, occurring more rapidly at higher temperature . Sodium azide and mannitol inhibited S-nitrosothiol/H2O2-induced strand breaks in DNA . Catalase inhibited damage to DNA in a concentration-dependent manner whereas both superoxide dismutase and a yeast protector protein did not prevent damage to DNA . It is suggested that observed strand breaks in DNA are mediated by hydroxyl radicals arising from the reaction between H2O2 and thiyl radicals generated by homolytic decomposition of S-nitrosothiol. Proc Natl Acad Sci U S A, 1994 Apr 26, 91(9), 3593 - 7 A recombinant bisphosphoglycerate mutase variant with acid phosphatase homology degrades 2,3-diphosphoglycerate; Garel MC et al.; To date no definite and undisputed treatment has been found for sickle cell anemia, which is characterized by polymerization of a deoxygenated hemoglobin mutant (HbS) giving rise to deformed erythrocytes and vasoocclusive complications . Since the erythrocyte glycerate 2,3-bisphosphate (2,3-DPG) has been shown to facilitate this polymerization, one therapeutic approach would be to decrease the intraerythrocytic level of 2,3-DPG by increasing the phosphatase activity of the bisphosphoglycerate mutase (BPGM; 3-phospho-D-glycerate 1,2-phosphomutase, EC 5.4.2.4) . For this purpose, we have investigated the role of Gly-13, which is located in the active site sequence Arg9-His10-Gly11-Glu12-Gly13 in human BPGM . This sequence is similar to the Arg-His-Gly-Xaa-Arg* sequence of the distantly related acid phosphatases, which catalyze as BPGM similar phosphoryl transfers but to a greater extent . We hypothesized that the conserved Arg* residue in acid phosphatase sequences facilitates the phosphoryl transfer . Consequently, in human BPGM, we replaced by site-directed mutagenesis the corresponding amino acid residue Gly13 with an Arg or a Lys . In another experiment, we replaced Gly13 with Ser, the amino acid present at the corresponding position of the homologous yeast phosphoglycerate mutase (D-phosphoglycerate 2,3-phosphomutase, EC 5.4.2.1) . Mutation of Gly13 to Ser did not modify the synthase activity, whereas the mutase and the phosphatase were 2-fold increased or decreased, respectively . However, replacing Gly13 with Arg enhanced phosphatase activity 28.6-fold, whereas synthase and mutase activities were 10-fold decreased . The presence of a Lys in position 13 gave rise to a smaller increase in phosphatase activity (6.5-fold) but an identical decrease in synthase and mutase activities . Taken together these results support the hypothesis that a positively charged amino acid residue in position 13, especially Arg, greatly activates the phosphoryl transfer to water . These results also provide elements for locating the conserved Arg* residue in the active site of acid phosphatases and facilitating the phosphoryl transfer . The implications for genetic therapy of sickle cell disease are discussed. J Mol Biol, 1994 Apr 22, 238(1), 128 - 30 Crystallization and preliminary X-ray analysis of phenol hydroxylase from Trichosporon cutaneum; Enroth C et al.; Recombinant phenol hydroxylase from the soil yeast Trichosporon cutaneum has been crystallized with PEG 4000 as precipitant . The crystals are monoclinic, space group P2(1) with cell dimensions a = 101.8 A, b = 153.0 A, c = 116.0 A and beta = 114.8 degrees . The crystal asymmetric unit most likely contains two dimers of phenol hydroxylase corresponding to a packing density in the crystals of 2.54 A 3/Da . The self-rotation function is consistent with the packing of two dimers in the asymmetric unit . The observed diffraction pattern extends beyond 2.8 A resolution and the crystals are well suited for structural analysis by X-ray diffraction methods. Eur J Biochem, 1994 Apr 15, 221(2), 847 - 54 Inhibition and activation studies on sheep liver sorbitol dehydrogenase; Lindstad RI et al.; Reversible inhibition and activation, as well as protection against affinity labelling with DL-2-bromo-3-(5-imidazolyl)propionic acid, of sheep liver sorbitol dehydrogenase have been studied . The results presented are discussed in terms of enzyme active-site properties and may have potential applications for drug design . Kinetics with mainly sorbitol competitive inhibitors reveals that aliphatic thiols are generally the most potent inhibitors of enzyme activity . Inhibition and inactivation by heterocyclics parallel that seen previously with sorbitol dehydrogenase from other sources as well as with alcohol dehydrogenase from yeast . However, there are significant differences in relation to the structurally similar horse liver alcohol dehydrogenase, as the catalytic zinc of sorbitol dehydrogenase is more easily removed by chelating molecules . Several aldose reductase inhibitors are shown to also inhibit sorbitol dehydrogenase, but at concentrations unlikely to be reached clinically . Enzyme activation has been observed with various compounds, in particular halo-alcohols and detergents . Several inhibitors provide competitive protection against enzyme inactivation by DL-2-bromo-3-(5-imidazolyl)propionic acid . This enables the dissociation constants for binary enzyme-inhibitor complexes to be determined . NADH protects noncompetitively against inactivation . The presence of some binary and ternary enzyme-NADH complexes is indicated from fluorescence emission spectra, as a shift in the fluorescence maximum and intensity is observed due to their formation. Biochem Biophys Res Commun, 1994 Apr 15, 200(1), 202 - 5 The putative fifth human serum amyloid A protein (SAA)-related gene "SAA5" is defined by SAA3; Sellar GC et al.; The four well characterised members of the human serum amyloid A protein (SAA) gene family are clustered on chromosome 11p15.1 . The acute phase SAA genes, SAA1 and SAA2, are hyperinducible in response to inflammatory stimuli, whereas SAA4 is only minimally induced, and SAA3 is a pseudogene . We recently demonstrated that the GSAA4 sequence, reported by others (Sack, G.H . Jr . and Talbot, C.C . Jr., 1992 . Biochem . Biophys . Res . Comm . 183, 362-366), and misidentified as corresponding to the SAA4 locus, maps to the 11p15 region and speculated that it may be in close proximity to a distinct fifth SAA locus: "SAA5" . In this report we have used vectorette PCR in combination with direct sequencing and computer based homology searches of the nucleotide sequence databases to establish that the putative fifth SAA-related locus, "SAA5", is defined by SAA3 and therefore does not represent a distinct SAA gene. Structure, 1994 Apr 15, 2(4), 293 - 308 The sequence, crystal structure determination and refinement of two crystal forms of lipase B from Candida antarctica; Uppenberg J et al.; BACKGROUND: Lipases constitute a family of enzymes that hydrolyze triglycerides . They occur in many organisms and display a wide variety of substrate specificities . In recent years, much progress has been made towards explaining the mechanism of these enzymes and their ability to hydrolyze their substrates at an oil-water interface . RESULTS: We have determined the DNA and amino acid sequences for lipase B from the yeast Candida antarctica . The primary sequence has no significant homology to any other known lipase and deviates from the consensus sequence around the active site serine that is found in other lipases . We have determined the crystal structure of this enzyme using multiple isomorphous replacement methods for two crystal forms . Models for the orthorhombic and monoclinic crystal forms of the enzyme have been refined to 1.55 A and 2.1 A resolution, respectively . Lipase B is an alpha/beta type protein that has many features in common with previously determined lipase structures and other related enzymes . In the monoclinic crystal form, lipid-like molecules, most likely beta-octyl glucoside, can be seen close to the active site . The behaviour of these lipid molecules in the crystal structure has been studied at different pH values . CONCLUSION: The structure of Candida antarctica lipase B shows that the enzyme has a Ser-His-Asp catalytic triad in its active site . The structure appears to be in an 'open' conformation with a rather restricted entrance to the active site . We believe that this accounts for the substrate specificity and high degree of stereospecificity of this lipase. Proc Natl Acad Sci U S A, 1994 Apr 12, 91(8), 3151 - 5 Mutational analysis of the N-terminal topogenic signal of watermelon glyoxysomal malate dehydrogenase using the heterologous host Hansenula polymorpha; Gietl C et al.; We have studied the significance of the N-terminal presequence of watermelon (Citrullus vulgaris) glyoxysomal malate dehydrogenase {gMDH; (S)-malate:NAD+ oxidoreductase; EC 1.1.1.37} in microbody targeting . The yeast Hansenula polymorpha was used as heterologous host for the in vivo expression of various genetically altered watermelon MDH genes, whose protein products were localized by immunocytochemical techniques . It is shown that the presequence of gMDH is essential and sufficient for peroxisomal targeting; it can target the mature part of the mitochondrial MDH to microbodies, whereas deletion of the presequence results in accumulation of the mature form of gMDH in the cytosol . Alignment of the N termini of several peroxisomal proteins that are assumed to contain a peroxisomal targeting signal at the N terminus (PTS2) suggested the consensus seqence RL-X5-HL . A similar motif is present in the presequence of watermelon gMDH--namely, 10RI-X5-17HL . Mutational analysis revealed that substitutions of 10RI into DD or 17HL into DE destroyed the topogenic information, whereas substitutions of 25M into I and 26EE into LV did not . By combining our data with recent analyses of others on the presequences of mammalian thiolases, it is concluded that the peroxisomal targeting information of PTS2 is contained in the consensus sequence RL/I-X5-HL . In contrast to the higher plant and mammals, the Hansenula yeast peroxisomes seem to lack an enzyme capable of removing microbody presequences of higher eukaryotes. Gene, 1994 Apr 8, 141(1), 145 - 6 A gene from the hypotrichous ciliate Stylonychia lemnae coding for a protein with homology to cyclin B; Maercker C et al.; A nucleotide (nt) sequence of a DNA molecule from Stylonychia lemnae with an open reading frame encoding a protein showing homology to cyclin B has been determined . The DNA molecule is 3791-nt long and the deduced 444-amino-acid (aa) sequence shares about 30% identity with the sequences of two yeast cyclin-B homologs over a length of about 210 aa. Proteins, 1994 Apr, 18(4), 390 - 3 Lobster enolase crystallized by serendipity; Duquerroy S et al.; An unknown protein crystallized from a lobster muscle preparation in which arginine kinase was the majority component . It was identified as enolase by peptide sequencing and activity testing, and a SIRAS electron density map showed its three-dimensional structure to be very similar to that of yeast enolase. Curr Opin Obstet Gynecol, 1994 Apr, 6(2), 184 - 9 Fertilization and implantation; Zilberstein M et al.; Recent advances in seemingly remote areas of investigation, i.e . yeast cell cycle research and DNA amplifications, have opened spectacular avenues for understanding reproduction . The new insights on the single cell and subcellular level of processes, such as egg maturation, sperm-egg interaction and implantation enhance, immensely, the power of assisted fertilization . These techniques, have become the mainstay of infertility therapy . This review focuses on the recent developments in these areas. Am J Physiol, 1994 Apr, 266(4 Pt 1), L319 - 53 Transgenic models for the study of lung biology and disease; Ho YS; Transgenic models provide a means of understanding the molecular mechanisms for the temporal, spatial, and stimulus-responsive regulation of gene expression in vivo and importantly the pathophysiological consequences of the altered expression of a normal or mutated gene . To facilitate the application of transgenic models in lung research, this review describes several practical considerations in generation of transgenic mice . The potential of transgenic models in lung research is also illustrated by depicting the current models in lung research including those for understanding lung gene regulation, tumorigenesis, mutation detection, antioxidant defense, emphysema, fibrosis, and hypertension . The impact of important new development of producing transgenic mice carrying large fragments of DNA contained in yeast artificial chromosomes to achieve proper control of transgene expression and gene targeting technology is also discussed . It is anticipated that transgenic models will provide invaluable information in future lung research. Jikken Dobutsu, 1994 Apr, 43(2), 199 - 207 {Feeding experiment on laboratory-bred male cynomolgus monkeys . II . Hematological and serum biochemical studies}; Yoshida T et al.; The effects of restricted feeding on hematological and serum biochemical values were studied in laboratory-bred male cynomolgus monkeys (Macaca fascicularis) aged between one year and four years . In animals younger than 2.5 years old, the amount of commercial diet (Type AS, Oriental Yeast Co . Ltd.) given per day was restricted to 50 g (group A), 30 g (group B) or 20 g (group C) and increased to 100 g (group A), 50 g (group B) or 40 g (group C) at 2.5 years old . Throughout the experimental period, about 100 g per day of apples and oranges were fed to the animals . Differences in red blood cell counts, hematocrit values and hemoglobin concentrations between groups A and C were detected in the middle period of the experiment . The function of hematogenesis may be suppressed by restricted feeding . A significant increase in serum triglyceride concentration in group C and slight increase in group B were observed during the second trimester of the experiment . A decrease in serum alkaline phosphatase activities in group C during the same period suggests that restricted feeding has a suppressive effect on bone growth . The results obtained by hematological and serum biochemical observations in this study are in good agreement with the previous somatometric study {10}. Jikken Dobutsu, 1994 Apr, 43(2), 173 - 80 {A feeding experiment on laboratory-bred male cynomolgus monkeys . I . Morphometrical study}; Shimizu T et al.; Effects of restricted feeding on the growth of laboratory-bred male cynomolgus monkeys (Macaca fascicularis) aged between one year and four years were studied morphometrically . A series of 11 variables representing physical elements were measured . Before 2.5 years of age, the amount of commercial diet (Type AS, 382kcal/100g and 28.1% of crude protein, Oriental Yeast Co . Ltd.) per day was restricted to 50g (group A), 30g (group B) or 20g (group C) and increased to 100g (group A), 50g (group B) or 40g (group C) respectively at 2.5 years old . Throughout the experimental period ca 100g of apples and oranges per day were provided . Significant differences in body weight between group A and C were detected excepting the early period of the experiment . Although the slight suppression in weight gain was observed in group B during the second trimester of the experiment, body weight increased gradually after increasing the amount of food and no significant difference from A was observed at four years of age . The minimum requirement of diets is judged to be 30g/day before two years old and 50g/day over two years old in the laboratory-bred male cynomolgus monkey . Moreover, the suppressive effects of restricted feeding were most significant on the growth of the limbs and, secondarily, on the growth of the trunk . Practically no effect on the growth of the head was observed. J Parasitol, 1994 Apr, 80(2), 225 - 31 In vitro culture of equine strongylidae to the fourth larval stage in a cell-free medium; Chapman MR et al.; An efficient and reliable method is described for the culture of equine strongyles from the third (L3) to the fourth (L4) larval stage . Medium consists of 50% fetal calf serum and 50% NCTC with additions of L-glutamine, NaHCO3, yeast extract, bactopeptone, and dextrose . The gas phase used is of prime importance; it is a mixture of 10% CO2, 5% O2, and 85% N2 . Strongylus vulgaris, Strongylus edentatus, Strongylus equinus, Triodontophorus brevicauda, Triodontophorus serratus, Triodontophorus tenuicollis, Oesophagodontus robustus, Cylicocyclus insigne, and mixed species of cyathostomes were cultured to the L4 stage . Oesophagodontus robustus was cultured to the fifth larval stage . Depending on species, 44-95% of Strongylinae L3 inoculated into this system molted to L4 . Although some development of the Cyathostominae L3 occurred, only a small portion (1%) completed ecdysis to L4 . Viability in cultures of all species remained high (> 60-70% larvae surviving) for at least 4 wk (cyathostomes) and as long as 6 mo (S . edentatus) . The addition of equine hemin to cultures of S . vulgaris and O . robustus L4 enhanced development and prolonged viability of these larvae . Hemin had no effect on cultures of S . edentatus or S . equinus, and it was not tested in cultures of other species. EMBO J, 1994 Apr 1, 13(7), 1620 - 7 Pheromones trigger filamentous growth in Ustilago maydis; Spellig T et al.; Cell recognition and mating in the smut fungus Ustilago maydis have been proposed to involve specific pheromones and pheromone receptors . The respective structural genes are located in the a mating type locus that exists in the alleles a1 and a2 . We demonstrate that binding of pheromone to the receptor can induce a morphological switch from yeast-like to filamentous growth in certain strains . Using this as biological assay we were able to purify both the a1 and a2 pheromone . The structure of the secreted pheromones was determined to be 13 amino acids for a1 and nine amino acids for a2 . Both pheromones are post-translationally modified by farnesylation and carboxyl methyl esterification of the C-terminal cysteine . An unmodified a1 peptide exhibits dramatically reduced activity . The pheromone alone is able to induce characteristic conjugation tubes in cells of opposite mating type and confers mating competence; even cells of the same mating type undergo fusion . We discuss the role of pheromones in initiating filamentous growth and pathogenic development. Arterioscler Thromb, 1994 Apr, 14(4), 534 - 41 The human apolipoprotein(a)/plasminogen gene cluster contains a novel homologue transcribed in liver; Byrne CD et al.; Lipoprotein(a) is an atherogenic lipoprotein whose function and plasma concentration reflect the structure and regulation of the apolipoprotein(a) gene . Apolipoprotein(a) is a close homologue of plasminogen, and their genes are tightly linked on chromosome 6 . To further characterize these genes, we analyzed overlapping human genomic yeast artificial chromosome clones, which revealed a cluster of four highly homologous genes encoding apolipoprotein(a), plasminogen, and two apolipoprotein(a)-related genes (rg) or pseudogenes . Hybridization analysis and reverse transcriptase polymerase chain reaction showed that one of these novel genes, designated apolipoprotein(a)rg-C, has a domain structure similar to apolipoprotein(a) and is transcribed in human liver . Three additional homologues designated as plasminogen-related genes are shown to be unlinked to this gene cluster and reside on chromosomes 2 and 4. J Biol Chem, 1994 Apr 1, 269(13), 9493 - 9 ATP-dependent chaperoning activity of reticulocyte lysate; Schumacher RJ et al.; We have developed an assay for chaperone-mediated protein renaturation using thermally denatured Firefly luciferase . Dilution of denatured luciferase (> 99% loss of activity) into reticulocyte lysate typically results in recovery of 5-15% activity . Addition of an ATP-regenerating system increases yields to > 60%, while heat shock or the addition of denatured proteins inhibits the chaperoning activity . Reticulocyte lysate contains abundant quantities of the heat shock proteins, hsp90 and hsp70, and a 60-kDa protein homologous to the yeast stress protein, STI1 . Immune isolated samples of these three proteins support recovery of up to 35% of luciferase activity in an ATP-dependent manner, suggesting that these or associated proteins are involved in the renaturation of luciferase . Furthermore, we observed a correlation between luciferase renaturation activity and the levels of hsp70 and hsp90 in reticulocyte lysate preparations . Purified hsp90 and hsp70, along with an ATP-regenerating system, are able to renature luciferase to greater than 20% of its original activity . This renaturation is most efficient when hsp90 and hsp70 are at about a 2:1 ratio and at concentrations similar to those found in reticulocyte lysate . This study provides evidence for an ATP-dependent chaperoning activity in reticulocyte lysate that involves a cooperative action of hsp70 and hsp90. Blood, 1994 Apr 1, 83(7), 1922 - 8 Detection of chimeric BCR-ABL genes on bone marrow samples and blood smears in chronic myeloid and acute lymphoblastic leukemia by in situ hybridization; Bentz M et al.; The presence of BCR-ABL fusion genes has important diagnostic and prognostic implications in chronic myeloid leukemia (CML) and acute lymphoblastic leukemia (ALL) . The CML-specific chimeric BCR-ABL gene with a break involving the major breakpoint cluster region (M-bcr) of the BCR-gene has been detected by means of fluorescence in situ hybridization (FISH) . In this study, we present a FISH protocol that allows the detection of breaks in both the major and the minor breakpoint cluster region (m-bcr) . Three hybridization signals of D107F9, a yeast artificial chromosome (YAC)-derived probe spanning the breakpoint regions of the BCR gene, were indicative of the translocation events . To increase the specificity further, this probe was combined with cos-abl 8, a cosmid probe flanking the breakpoint within the ABL gene for dual-color hybridization . Samples of 21 patients with CML, the ALL-derived cell line SUP-B15, and of seven patients with Philadelphia chromosome (Ph1)-positive ALL (three of them with breakpoints within m-bcr) were examined . BCR-ABL fusion was detected in all cases with high specificity (false-positive nuclei: mean, 0.1%) . On cytogenetic preparations, the percentages of BCR-ABL-positive interphase cells ranged from 53% to 91% . Comparable efficiencies were achieved on blood smears . In conclusion, hybridization with D107F9 and cos-abl 8 allows unambiguous diagnosis of BCR-ABL genes and is likely to become an important tool for the monitoring of therapies in patients with CML and ALL. Am J Hum Genet, 1994 Apr, 54(4), 687 - 94 Use of genetic and physical mapping to locate the spinal muscular atrophy locus between two new highly polymorphic DNA markers; Clermont O et al.; The gene for autosomal recessive forms of spinal muscular atrophy (SMA) has recently been mapped to chromosome 5q13, within a 4-cM region between the blocks D5S465/D5S125 and MAP-1B/D5S112 . We identified two new highly polymorphic microsatellite DNA markers--namely, AFM265wf5 (D5S629) and AFM281yh9 (D5S637)--which are the closest markers to the SMA locus . Multilocus analysis by the location-score method was used to establish the best estimate of the SMA gene location . Our data suggest that the most likely location for SMA is between locus D5S629 and the block D5S637/D5S351/MAP-1B/D5S112/D5S357 . Genetic analysis of inbred SMA families, based on homozygosity by descent and physical mapping using mega-YACs, gave additional information for the loci order as follows: cen-D5S6-D5S125/D5S465-D5S435-D5S629-SMA-+ ++D5S637-D5S351-MAP-1B/D5S112-D5S357- D5S39-tel . These data give the direction for bidirectional walking in order to clone this interval and isolate the SMA gene. J Biochem (Tokyo), 1994 Apr, 115(4), 693 - 700 Improving cytochrome c function by protein engineering?: studies of site-directed mutants of the human protein; Wallace CJ et al.; We have expressed the gene for human cytochrome c, and six mutants of the native sequence, in yeast defective in its own iso-1-cytochrome c gene . All constructs support strong growth in strict aerobic metabolism, and substantial amounts of protein could be extracted from permeabilized cells . The purified analogs, Cys14Ala, Gly37Arg, Arg38Lys, Arg38Gly, Gly84Ser, and Thr28Ile,Gly84Ser, were examined for changes in functional properties, since the majority of these residues are strongly or absolutely conserved . Indeed, although growth rates of the host yeast strains were very similar, there was great divergence in both physicochemical and biological properties, which have been rationalized in terms of changes to the stability of the cytochrome fold, and to the dipole moment of the protein . Interestingly, although modification of electrostatic properties in some mutants can apparently produce a twofold increase in electron transfer efficiency, such changes are not evolutionarily acceptable . The "improvement" is illusory . We suggest that an associated decrease in the stability of the heme crevice offsets any advantage of increased transfer rates. Hum Mol Genet, 1994 Apr, 3(4), 629 - 33 High resolution ordering of YAC contigs using extended chromatin and chromosomes; Haaf T et al.; The general usefulness of fluorescence in situ hybridization (FISH) for the physical mapping of the human genome is greatly enhanced by improved DNA resolution . Several techniques have been described for decondensing or stretching interphase chromatin in a linear fashion, allowing long-range FISH mapping in finer detail . Highly extended linear chromatin can be hybridized over physical distances of at least several megabases, possibly over the whole length of a chromosome . By multi-color FISH, we have determined the order and overlaps of YACs from a 5q34-35 contig . Cosmids can be localized within larger YAC clones . Extended chromatin mapping can be applied as an adjunct ordering technique in genome studies. Nat Genet, 1994 Apr, 6(4), 379 - 83 Long-range mapping of gaps and telomeres with RecA-assisted restriction endonuclease (RARE) cleavage; Ferrin LJ et al.; RecA-assisted restriction endonuclease (RARE) cleavage is a method to perform sequence-specific cleavage of genomic DNA, and is useful in physical mapping studies . After making two modifications, we have applied this method to mapping large regions of DNA in several cell types, including a notorious gap near the Huntington disease (HD) locus on chromosome 4 . RARE cleavage fragments were analysed by pulsed field gel electrophoresis and Southern blotting and the distances between cleavage sites determined with accuracy . Using RARE cleavage, the gap measured was less than 60 kilobases in length . RARE cleavage is also a straightforward technique to map the distance from a marker to a telomere . The terminal 1.7 megabases of several HD and control cell lines were mapped with no large differences between cell lines in this region. Mol Biol Cell, 1994 Apr, 5(4), 413 - 21 Direct interaction of v-Src with the focal adhesion kinase mediated by the Src SH2 domain; Xing Z et al.; The recently described focal adhesion kinase (FAK) has been implicated in signal transduction pathways initiated by cell adhesion receptor integrins and by neuropeptide growth factors . To examine the mechanisms by which FAK relays signals from the membrane to the cell interior, we carried out a series of experiments to detect potential FAK interactions with proteins containing Src homology 2 (SH2) domains that are important intracellular signaling molecules . Using v-Src-transformed NIH3T3 cells, we showed that FAK was present in the immune-complex precipitated by anti-Src antibody, suggesting potential interaction of FAK with v-Src in vivo . We also showed potentially direct interaction of FAK with v-Src in vivo using the yeast two-hybrid system . Using recombinant FAK expressed in insect cells and bacterial fusion proteins containing Src SH2 domains, we showed direct binding of FAK to the Src SH2 domain but not to the SH3 domain in vitro . A kinase-defective mutant of FAK, which is not autophosphorylated, did not interact with the Src SH2 domain under the same conditions, suggesting the involvement of the FAK autophosphorylation sites . Treatment of FAK with a protein-tyrosine phosphatase decreased its binding to the Src SH2 domain, whereas autophosphorylation in vitro increased its binding . These results confirm the importance of FAK autophosphorylation sites in its interaction with SH2 domain-containing proteins . Taken together, these results suggest that FAK may mediate signal transduction events initiated on the cell surface by kinase activation and autophosphorylation that result in its binding to other key intracellular signaling molecules. J Biol Chem, 1994 Apr 1, 269(13), 9590 - 7 Structure and organization of mouse GlcNAc-1-phosphate transferase gene; Rajput B et al.; The gene encoding UDP-GlcNAc:dolichol phosphate N-acetylglucosamine-1-phosphate transferase (GPT), the enzyme that initiates the pathway for the biosynthesis of asparagine-linked glycoproteins, was isolated and characterized . Southern blot analyses demonstrated a single copy gene for GPT . The gene spans about 7.5 kilobase pairs of DNA and is divided into 9 exons by 8 introns . All the introns are found in the coding region, and most of them occur in segments separating the putative membrane-spanning domains . The exon/intron organization of the gene also correlates with the presence of several highly conserved regions of potential functional importance among yeast, leishmania, hamster, and mouse enzymes . Primer extension and reverse transcription-polymerase chain reaction analyses suggested the presence of several potential transcription start sites, with the closest one being approximately 200 base pairs upstream from the translation initiation codon . The 5'-flanking region lacks a typical TATA box, but is high in GC content and contains two putative Sp1 binding sites (GC boxes), consistent with promoters described for housekeeping genes . The 3'-end reverse transcription-polymerase chain reaction analysis indicated that the first of the two polyadenylation sites was used predominantly, in agreement with a approximately 2.0-kilobase pair GPT message seen on Northern blots of RNA from a wide variety of mouse tissues . This is the first report of cloning of a gene for an enzyme of the dolichol cycle in higher eukaryotes . A novel finding of this study is the observation of a G-->A change between the genomic sequence and nucleotide 280 in the cDNA . This could have important implications as an RNA editing mechanism for regulating the expression of the gene and therefore, protein N-glycosylation . A previous study (11) had shown that the activity of GPT was developmentally regulated in mouse mammary gland, with possible involvement by the hormone prolactin . The availability of the GPT gene with its promoter should facilitate future studies on delineating the mechanism for the hormonal regulation of GPT. Radiother Oncol, 1994 Apr, 31(1), 1 - 13 The molecular basis for cell cycle delays following ionizing radiation: a review; Maity A et al.; Exposure of a wide variety of cells to ionizing (X- or gamma-) irradiation results in a division delay which may have several components including a G1 block, a G2 arrest or an S phase delay . The G1 arrest is absent in many cell lines, and the S phase delay is typically seen following relatively high doses (> 5 Gy) . In contrast, the G2 arrest is seen in virtually all eukaryotic cells and occurs following high and low doses, even under 1 Gy . The mechanism underlying the G2 arrest may involve suppression of cyclin B1 mRNA and/or protein in some cell lines and tyrosine phosphorylation of p34cdc2 in others . Similar mechanisms are likely to be operative in the G2 arrest induced by various chemotherapeutic agents including nitrogen mustard and etoposide . The upstream signal transduction pathways involved in the G2 arrest following ionizing radiation remain obscure in mammalian cells; however, in the budding yeast the rad9 gene and in the fission yeast the chk1/rad27 gene are involved . There is evidence indicating that shortening of the G2 arrest results in decreased survival which has led to the hypothesis that during this block, cells repair damaged DNA following exposure to genotoxic agents . In cell lines examined to date, wildtype p53 is required for the G1 arrest following ionizing radiation . The gadd45 gene may also have a role in this arrest . Elimination of the G1 arrest leads to no change in survival following radiation in some cell lines and increased radioresistance in others . It has been suggested that this induction of radioresistance in certain cell lines is due to loss of the ability to undergo apoptosis . Relatively little is known about the mechanism underlying the S phase delay . This delay is due to a depression in the rate of DNA synthesis and has both a slow and a fast component . In some cells the S phase delay can be abolished by staurosporine, suggesting involvement of a protein kinase . Understanding the molecular mechanisms behind these delays may lead to improvement in the efficacy of radiotherapy and/or chemotherapy if they can be exploited to decrease repair or increase apoptosis following exposure to those agents. Genomics, 1994 Apr, 20(3), 463 - 7 Organization of the human annexin V (ANX5) gene; Cookson BT et al.; We characterized the region of human chromosome 4q26-q28 that contains the gene encoding annexin V (placental anticoagulant protein I), a member of a family of calcium-dependent phospholipid binding proteins . A total of 14.5 kb, containing 9 introns, could be directly amplified from genomic DNA; the remainder was characterized from genomic clones in phage lambda and a yeast artificial chromosome . The gene was mapped with restriction enzymes BamHI, EcoRI, HindIII, SacI, StuI, and XbaI; the transcribed region spans 28 kb and contains 13 exons (44 tp 530 bp in size) and 12 introns (0.23 to 8.8 kb in size) . Several putative transcription factor binding sites are present in the 5'-region, but the promoter has no recognizable TATA box . This study will facilitate further analysis of the functions of annexin V and its role in disease. Genomics, 1994 Apr, 20(3), 341 - 6 A 2-Mb YAC contig encompassing three loci (DXF34, DXS14, and DXS390) that lie between Xp11.2 translocation breakpoints associated with incontinentia pigmenti type 1; Reed V et al.; We demonstrate that all the repeat elements representing the conserved loci DXF34 and DXS390 lie between the X;9 and the X;17 translocation breakpoints associated with incontinentia pigmenti type 1 (IP1) . Sequence-tagged sites (STSs) at DXF34S1, DXS14, and DXS390 have been used to isolate YAC clones containing these loci, and a contig of approximately 2 Mb has been constructed . Patterns of hybridization observed in the YAC clones indicate that DXS390 comprises two distinct regions (A and B) . The STS at DXS390 detects the A region and includes a polymorphic CA repeat (PIC = 0.25) . This expansion of the cloned region around DXF34 and DXS390 will enable the isolation of additional conserved sequences that will help in understanding both the lesions underlying the pathogenesis of IP1 and the size and extent of the man-mouse homologous block defined by DXF34. Curr Opin Genet Dev, 1994 Apr, 4(2), 298 - 304 Pre-mRNA splicing; Newman AJ; Information from yeast and mammalian pre-mRNA splicing systems has advanced our understanding of the roles of protein factors in the early steps of spliceosome assembly . New results on the stereochemistry of nuclear pre-mRNA splicing and data on the transposition of Group II self-splicing introns in vivo have fuelled the long-running debate on the evolution of introns and RNA splicing. Curr Opin Genet Dev, 1994 Apr, 4(2), 236 - 44 The role of activators in assembly of RNA polymerase II transcription complexes; Hori R et al.; The past year has provided new insights into the biochemical mechanism of gene activation . Key discoveries include the finding that TFIIA plays an important regulatory role in transcription complex assembly, the TBP-associated factors are direct targets of at least two classes of activator, and a largely pre-assembled transcription complex has been isolated from yeast cells, challenging the step-wise assembly pathway . This review also presents an update on the argument that TFIIB is the target of VP16 and insights into the energetic role of ATP in RNA polymerase II initiation. Protein Eng, 1994 Apr, 7(4), 531 - 5 Variability within the Candida rugosa lipases family; Lotti M et al.; Several fungi secrete lipase isozymes differing in biochemical properties and in some cases in substrate specificity . In the yeast Candida rugosa, a family of related genes encodes for multiple lipase proteins, highly homologous in sequence but partially different in the regions interacting with the substrate molecule . Analysis of these substitutions performed on the basis of multiple alignments and using a 3-D model of the enzyme, allows identification of a restricted number of amino acids possibly involved in substrate specificity of Candida lipases. Plant Physiol, 1994 Apr, 104(4), 1359 - 70 Structural organization of the spinach endoplasmic reticulum-luminal 70-kilodalton heat-shock cognate gene and expression of 70-kilodalton heat-shock genes during cold acclimation; Anderson JV et al.; The 70-kD heat-shock proteins (HSP70s) are encoded by a multigene family in eukaryotes . In plants, the 70-kD heat-shock cognate (HSC70) proteins are located in organellar and cytosolic compartments of cells in most tissues . Previous work has indicated that HSC70 proteins of spinach (Spinacia oleracea) are actively synthesized during cold-acclimating conditions . We have isolated, sequenced, and characterized cDNA and genomic clones for the endoplasmic reticulum (ER) luminal HSC70 protein (immunoglobulin heavy chain-binding protein; BiP) of spinach . The spinach ER-luminal HSC70 is a constitutively expressed gene consisting of eight exons . Spinach BiP mRNA appears to be up-regulated during cold acclimation but is not expressed during water stress or heat shock . In contrast to the differential regulation of mRNA, the ER-luminal HSC70 protein levels remain constant in response to various environmental stresses . Two other members of the spinach 70-kD heat-shock (HS70) multigene family also show differential expression in response to a variety of environmental stresses . A constitutively expressed cytosolic HSC70 protein in spinach appears also to be up-regulated in response to both cold-acclimating and heat-shock treatments . Spinach also contains a cold-shock-induced HS70 gene that is not expressed during heat shock or water stress . Since HSP70s are considered to be involved with the chaperoning and folding of proteins, the data further support the concept that they may be important for maintaining cellular homeostasis and proper protein biogenesis during cold acclimation of spinach. J Trop Pediatr, 1994 Apr, 40(2), 104 - 7 Female children respond to recombinant hepatitis B vaccine with a higher titre than male; Fang JW et al.; One-hundred and eighty Chinese children {age range 5 months to 12 years, seronegative for all hepatitis B virus (HBV) markers} of parents seropositive for HBV surface antigen (HBsAg) were randomized to receive doses of either 10 or 20 micrograms of recombinant yeast-derived HBV vaccine at intervals of 0, 1, and 6 months . Six children defaulted and three other children (1.7 per cent) seroconverted to anti-HBc positivity without detectable HBsAg in the serum . All other children attained an anti-HBs titre of > 10 mlU/ml after three doses . Both 10 and 20 micrograms/dose regime gave a similar geometric mean titre (GMT) of anti-HBs . Children aged 0-4 responded with a similar titre compared with children aged > 4 . Female children responded with a significantly higher GMT than male children and this was due to a high proportion of female children with higher peak titres . No major side-effects were encountered . We conclude that recombinant HB vaccine is highly immunogenic, well tolerated and equally effective with doses of both 10 and 20 micrograms and that girls responded with a higher anti-HBs titre compared with boys. Plant J, 1994 Apr, 5(4), 459 - 67 FIL2, an extracellular Leucine-Rich Repeat protein, is specifically expressed in Antirrhinum flowers; Steinmayr M et al.; The expression of the Antirrhinum gene FIL2 is affected in mutants of the homeotic transcription factor DEFICIENS . Northern and Western blot analyses showed that FIL2 in wild-type Antirrhinum flowers is expressed weakly in the petals and more abundantly in the reproductive organs; the gene is active in the filaments and anthers of stamens, and in the stigma and transmitting tissue of the carpels . The FIL2 protein is glycosylated with high mannose type glycan chains and is located in the middle lamella of the extracellular matrix . The amino acid sequence contains 10 tandem repeats, the composition of which is similar to the Leucine-Rich Repeat (LRR) motif found in mammals, Drosophila and yeast . The possibility that FIL2 might be a component of a cellular signalling mechanism, involving LRR-mediated protein-protein interactions is discussed. Genes Dev, 1994 Apr 1, 8(7), 783 - 95 Abl protein-tyrosine kinase selects the Crk adapter as a substrate using SH3-binding sites; Ren R et al.; To understand the normal and oncogenic functions of the protein-tyrosine kinase Abl, the yeast two-hybrid system has been used for identifying proteins that interact with it . One interacting protein is Crk-I, an SH3/SH2-containing adapter protein that was originally identified as the oncogenic element in the avian sarcoma virus CT10 . Direct interaction between the Crk-I SH3 and Abl at novel, approximately 10 amino acid sites just carboxy-terminal to the Abl kinase domain occurs in vitro and in mammalian cells . There is a nearby site specific for binding another adapter, Nck, and these sites also bind Grb-2 . When bound to Abl, Crk-I was phosphorylated on tyrosine . Thus, the SH3-binding sites on Abl serve as substrate recognition sites for the relatively nonspecific kinase of Abl . In Crk-I-transformed cells, Crk-I associates with endogenous c-Abl and is phosphorylated on tyrosine . The association of Crk and Abl suggests that Abl could play a role in v-Crk and Crk-I transformation and that normal Abl function may be partly mediated through bound adapter molecules. Boll Soc Ital Biol Sper, 1994 Apr, 70(4), 83 - 8 Degradation of aromatic compounds by Trichosporon sp; Caselli L et al.; When cultured in liquid media, samples of the yeast Trichosporon grow readily and degrade phenol; glutamate was found to stimulate both fungal growth and phenol catabolism, with a distinctive lag . In addition, this same strain grows in the presence of 2-chloro-phenol and 2-methyl, 4-chlorophenol (which are also degraded) and in the presence of 2-nitrophenol, 4-nitrophenol, 2,4-dinitrophenol, which are either degraded or not, as in the case of 4-nitrophenol . The kinetics of both growth and of aromatic catabolism is suggestive of inductive phenomena for key metabolic enzymes. Neuron, 1994 Apr, 12(4), 707 - 16 The exocytotic fusion pore and neurotransmitter release; Monck JR et al.; Membrane fusion is ubiquitous in biological systems, occurring in the simplest of unicellular eukaryotes as well as higher eukaryotes . As soon as the first primitive eukaryotic cell utilized a lipid bilayer as an outer membrane, membrane fusion (and fission) became necessary for the traffic of material from the outside to the inside, the inside to the outside, and between different intracellular membrane-bounded compartments . The earliest cells would have made use of the intrinsic ability of lipid bilayers to fuse under certain conditions . Although this fusogenic property of bilayers has been known for some time, it is has become clear only relatively recently that two phospholipid bilayers will fuse spontaneously, owing to a hydrophobic force, when the bilayers are brought close together under conditions of membrane tension or high curvature (Helm and Israelachvili, 1993) . The primeval cell would have used proteins to develop the appropriate architecture in which such fusion would occur in a regulated manner . During the course of evolution, ever more sophisticated ways of regulating this basic process would evolve, but the underlying fusion mechanism would remain unchanged . We have proposed that a macromolecular scaffold of proteins is responsible for bringing the plasma membrane close to the secretory granule membranes and creating the architecture that enables the hydrophobic force to cause fusion (Figure 1; Nanavati et al., 1992; Monck and Fernandez, 1992; Oberhauser and Fernandez, 1993) . Evidence is now accumulating that there are several highly conserved families of proteins associated with vesicle fusion events, from yeast to mammalian cells, and with intracellular traffic, as well as with regulated exocytosis and synaptic transmission (Bennett and Scheller, 1993; Sollner et al., 1993; Sudhof et al., 1993) . The molecular structures (or scaffolds) that regulate membrane fusion are likely to contain related proteins and share certain fundamental properties. Infect Agents Dis, 1994 Apr-Jun, 3(2-3), 77 - 84 Cis-acting signals and trans-acting factors involved in influenza virus RNA synthesis; O'Neill RE et al.; Influenza A virus RNA replication and expression is directed from cis-acting sequences present on the viral RNAs with the help of trans-acting factors encoded by the virus . Ribonucleoprotein (RNP) complexes reconstituted from synthetic cDNA-derived RNA and purified viral proteins have facilitated the dissection of these cis-acting signals and trans-acting factors . Prior to these studies influenza viruses and other negative-strand RNA viruses were refractory to molecular genetic manipulations . These reverse genetic studies have helped in defining the promoter and polyadenylation signals required for viral RNA synthesis . Studies involving the use of reconstituted RNP complexes have revealed that the viral proteins PB1, PB2, PA, and the nucleoprotein (NP) are necessary for replication and expression of influenza virus RNA . Inroads have also been made in determining the cellular proteins that participate in influenza virus gene expression and replication . The yeast interactive trap system has been used to identify and clone a gene (NPI-1), which encodes a protein that interacts with the influenza virus NP suggesting that this cellular protein is a trans-acting factor functioning in viral RNA synthesis. Biosci Biotechnol Biochem, 1994 Apr, 58(4), 639 - 43 Formation of beta-galactosyl compounds of arabinosylcytosine in growing culture of Sporobolomyces singularis; Suzuki Y et al.; Two new compounds of 1-beta-D-arabinofuranosylcytosine (ara-C) were found to be produced in a high yield in a culture filtrate of Sporobolomyces singularis, when grown on a medium containing lactose and ara-C . The compounds I and II were obtained as white needle crystals from the culture filtrate by preparative paper chromatography, gel-filtration on Sephadex G-10 and Toyopearl HW-40S, lyophilization, and ethanol treatment . The compounds I and II were identified as 3'-O-(beta-D-galactopyranosyl)-ara-C and 3'-O-{beta-D-galactopyranosyl-(1-->4)-O-beta-D-galactopyranosyl}-ara-C, respectively, on the basis of the various experimental results, viz., elementary analyses, UV, IR, 1H-, and 13C-NMR spectra, and products by hydrolysis with alpha- and beta-galactosidases . Also, the yeast produced a large amount of 3'-O-beta-galactosyl compounds of adenosine and inosine in the culture filtrate when grown on a medium containing lactose and their ribonucleosides. J Biol Chem, 1994 Apr 1, 269(13), 9562 - 7 Role of gamma 87 Gln in the inhibition of hemoglobin S polymerization by hemoglobin F; Adachi K et al.; Previous studies suggested that gamma 87 Gln in hemoglobin (Hb) F is an important site for promoting inhibition of Hb S (alpha 2 beta 2(6 Glu-->Val) polymerization by Hb F . We engineered and isolated the double mutant (Hb alpha 2 beta 2(6 Glu-->Val,87 Thr-->Gln) using a yeast expression system and characterized polymerization properties of this modified tetramer in an effort to clarify the role of Gln at position 87 in inhibiting Hb S polymerization . Electrophoretic mobility and absorption spectra of this double mutant were the same as that of Hb S, while oxygen affinity was higher, and effects of organic phosphates on oxygen affinity were reduced . The deoxy form of the double mutant showed a characteristic delay time prior to polymerization in vitro . The critical concentration for polymerization of the double mutant was about 1.5 times higher than Hb S, and delay and polymerization times were much longer than Hb S at the same hemoglobin concentrations . The logarithmic plot of delay time versus hemoglobin concentration for the double mutant showed a straight line that was intermediate between lines for AS and FS mixtures . These results and those of kinetics of polymerization of Hb S/double mutant mixtures indicate that substitution of Gln for Thr at beta 87 in Hb S prolongs delay time and inhibits polymerization, although the double mutant forms polymers like Hb S. Gene, 1994 Mar 25, 140(2), 239 - 42 Isolation and characterization of the Drosophila melanogaster eIF-2 alpha gene encoding the alpha subunit of translation initiation factor eIF-2; Qu S et al.; Genomic and cDNA clones encoding the Drosophila melanogaster alpha-subunit of translational initiation factor 2 (eIF-2 alpha) were isolated . The D . melanogaster eIF-2 alpha gene encodes a 341 amino-acid (aa) protein that shares 57 and 44% identity to its human and yeast homologues, respectively . The regulatory phosphorylation site at Ser50 is embedded in a segment of 19 conserved aa residues . Analysis of the genomic DNA and cDNA clones indicated that eIF-2 alpha is a single-copy gene and its coding region is interrupted by a 260-bp intron . The D . melanogaster eIF-2 alpha mRNA is 1350 nt in length and is expressed throughout development. J Biol Chem, 1994 Mar 25, 269(12), 9319 - 24 Isolation of a cDNA encoding 5T4 oncofetal trophoblast glycoprotein . An antigen associated with metastasis contains leucine-rich repeats; Myers KA et al.; The monoclonal antibody 5T4 defines a human oncotrophoblast antigen expressed by a variety of carcinomas but with a restricted pattern of expression in normal adult tissues . The 5T4 antigen has been isolated from term placenta as a 72-kDa glycoprotein consisting of a 42-kDa core protein with extensive N-linked glycosylation . A cDNA has been isolated from a human placental library using pools of oligonucleotides based on amino acid sequence obtained from purified 5T4 molecules . The predicted open reading frame encodes a protein of 420 amino acids with a molecular mass of 46 kDa and 8 potential N-glycosylation sites . There are N- and C-terminal hydrophobic segments corresponding to putative signal and membrane anchorage sequences, respectively . Northern analysis has demonstrated a major 2.5-kilobase mRNA present in cell lines serologically reactive with the monoclonal antibody 5T4 . Comparison of the 5T4 protein sequence with current sequence data bases has identified the presence of leucine-rich repeats, which are found in a variety of proteins from yeast, insects, and mammals . The 5T4 antigen expression is strongly associated with metastasis in colorectal and gastric cancer, and, hence, the possible functions of the gene product and its relationship to tumor growth and progression are discussed. J Biol Chem, 1994 Mar 25, 269(12), 9147 - 54 A highly active ubiquinol-cytochrome c reductase (bc1 complex) from the colorless alga Polytomella spp., a close relative of Chlamydomonas . Characterization of the heme binding site of cytochrome c1; Gutierrez-Cirlos EB et al.; The alga Polytomella spp . offers extraordinary advantages in the preparation of mitochondria since it lacks chloroplasts and a cell wall . In this work the mitochondrial bc1 complex from Polytomella spp . was solubilized and purified by ion exchange chromatography . The complex was found to be composed of 10 polypeptides and exhibited high rates of ubiquinol-cytochrome c oxidoreductase activity (> 300 s-1) sensitive to antimycin and myxothiazol . The molecular mass of the bc1 complex from Polytomella spp . was assayed by gel filtration and estimated to be of 256,300 Da . Therefore, this complex exhibits the unique property of behaving as a monomer . Amino-terminal sequencing of cytochrome c1 identified 7 residues, from which a deoxyoligonucleotide was designed . A second deoxyoligonucleotide was constructed based on a highly conserved region of the c1 type cytochromes . With these probes, a fragment of the cytochrome c1 gene was amplified by polymerase chain reaction and sequenced . The deduced sequence of the apoprotein exhibited a consensus binding site CXXCH . The data suggest that the cytochrome c1 from Polytomella spp . differs from other protoctists like Crithidia and Euglena, i.e . it exhibits a heme binding domain structurally related to the bovine, yeast, and Neurospora c1 type cytochromes. J Biol Chem, 1994 Mar 25, 269(12), 9030 - 7 Histidyl phosphorylation and dephosphorylation of P36 in rat liver extract; Motojima K et al.; Protein histidine kinase (Motojima, K., and Goto, S . (1993) FEBS Lett . 319, 75-79) and phosphatase in rat liver extract were characterized . The histidine kinase was recovered mostly in the membrane and the phosphatase in the soluble fraction . The kinase and its substrate 36-kDa protein (P36) were co-solubilized from the membrane under conditions in which most of the other kinases, and their substrate proteins were not solubilized . The solubilized kinase and P36 were co-eluted after high pressure liquid chromatography gel filtration, showing an apparent molecular mass of 70-75 kDa . They were also co-eluted after ion exchange chromatography . These characteristics, together with its complete resistance to genistein, indicate that the rat liver histidine kinase is not cognate to the yeast enzyme (Huang, J., Nasr, M., Kim, Y., and Matthews, H.R . (1992) J . Biol . Chem . 267, 15511-15515) . The phosphatase that dephosphorylates histidyl-phosphorylated P36 was also studied using rat liver subcellular fractions and in vitro phosphorylated P36 as the substrate . The characteristics of the phosphatase, that is, 1) Mg2+ requirement for activity, 2) apparent molecular mass of 45 kDa by high performance liquid chromatography gel filtration, and 3) resistance to 100 microM okadaic acid, suggest that the primary phosphatase active in vitro is protein phosphatase 2C. J Biol Chem, 1994 Mar 25, 269(12), 8623 - 6 In chromaffin cells, the mammalian Sec1p homologue is a syntaxin 1A-binding protein associated with chromaffin granules; Hodel A et al.; Membrane proteins of the synaptic vesicle and the presynaptic plasma membrane together with soluble proteins form a secretory fusion complex conserved from yeast to neurons (Sollner, T., Whiteheart, S . W., Brunner, M., Erdjument-Bromage, H., Geromanos, S., Tempst, P., and Rothman, J . E . (1993) Nature 362, 318-324) . Two of the membrane proteins have been localized in chromaffin cells, which secrete catecholamines stored in chromaffin granules . Syntaxin 1A and 1B are found in a plasma membrane-enriched fraction, whereas synaptobrevin is concentrated on the granules . Recombinant syntaxin 1A has been used in an affinity chromatography assay to isolate syntaxin receptor proteins of the chromaffin granules . Solubilized granule membranes contain a single protein with high affinity for syntaxin 1A . Sequencing revealed partial homology with Sec1p, a hydrophilic yeast protein acting late in the secretory process . Genetic suppressor analyses predicted the interaction of Sec1p with Sso1p, a yeast homologue of syntaxin 1A, and with Sec4p, a homologue of rab3A (Aalto, M., Ronne, H., and Keranen, S . (1993) EMBO J . 12, 4095-4104) . Although rab3A is present on chromaffin granules, we did not detect it bound to syntaxin 1A together with the mammalian Sec1p homologue (mSec1) . The mSec1 peptide sequences are almost identical with respective sequences of a soluble protein, termed Munc-18, reported to be the only brain protein with affinity for recombinant syntaxin 1A (Hata, Y., Slaughter, C . A., and Sudhof, T . C . (1993) Nature 366, 347-351) . The mSec1/Munc-18 may be a receptor protein for syntaxin 1A on the transmitter vesicles mediating their interaction with the plasma membrane in docking and fusion. Biochemistry, 1994 Mar 22, 33(11), 3229 - 36 Human, mouse, and rat calnexin cDNA cloning: identification of potential calcium binding motifs and gene localization to human chromosome 5; Tjoelker LW et al.; Calnexin is a 90-kDa integral membrane protein of the endoplasmic reticulum (ER) . Calnexin binds Ca2+ and may function as a chaperone in the transition of proteins from the ER to the outer cellular membrane . We have purified human calnexin in association with the human interferon-gamma receptor and cloned calnexin cDNA from placenta . Fragments of calnexin have been prepared as glutathione S-transferase fusion proteins and analyzed for their abilities to bind 45Ca2+ and ruthenium red . A subdomain containing four internal repeats binds Ca2+ with the highest affinity . This sequence is highly conserved when compared to calreticulin (a luminal ER protein), an Onchocerca surface antigen, and yeast and plant calnexin homologues . Consequently, this sequence represents a conserved motif for the high-affinity binding of Ca2+, which is clearly distinct from the "E-F hand" motif . An adjacent subdomain, also highly conserved and containing four internal repeats, fails to bind Ca2+ . The carboxyl-terminal, cytosolic domain is highly charged and binds Ca2+ with moderate affinity, presumably by electrostatic interactions . The calnexin amino-terminal domain (residues 1-253) also binds Ca2+, in contrast to the amino-terminal domain of calreticulin, which is relatively less acidic . We have also determined the cDNA sequences of mouse and rat calnexins . Comparison of the known mammalian calnexin sequences reveals very high conservation of sequence identity (93-98%), suggesting that calnexin performs important cellular functions . The gene for human calnexin is located on the distal end of the long arm of human chromosome 5, at 5q35. J Biol Chem, 1994 Mar 18, 269(11), 8234 - 9 Identification of an essential cysteine residue in pyridoxal phosphatase from human erythrocytes; Gao G et al.; Pyridoxal-specific phosphatase purified from human erythrocytes was inactivated by a variety of thiol-specific reagents in a time- and concentration-dependent manner . The presence of pyridoxal phosphate, a substrate, or inorganic phosphate, a competitive inhibitor, protected the enzyme from inactivation . Phosphatase inactivated by disulfide reagents was reactivated by the addition of excess dithiothreitol, indicating that the inactivation was due to formation of a mixed disulfide between the reagent and a free cysteinyl residue at or near the active site of the enzyme . Incorporation of either 1 mol of 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), 0.6 mol of iodo{3H}acetate, or 0.6 mol of N-{3H}ethylmaleimide per mol of subunit led to complete inactivation of the enzyme . High concentration of phosphate prevented the incorporation of DTNB and iodo{3H}acetate . Amino acid analysis of carboxymethylated enzyme and DTNB titration of the denatured phosphatase indicated that there may be only 1 cysteinyl residue per subunit . Modification by iodoacetate did not affect the quaternary structure of the enzyme . The phosphatase modified by iodo{3H}acetate was subjected to trypsin digestion, and the resulting peptides were separated on a reverse phase C18 column . Two radioactive peaks were obtained and contained a peptide with the N-terminal sequence of Ala-Gln-Gly-Val-Leu-Phe-Asp-Cys(Cm)-Asp-Gly-Val-Leu-X-Asn-Gly . Most of the radioactivity was released with Cys(Cm) . These results indicate that the cysteinyl residue in this sequence is at or near the active site and is essential for activity . Residues 5-12 and 15 of this peptide are identical with a sequence of a yeast alkaline p-nitrophenylphosphatase, and the peptide has little homology with other mammalian phosphatases. Science, 1994 Mar 18, 263(5153), 1629 - 31 Coatomer interaction with di-lysine endoplasmic reticulum retention motifs; Cosson P et al.; Although signals for retention in the endoplasmic reticulum (ER) have been identified in the cytoplasmic domain of various ER-resident type I transmembrane proteins, the mechanisms responsible for ER retention are still unknown . Yeast and mammalian ER retention motifs interacted specifically in cell lysates with the coatomer, a polypeptide complex implicated in membrane traffic . Mutations that affect the ER retention capacity of the motifs also abolished binding of the coatomer . These results suggest a role for the coatomer in the retrieval of transmembrane proteins to the ER in both yeast and mammals. Biochim Biophys Acta, 1994 Mar 16, 1205(1), 29 - 38 A glutathione S-transferase (GST) isozyme from broccoli with significant sequence homology to the mammalian theta-class of GSTs; Lopez MF et al.; A novel glutathione S-transferase (GST) was purified from broccoli (Brassica oleracea var . italica) . Partial amino-acid sequencing indicated that the protein shared significant homology with several different plant GSTs from maize, silene, Dianthus, Nicotiana and Triticum, but little homology to yeast (Issatchenkia) GST . One region of the polypeptide near the N-terminal also shared significant homology to a region of rat 5-5, rat 12-12 and human theta-GST (collectively referred to as the theta-GST-class) but little structural homology to the common mammalian cytosolic GSTs (alpha-, mu- or pi-classes) . The broccoli GST was retained on a novel membrane based glutathione affinity matrix and displayed activity towards 1-chloro-2,4-dinitro-benzene (CDNB), a general GST substrate, as well as 4-nitrophenethyl bromide, a marker substrate for the theta-class of GSTs . The characteristics of the broccoli GST potentially define it as a member of the theta-class . This is consistent with the view that the theta-class may have arisen prior to the divergence of animals and plants while the mammalian mu-, pi- and alpha-classes evolved after the two kingdoms were established. Biochem Biophys Res Commun, 1994 Mar 15, 199(2), 862 - 8 Chromosomal assignment of the human SA gene to 16p13.11 and demonstration of its expression in the kidney; Samani NJ et al.; The SA gene is a novel gene of yet unknown function recently implicated in blood pressure regulation in rodent models of genetic hypertension . In this study we have located the human homologue of the SA gene to chromosome 16p13.11, by a combination of fluorescence in-situ hybridization and analysis of somatic cell hybrids carrying different segments of chromosome 16 . This should facilitate investigation of its role in the genetic tendency to hypertension in humans . Increased expression of the gene in the kidney may be the mechanism through which some allelic variants of the gene raise blood pressure in rodent models . In this study we also demonstrate that the SA gene is expressed in human kidneys. Biochem Biophys Res Commun, 1994 Mar 15, 199(2), 706 - 13 A novel heparin-binding protein, HBp15, is identified as mammalian ribosomal protein L22; Fujita Y et al.; A 15kDa-protein (HBp15) was purified from mouse submandibular gland and bovine brain by virtue of its heparin-binding property . The amino acid sequences of mouse and bovine HBp15 showed a high degree of homology to a sea urchin protein encoded by gene called "development specific protein 217." Using reverse transcription-polymerase chain reaction methods, cDNA clones for HBp15 were isolated from submandibular gland mRNA of mouse, human and pig, and sequenced . Database search of HBp15 showed that HBp15 also resembles yeast ribosomal protein YL31 in addition to the 217 protein . Using specific antibodies against HBp15, HBp15 was identified as mammalian ribosomal protein L22, for which no sequence information is available. Biochemistry, 1994 Mar 15, 33(10), 2830 - 7 Crystal structure of recombinant chicken triosephosphate isomerase-phosphoglycolohydroxamate complex at 1.8-A resolution; Zhang Z et al.; The crystal structure of recombinant chicken triosephosphate isomerase (TIM, E.C . 5.3.1.1) complexed with the intermediate analogue phosphoglycolohydroxamate (PGH) has been solved by the method of molecular replacement and refined to an R-factor of 18.5% at 1.8-A resolution . The structure is essentially identical to that of the yeast TIM-PGH complex {Davenport, R . C., et al . (1991) Biochemistry 30, 5821-5826} determined earlier and refined at comparable resolution . This identity extends to the high-energy conformations of the active-site residues Lys13 and Ser211, as well as the positions of several bound water molecules that are retained in the active site when PGH is bound . Comparison with the structure of uncomplexed chicken TIM shows that the catalytic base, Glu165, moves several angstroms when PGH binds . This movement may provide a trigger for a larger conformational change, one of 7 A, in a loop near the active site, which folds down like a lid to shield the bound inhibitor and catalytic residues from contact with bulk solvent . These same conformational changes were seen in crystalline yeast TIM upon binding of PGH; their occurrence here in a different crystal form of TIM eliminates the possibility that they are an artifact of crystal packing. Int J Cancer, 1994 Mar 15, 56(6), 900 - 5 Clofazimine alters the energy metabolism and inhibits the growth rate of a human lung-cancer cell line in vitro and in vivo; Sri-Pathmanathan RM et al.; The anti-leprosy drug Clofazimine is known to inhibit respiratory function and hence energy metabolism in yeast and in transformed fibroblasts . The aim of this study was to examine the effect of Clofazimine on the energy metabolism of a chemoresistant human non-small-cell bronchial-carcinoma cell line (WIL) and to determine whether this agent might inhibit the growth rate of this cell line in vitro and in vivo . Oxidative phosphorylation was estimated in vitro by measuring oxygen consumption polarographically and glycolysis was estimated from lactate production . In cells that had been pre-treated with an ATP synthetase inhibitor (oligomycin), the addition of Clofazimine resulted in an increase in oxygen consumption similar to that observed with 2,4-dinitrophenol, a classical inhibitor of oxidative phosphorylation . This inhibition of mitochondrial function was associated with an increase in lactate production . Cellular ATP levels were maintained, possibly indicating a compensatory increase in ATP production via glycolysis . Clofazimine was shown to have a direct cytotoxic effect in vitro with an ID50 of 10.2 microM . When Clofazimine was administered to athymic mice bearing WIL as a subcutaneous xenograft, tumour growth rate was significantly reduced, so that after 3 weeks, tumour size was one third that of controls (p < 0.01) . These results suggest that selective inhibition of tumour energy metabolism with agents such as Clofazimine is a potential novel approach to cancer treatment. Genomics, 1994 Mar 15, 20(2), 249 - 57 High-resolution genomic mapping of the three human replication protein A genes (RPA1, RPA2, and RPA3); Umbricht CB et al.; Human replication protein A (RPA) is a three-subunit protein that plays a central role in eukaryotic DNA replication, homologous recombination, and excision repair . We have previously reported the cloning and bacterial overexpression of the three RPA genes and have mapped them to chromosome 1 (RPA2), chromosome 7 (RPA3), and chromosome 17 (RPA1) . We have now obtained yeast strains with artificial chromosomes carrying the three human RPA genes and report the more detailed genomic mapping of RPA . RPA1 was mapped to chromosome 17p13.3 using a combination of PCR amplification of somatic cell hybrids and radiation hybrids containing chromosome 17 fragments . RPA2 was mapped to chromosome 1p35 by PCR amplification of somatic cell hybrids of chromosome 1 and by fluorescence in situ hybridization . RPA3 was mapped to chromosome 7p22 by Southern analysis and PCR amplification of somatic cell hybrids of chromosome 7 as well as fluorescence in situ hybridization . Since RPA is an essential component of major metabolic events affecting DNA, the physical mapping of the genes for it may help elucidate the biochemical basis of genetic disorders involving DNA metabolism. Genomics, 1994 Mar 15, 20(2), 231 - 7 Structure of the gene for the testis-specific proprotein convertase 4 and of its alternate messenger RNA isoforms; Mbikay M et al.; Proprotein convertase 4 (PC4) is a mammalian secretory serine endoproteinase similar to the yeast KEX2 gene product and specifically expressed in testicular germs cells . PC4 mRNA isoforms that vary in size and 3' coding sequence have been reported (N . G . Seitah, R . Day, J . Hamelin, A . Gaspar, M . W . Collard, and M . Chretien, 1992, Mol . Endocrinol . 6: 1559-1570) . To determine the origin of these various forms, the mouse PC4 gene was cloned and its organization determined . The structural gene is approximately 9.5 kb long . It contains 15 exons and 14 introns . The exon-intron organization is very similar to that of the genes for the related convertases furin, PC1, and PC2 . The upstream region carries several GGGCGG and three CCAAT but no TATAA motifs . Analysis of the 5' end of PC4 mRNA in the testis has led to the identification of two novel 5' splice variants that might encode a nonsecretory enzyme . The multiple forms of PC4 mRNA can all be explained by alternate splicing of primary transcripts of a single gene. J Biol Chem, 1994 Mar 11, 269(10), 7435 - 8 A single mutation in uncoupling protein of rat brown adipose tissue mitochondria abolishes GDP sensitivity of H+ transport; Murdza-Inglis DL et al.; The uncoupling protein is one of a family of mitochondrial transport proteins involved in energy metabolism . It dissipates oxidative energy to generate heat, either by catalyzing proton transport directly or by catalyzing fatty acid anion transport, thus enabling fatty acids to act as cycling protonophores . This transport process is tightly regulated by purine nucleotides . We have expressed uncoupling protein in yeast and examined its proton transport activity after its reconstitution into proteoliposomes . A directed change of Arg276 to Leu or Gln completely abolished nucleotide inhibition of protonophoretic action of the reconstituted mutant uncoupling proteins without affecting the transport process . Arg276 is the first residue of functional importance to be identified in uncoupling protein . Mutation of the homologous residue in the yeast ADP/ATP translocator prevented the growth of yeast on a nonfermentable carbon source, presumably by interfering with nucleotide exchange (Nelson, D . R., Lawson, J . E., Klingenberg, M., and Douglas, M . G . (1993) J . Mol . Biol . 230, 1159-1170) . Demonstration of the essential role of a single homologous residue in protein-nucleotide interaction within these two transporters is the first direct evidence that uncoupling protein and the ADP/ATP translocator belong to the same gene family. J Biol Chem, 1994 Mar 4, 269(9), 6498 - 505 Regulation of enzymatic activity by active site fatty acylation . A new role for long chain fatty acid acylation of proteins; Berthiaume L et al.; Methylmalonate semialdehyde dehydrogenase (MMSDH) is a mitochondrial enzyme which can be acylated by myristoyl-CoA analogs (Deichaite, I., Berthiaume, L., Peseckis, S . M., Patton, W . F., and Resh, M . D . (1993) J . Biol . Chem . 268, 13788-13747) . Here we describe the mechanisms which mediate regulation of the enzymatic activity of bovine MMSDH by long chain fatty acylation . The substrate specificity of the acylation reaction was measured in vitro using purified MMSDH and the coenzyme A derivative of an 125I-labeled long chain fatty acid (13-iodotridecanoate), an analog of myristoyl-CoA . Long chain fatty acyl CoAs (> 8 carbons) were able to inhibit radiolabeling of MMSDH . In order to study the physiological role of the acylation process in vivo, a system using highly purified mitochondria from COS-1 cells overexpressing MMSDH was exploited . MMSDH was shown to be processed properly, targeted to the mitochondrial fraction, and enzymatically active . The extent of fatty acylation of MMSDH as well as of other mitochondrial proteins was correlated with the mitochondrial energy level . Biochemical evidence as well as site-specific mutagenesis of cysteine 319 revealed that this highly conserved active site cysteine of MMSDH was the target of the fatty acylation . Another member of the aldehyde dehydrogenase family, yeast aldehyde dehydrogenase was also covalently modified by {125I}13-iodotridecanoyl-CoA and thereby inactivated . Furthermore, we demonstrate that glutamate dehydrogenase, an enzyme that has been previously shown to be strongly inhibited by palmitoyl-CoA, is fatty acylated by the 125I-labeled myristoyl-CoA analog . Our data suggest that attachment of long chain fatty acids to proteins is a new and potentially widespread type of enzyme regulation mechanism that we denote active site fatty acylation. J Biol Chem, 1994 Mar 4, 269(9), 6566 - 70 Human serum biotinidase . cDNA cloning, sequence, and characterization; Cole H et al.; Biotinidase (EC 3.5.1.12) catalyzes the hydrolysis of biocytin, the product of biotin-dependent carboxylase degradation, to biotin and lysine . Biotinidase deficiency is an inherited metabolic disorder of biotin recycling that is characterized by neurological and cutaneous abnormalities, and can be successfully treated with biotin supplementation . Sequences of tryptic peptides of the purified human serum enzyme were used to design oligonucleotide primers for polymerase chain reaction amplification from human hepatic total RNA to generate putative biotinidase cDNA fragments . Sequence analysis of a cDNA isolated from a human liver library by plaque hybridization with the largest cDNA probe revealed an open reading frame of 1629 bases encoding a protein of 543 amino acid residues, including 41 amino acids of a potential signal peptide . Comparison of the open reading frame with the known biotinidase tryptic peptides and recognition of the expressed protein encoded by this cDNA by monoclonal antibodies prepared against purified biotinidase demonstrated the identity of this cDNA . Southern analyses suggested that biotinidase is a single copy gene and revealed that human cDNA probes hybridized to genomic DNA from mammals, but not from chicken or yeast . Northern analysis indicated the presence of biotinidase mRNA in human heart, brain, placenta, liver, lung, skeletal muscle, kidney, and pancreas. Plant Mol Biol, 1994 Mar, 24(6), 969 - 72 Sequence of a plant cDNA from Vicia faba encoding a novel Ran-related GTP-binding protein; Saalbach G et al.; A clone obtained from a broad bean (Vicia faba) developing cotyledon cDNA library contained the complete coding sequence of a polypeptide with very high homology to the small GTP-binding proteins Ran from human cells and Spi1 from yeast . These proteins belong to the ras superfamily of proteins involved in different basic cellular processes . The Ran/Spi1 proteins interact with a protein bound to DNA (RCC1) and are thought to function in the regulation of the cell cycle . The amino acid sequence of the obtained plant Ran-homologue, designated Vfa-ran, is 74% and 76% identical to Ran and Spi1, respectively . The five functional, conserved domains of ras-related proteins are present in the Vfa-ran sequence . However, as in Ran/Spi1 the C-terminus of Vfa-ran is very acidic and lacks the Cys motif for isoprenylation . Northern blotting revealed a corresponding mRNA expression in broad bean roots, leaves, and cotyledons with the highest level in roots. Bone Marrow Transplant, 1994 Mar, 13(3), 333 - 4 Native valve endocarditis due to Candida parapsilosis: a late complication after bone marrow transplantation-related fungemia; Cancelas JA et al.; A case of Candida parapsilosis endocarditis observed 16 months after BMT is reported . The patient, a 35-year-old female with CML, suffered from Candida parapsilosis fungemia on day +22 after BMT . In spite of treatment with amphotericin B, fluconazole and catheter withdrawal, the same yeast was isolated > 1 year later from a vegetation on an old rheumatic mitral valve . Although the patient remained in complete cytogenetical and hematological remission, in vitro tests showed reduced phagocytic and chemotactic capacity of neutrophils and monocytes . This case stresses the need of prolonged therapy for patients with candidemia after BMT. Development, 1994 Mar, 120(3), 535 - 44 The Serrate locus of Drosophila and its role in morphogenesis of the wing imaginal discs: control of cell proliferation; Speicher SA et al.; The Drosophila gene Serrate encodes a transmembrane protein with 14 EGF-like repeats in its extracellular domain . Here we show that loss-of-function mutations in this gene lead to larval lethality . Homozygous mutant larvae fail to differentiate the anterior spiracles, exhibit poorly developed mouth-hooks and show a severe reduction in the size of the wing and haltere primordia, which is not due to cell death . The few homozygous mutant escapers that pupariate develop into pharate adults that almost completely lack wings and halteres . Clonal analysis in the adult epidermis demonstrates a requirement for Serrate during wing and haltere development . Targeted ectopic expression of Serrate in the imaginal discs using the yeast transcriptional activator Gal4 results in regionally restricted induction of cell proliferation, e.g . the ventral tissues in the case of the wings and halteres . The results suggest that the wild-type function of Serrate is required for the control of position-specific cell proliferation during development of meso- and metathoracic dorsal discs, which in turn exerts a direct effect on morphogenesis. J Clin Invest, 1994 Mar, 93(3), 944 - 50 17 beta-Estradiol inhibits expression of human interleukin-6 promoter-reporter constructs by a receptor-dependent mechanism; Pottratz ST et al.; We previously reported that 17 beta-estradiol inhibits cytokine-stimulated bioassayable IL-6 and the steady-state level of IL-6 mRNA . To determine the molecular basis of this effect, the transient expression of chloramphenicol acetyltransferase (CAT) reporter plasmid driven by the human IL-6 promoter was studied here in HeLa or murine bone marrow stromal cells (MBA 13.2) . 17 beta-estradiol (10(-8) M) completely suppressed stimulated CAT expression in HeLa cells cotransfected with IL-6/CAT constructs and a human estrogen receptor (hER) expression plasmid; but had no effect on reporter expression in HeLa cells not transfected with hER . 17 beta-estradiol also inhibited stimulated expression in MBA 13.2 cells (which express the estrogen receptor constitutively) without the requirement of cotransfection of the hER plasmid . The hormonal effects were indistinguishable between constructs containing a 1.2-kb fragment of the 5' flanking region of the IL-6 gene or only the proximal 225-bp fragment . However, yeast-derived recombinant hER did not bind to the 225-bp segment in DNA band shift assays, nor did the 225-bp fragment compete for binding of an estrogen response element oligonucleotide to yeast-derived estrogen receptor . These data suggest that 17 beta-estradiol inhibits the stimulated expression of the human IL-6 gene through an estrogen receptor mediated indirect effect on the transcriptional activity of the proximal 225-bp sequence of the promoter. Chemotherapy, 1994 Mar-Apr, 40(2), 92 - 8 In vitro susceptibility of fungal isolates of clinically important specimens to itraconazole, fluconazole and amphotericin B; Dermoumi H; The in vitro activity of itraconazole, fluconazole and amphotericin B was tested against 207 yeast strains and 3 Aspergillus fumigatus strains isolated from blood . The other 42 A . fumigatus strains were selected from respiratory tract infections . A microdilution method was employed to determine the inhibitory concentrations to restrain 70% of isolate growth (IC30) . The inhibition concentrations of amphotericin B are all around the value 0.25 mg/l with a deviation of two concentration degrees, indicating sensitivity of all strains . The test results of itraconazole showed the majority of strains in the range of sensitivity (200 isolates) and low sensitivity (44 isolates) . Five of 19 Candida glabrata strains demonstrated resistance to itraconazole . The results of fluconazole showed a good sensibility of C . albicans, but 4.5% of strains were resistant . Low sensitivity (1 strain) and resistance (24 strains) were evaluated for C . glabrata and C . krusei isolates . No strain of A . fumigatus was inhibited by fluconazole . The data show the necessity of in vitro tests. RN, 1994 Mar, 57(3), 44 - 7 Teaching teens about condoms; Bolus J; PIP: Detailed information was given which would be useful in advising adolescents about condoms and safe sexual practices . The concern is for protection from HIV infection and sexually transmitted diseases . 3% of the HIV-infected and sexually transmitted diseases . 3% of the HIV-infected population are currently adolescents . In teaching about condoms it was stressed that adolescents expect a willingness and openness in maintaining a discussion about all aspects of sex, and if the teacher is uneasy, then another teacher should be presenting the information and instruction . Important points were that AIDS is most likely to be transmitted through anal sex, and that even HIV-infected partners should use condoms, and that condoms should be used for protection regardless of sexual practices . Latex condoms are the most effective in preventing diseases are porous . US-made condoms meet strict standards and have expiration dates clearly marked on the box and on the condom wrapper . The safest are latex condoms with nonoxynol-9 as a lubricant . Latex condoms deteriorate in heat, sunlight, and moisture and should be carried in a breast pocket or purse . Never use condoms from an unsealed package . Instructions for use were provided in a description and in drawings . Females with yeast infections are advised to refrain from sex until the infection is cleared up because of the increased risk of infection; use plenty of spermicide when having sex during yeast infections . Allergies to latex or spermicides can be handled by applying a lambskin condom over the latex one or by applying a condom without spermicidal lubricant over a lubricated one . For a man's allergy, use the lambskin first and then the latex . After ejaculation, withdrawal should be immediate to prevent semen from leaking out . Holding the rim will assure that the condom does not slip off . Three ways of safe condom removal were recommended . Hands and genitals should be washed before putting on a new condom . Nonlubricated male (or female) condoms should be used for oral sex . Food placed on genitals should be free from oils . Biting and nibbling can puncture latex . Lubricated condoms should be used for anal sex . Advice on insertion of female condoms and the warning that effectiveness is unknown was provided . Hum Genet, 1994 Mar, 93(3), 291 - 4 Refining the genetic map for the region flanking the X-linked hypophosphataemic rickets locus (Xp22.1-22.2); Rowe PS et al.; We have screened fourteen kindreds with X-linked hypophosphataemic rickets with four microsatellite markers, viz AFM163yh2, DXS999 (AFM234yf12), DXS443 and DXS365, in order to refine the genetic map flanking the gene, and to define a close flanking interval for the construction of a yeast artificial chromosome (YAC) and cosmid contig . The genetic data were enhanced after the isolation of a large 1.2-megabase YAC derived from AFM163yh2, in which marker DXS274 was present but not DXS365 or DXS443 . Against HYP, DXS365, AFM163yh2 and DXS443 showed no recombinants (Zmax = 18.1, Zmax = 9.9, and Zmax = 16.0 respectively) . DXS999 gave Zmax = 9.6 at 4% recombination and lies distal to HYP but proximal to DXS197 and DXS43 . The disease gene and markers AFM163yh2 and DXS365 are flanked by DXS443 and DXS274 . Combining the genetic and physical data, we are able to propose the following gene marker order: Xptel-DXS43-DXS197-DXS999-DXS443-{(DXS3 65-AFM163yh2), HYP}-DXS274-DXS41-Xcen. Hum Genet, 1994 Mar, 93(3), 229 - 35 Multicolor fluorescence in situ hybridization on metaphase chromosomes and interphase Halo-preparations using cosmid and YAC clones for the simultaneous high resolution mapping of deletions in the dystrophin gene; Tocharoentanaphol C et al.; We report on multicolor fluorescence in situ hybridization protocols for the simultaneous visualization of deletion-prone regions for carrier detection of Duchenne/Becker (DMD/BMD) muscular dystrophy . Cosmid and yeast artificial chromosome (YAC) clones specific for preferentially deleted subregions of the dystrophin gene were labeled differentially and detected with three different fluorochromes using digital imaging microscopy . This approach allows for an assessment of the carrier status of female relatives even in families where no index patient is available . Cosmid and YAC clones, and different probe-generation protocols are compared with respect to their feasibility for carrier detection . The use of histone-depleted interphase nuclei (Halo-preparations) for deletion mapping is demonstrated and shown to have a resolution power of 5 kb. Blood, 1994 Mar 1, 83(5), 1348 - 54 DNA rearrangements proximal to the EVI1 locus associated with the 3q21q26 syndrome; Levy ER et al.; Specific rearrangements involving 3q21 and 3q26 are well documented in acute myeloid leukemia (AML) . Aberrant expression of the Ecotropic virus integration-1 (EVI1) gene, located at 3q26, has been reported in individuals with AML and translocations or inversions of chromosome 3 long arm . We have studied six individuals with AML and inv(3)(q21q26) for disruptions to the EVI1 locus by in situ hybridization and long-range mapping . EVI1 transcripts have been detected in the blast cells of the two individuals available for expression studies . We derived a YAC containing the EVI1 gene and showed that it crossed the 3q26 inversion breakpoints in three of four cases examined . Pulsed field analysis detected aberrant fragments 3' of the EVI1 gene in all six patients . The orientation of the gene was established and the locations of the breakpoints were refined by in situ hybridization using phage clones from this region. Am J Hum Genet, 1994 Mar, 54(3), 526 - 34 A radiation hybrid map of the BRCA1 region; O'Connell P et al.; A locus on chromosome 17q, designated "BRCA1," has been identified as a predisposition gene for breast cancer . A panel of chromosome 17-specific radiation-reduced somatic cell hybrid clones has been assembled for high-resolution mapping of chromosome 17 . A series of 35 markers, known to span the BRCA1 locus, were tested against this hybrid panel by PCR assays . Statistical analysis of these data yields a BRCA1 radiation hybrid map at a density sufficient to initiate YAC cloning and pulsed-field gel electrophoretic mapping of the candidate region . In addition, many of the markers reveal genetic polymorphisms and may be tested in breast cancer families and in loss-of-heterozygosity studies of sporadic breast cancers to better define the BRCA1 gene candidate region. Am J Hum Genet, 1994 Mar, 54(3), 516 - 25 Genetic mapping of the BRCA1 region on chromosome 17q21; Albertsen H et al.; Chromosome 17q21 harbors a gene (BRCA1) associated with a hereditary form of breast cancer . As a step toward identification of this gene itself we developed a number of simple-sequence-repeat (SSR) markers for chromosome 17 and constructed a high-resolution genetic map of a 40-cM region around 17q21 . As part of this effort we captured genotypes from five of the markers by using an ABI sequencing instrument and stored them in a locally developed database, as a step toward automated genotyping . In addition, YACs that physically link some of the SSR markers were identified . The results provided by this study should facilitate physical mapping of the BRCA1 region and isolation of the BRCA1 gene. Mol Biochem Parasitol, 1994 Mar, 64(1), 95 - 110 Expression of heat shock protein 83 in Leishmania is regulated post-transcriptionally; Argaman M et al.; Mechanisms for regulation of heat shock protein (hsp) 83 expression were examined in Leishmania amazonesis . Transcripts of hsp83 accumulated upon temperature elevation; however, in contrast to non-protozoan eukaryotes (i.e . Drosophila, yeast, avian or human cells), no transcriptional activation was observed . The increase in the hsp83 mRNA level evolved from temperature induced variations in mRNA turn-over: the hsp83 transcript was rapidly degraded at normal temperatures, whereas heat shock led to its stabilization . The quick decay of the mRNA at lower temperatures was dependent on active protein synthesis . A similar pattern of regulation was observed for the transfected chloramphenicol acetyltransferase (CAT) gene, which was flanked by sequences from the hsp83 intergenic region (IR), and cloned into the pX transfection vector (pX-ICI) . CAT mRNA was abundant at normal temperatures and further accumulated upon temperature elevation . The altered turn-over rates of CAT mRNA at the different temperatures were observed only in the presence of flanking hsp83 IR sequences . The increase in temperature also affected translational regulation of hsps, and synthesis of hsp83 was more efficient at 35 degrees C than at 26 degrees C . However, the effect of translation was transient, and the steady state level of the protein was hardly altered. Virus Genes, 1994 Mar, 8(2), 151 - 8 Chilo iridescent virus encodes a putative helicase belonging to a distinct family within the "DEAD/H" superfamily: implications for the evolution of large DNA viruses; Sonntag KC et al.; The complete nucleotide sequence of the EcoRI DNA fragment M (7099 bp; 0.310-0.345 map units) of the genome of insect iridescent virus type 6--Chilo iridescent virus (CIV)--was determined . A 606 codon open reading frame located in this region encoded a protein (p69) related to a distinct family of putative DNA and/or RNA helicases belonging to the "DEAD/H" superfamily . Unique sequence signatures were derived that allowed selective retrieval of the putative helicases of the new family from amino acid sequence databases . The family includes yeast, Drosophila, mammalian, and bacterial proteins involved in transcription regulation and in repair of damaged DNA . It is hypothesized that p69 of CIV may be a DNA or RNA helicase possibly involved in viral transcription . A distant relationship was observed to exist between this family of helicases and another group of proteins that consists of putative helicases of poxviruses, African swine fever virus, and yeast mitochondrial plasmids . It is shown that p69 of CIV is much more closely related to cellular helicases than any of the other known viral helicases . Phylogenetic analysis suggested an independent origin for the p69 gene and the genes encoding other viral helicases. Mycopathologia, 1994 Mar, 125(3), 157 - 62 Comparison of four media for the isolation of Aspergillus flavus group fungi; Cotty PJ; Four agar media used to isolate aflatoxin producing fungi were compared for utility in isolating fungi in the Aspergillus flavus group from agricultural soils collected in 15 fields and four states in the southern United States . The four media were Aspergillus flavus and parasiticus Agar (AFPA, 14), the rose bengal agar described by Bell and Crawford (BCRB; 3), a modified rose bengal agar (M-RB), and Czapek's-Dox Agar supplemented with the antibiotics in BC-RB (CZ-RB) . M-RB was the most useful for studying the population biology of this group because it permitted both identification of the greatest number of A . flavus group strains and growth of the fewest competing fungi . M-RB supported an average of 12% more A . flavus group colonies than the original rose bengal medium while reducing the number of mucorales colonies and the number of total fungi by 99% and 70%, respectively . M-RB was successfully employed to isolate all three aflatoxin producing species, A . flavus, A . parasiticus and A . nomius, and both the S and L strains of A . flavus . M-RB is a defined medium without complex nitrogen and carbon sources (e.g . peptone and yeast extract) present in BC-RB . M-RB should be useful for studies on the population biology of the A . flavus group. Mol Microbiol, 1994 Mar, 11(5), 897 - 902 Disruption of two genes for chitin synthase in the phytopathogenic fungus Ustilago maydis; Gold SE et al.; The phytopathogenic fungus Ustilago maydis exhibits a dimorphic transition in which non-pathogenic, yeast-like cells mate to form a pathogenic, filamentous dikaryon . Northern analysis indicated that two chitin synthase genes, chs1 and chs2, from U . maydis are expressed at similar levels in yeast-like cells and in cells undergoing the mating reaction leading to the filamentous cell type . A mutation was constructed in each of the chitin synthase genes by targeted gene disruption . Each mutant showed a reduction in the level of trypsin-activated enzyme activity, compared with a wild-type strain, but retained the wild-type morphology, the ability to mate and the ability to form the filamentous pathogenic cell type. Genomics, 1994 Mar 1, 20(1), 75 - 83 Identification of YAC and cosmid clones encompassing the ZFX-POLA region using irradiation hybrid cell lines; Francis F et al.; The human Xp21.3-p22.1 region is poorly mapped relative to other X chromosome regions . To target cosmid and YAC clones specifically from Xp21.3-p22.1 for rapid contig construction, a hybridization-based screening approach using irradiation hybrids has been used . Alu-PCR products generated from hybrid lines containing small overlapping fragments from Xp21-p22 were hybridized to an X chromosome cosmid library, and cosmids predicted by their hybridization pattern to map to the region of interest were analyzed by fluorescence in situ hybridization (FISH) . Hybridization of the cosmids in pools to gridded YAC libraries identified 15 YACs, which were verified and tested for chime