Yeasts

Proc Natl Acad Sci U S A, 1995 Apr 25, 92(9), 3819 - 23
The hepatitis B virus X protein targets the basic region-leucine zipper domain of CREB; Williams JS et al.; The X gene product encoded by the hepatitis B virus, termed pX, is a promiscuous transactivator of a variety of viral and cellular genes under the control of diverse cis-acting elements . Although pX does not appear to directly bind DNA, pX-responsive elements include the NF-kappa B, AP-1, and CRE (cAMP response element) sites . Direct protein-protein interactions occur between viral pX and the CRE-binding transcription factors CREB and ATF . Here we examine the mechanism of the protein-protein interactions occurring between CREB and pX by using recombinant proteins and in vitro DNA-binding assays . We demonstrate that pX interacts with the basic region-leucine zipper domain of CREB but not with the DNA-binding domain of the yeast transactivator protein Gal4 . The interaction between CREB and pX increases the affinity of CREB for the CRE site by an order of magnitude, although pX does not alter the rate of CREB dimerization . Methylation interference footprinting reveals differences between the CREB DNA and CREB-pX DNA complexes . These experiments demonstrate that pX titers the way CREB interacts with the CRE DNA and suggest that the basic, DNA-binding region of CREB is the target of pX . Transfection assays in PC12 cells with the CREB-dependent somatostatin promoter demonstrate a nearly 15-fold transcriptional induction after forskolin stimulation in the presence of pX . These results support the significance of the CREB-pX protein-protein interactions in vivo.

Proc Natl Acad Sci U S A, 1995 Apr 25, 92(9), 3702 - 6
Expressed cadherin pseudogenes are localized to the critical region of the spinal muscular atrophy gene; Selig S et al.; Low-copy repeats have been associated with genomic rearrangements and have been implicated in the generation of mutations in several diseases . Here we characterize a subset of low-copy repeats in the spinal muscular atrophy (SMA) region in human chromosome 5q13 . We show that this repeated sequence, named c41-cad, is a highly expressed pseudogene derived from an intact neuronal cadherin gene, Br-cadherin, situated on 5p13-14 . Br-cadherin is expressed specifically in the brain, whereas the c41-cad transcripts are 10-15 times more abundant and are present in all tissues examined . We speculate that the c41-cad repeats, separately or in concert with other repeats in the SMA region, are involved in the pathogenesis of SMA by promoting rearrangements and deletions.

FEBS Lett, 1995 Apr 24, 363(3), 299 - 303
Isozyme hybrids within the protruding third loop domain of the barley alpha-amylase (beta/alpha)8-barrel . Implication for BASI sensitivity and substrate affinity; Juge N et al.; Barley alpha-amylase isozymes AMY1 and AMY2 contain three structural domains: a catalytic (beta/alpha)8-barrel (domain A) with a protruding loop (domain B; residues 89-152) that binds Ca2+, and a small C-terminal domain . Different parts of domain B secure isozyme specific properties as identified for three AMY1-AMY2 hybrids, obtained by homeologous recombination in yeast, with crossing-over at residues 112, 116, and 144 . The AMY1 regions Val90-Thr112 and Ala145-Leu161 thus confer high affinities for the substrates alpha-D-maltoheptaoside and amylose, respectively . Leu117-Phe144, and to a lesser degree Ala145-Leu161, are critical for the stability at low pH characteristic of AMY1 and for the sensitivity to barley alpha-amylase/subtilisin inhibitor specific to AMY2.

Proc R Soc Lond B Biol Sci, 1995 Apr 22, 260(1357), 73 - 8
Thermal evolution of growth efficiency in Drosophila melanogaster; Neat F et al.; Drosophila melanogaster shows geographic clines in body size, with genetically larger flies being found further from the equator and at higher altitudes . In the laboratory, evolution at lower temperatures results in genetically larger flies, and development at low temperature increases adult body size . This study demonstrates that when newly hatched larvae from laboratory temperature selection lines were raised on fixed amounts of food (yeast) at the same temperature, larvae from the lines with the cold evolutionary history required less food to produce a given size of adult . Larvae from both high- and low-temperature selection lines required more food, however, to make a given size of adult when grown in the cold than when grown in the hot . The opposite associations between growth efficiency and adult body size seen with evolution or development at low temperature are puzzling, and suggest that different mechanisms may underlie the size changes . Since environmental and evolutionary effects of temperature on body size seem to be widespread among ectotherms, some basic aspects of thermal physiology must be involved.

Cell, 1995 Apr 21, 81(2), 279 - 88
A 20S complex containing CDC27 and CDC16 catalyzes the mitosis-specific conjugation of ubiquitin to cyclin B; King RW et al.; Cyclin B is degraded at the onset of anaphase by a ubiquitin-dependent proteolytic system . We have fractionated mitotic Xenopus egg extracts to identify components required for this process . We find that UBC4 and at least one other ubiquitin-conjugating enzyme can support cyclin B ubiquitination . The mitotic specificity of cyclin ubiquitination is determined by a 20S complex that contains homologs of budding yeast CDC16 and CDC27 . Because these proteins are required for anaphase in yeast and mammalian cells, we refer to this complex as the anaphase-promoting complex (APC) . CDC27 antibodies deplete APC activity, while immunopurified CDC27 complexes are sufficient to complement either interphase extracts or a mixture of recombinant UBC4 and the ubiquitin-activating enzyme E1 . These results suggest that APC functions as a regulated ubiquitin-protein ligase that targets cyclin B for destruction in mitosis.

Cell, 1995 Apr 21, 81(2), 215 - 22
The peptide repeat domain of nucleoporin Nup98 functions as a docking site in transport across the nuclear pore complex; Radu A et al.; We report the cDNA deduced primary structure of a wheat germ agglutinin-reactive nuclear pore complex (NPC) protein of rat . The protein, termed Nup98 (for nucleoporin of 98 kDa), contains numerous GLFG and FG repeats and some FXFG repeats and is thus a vertebrate member of a family of GLFG nucleoporins that were previously discovered in yeast . Immunoelectron microscopy showed Nup98 to be asymmetrically located at the nucleoplasmic side of the NPC . Nup98 functions as one of several docking site nucleoporins in a cytosolic docking activity-mediated binding of a model transport substrate . The docking site of Nup98 was mapped to its N-terminal half, which contains all of the peptide repeats . A recombinant segment of this region depleted the docking activity of cytosol . We suggest that the peptide repeat domain of Nup98, together with peptide repeat domains of other nucleoporins, forms an array of sites for mediated docking of transport substrate, and that bidirectional transport across the NPC proceeds by repeated docking and undocking reactions.

Nature, 1995 Apr 20, 374(6524), 731 - 3
Cloning of a bcl-2 homologue by interaction with adenovirus E1B 19K; Farrow SN et al.; A number of DNA viruses carry apoptosis-inhibiting genes which enable the virus to escape from the host response . The adenovirus E1B 19K protein can inhibit apoptosis induced by E1A, tumour-necrosis factor-alpha, FAS antigen and nerve growth factor deprivation . The molecular basis of this inhibition remains poorly understood, but the fact that protection is seen in the absence of other viral proteins suggests that E1B 19K targets cellular proteins . We report here the identification of three cellular proteins that bind E1B 19K . One of these is a new member of the bcl-2 family, which we have called bak (for bcl-2 homologous antagonist/killer) . This protein, which is expressed in a wide variety of cell types, binds to E1B 19K and to the Bcl-2 homologue Bcl-XL (ref . 17) in yeast . In addition, overexpression of bak in sympathetic neurons deprived of nerve growth factor accelerates apoptosis and blocks the protective effect of co-injected E1B 19K.

Biochemistry, 1995 Apr 18, 34(15), 5259 - 68
Structural and functional effects of multiple mutations at distal sites in cytochrome c; Lo TP et al.; Multiple mutations at distally located sites have been introduced into yeast iso-1 cytochrome c to determine the contributions of three amino acids to the structural and functional properties of this protein . The mutant proteins, for which high-resolution structures were determined, included all possible combinations of the substitutions Arg38Ala, Asn52Ile, and Phe82Ser . Arg38, Asn52, and Phe82 are all conserved in the primary sequences of eukaryotic cytochromes c and have been shown to significantly affect several properties of these proteins including protein stability, heme reduction potential, and oxidation state dependent conformational changes . The present studies show that the structural consequences of each amino acid substitution in combinatorial mutant proteins were similar to those observed in the related single-mutant proteins, and therefore no synergistic effect between mutation sites was observed for this feature . With respect to protein stability, the effect of individual mutations can be understood from the structural changes observed for each . It is found that stability effects of the three mutation sites are independent and cumulative in multiple-mutant proteins . This reflects the independent nature of the structural changes induced at the three distally located mutation sites . In terms of heme reduction potential two effects are observed . For substitution of Phe82 by serine, the mechanism by which reduction potential is lowered is different from that occurring at either the Arg38 or the Asn52 site and is independent of residue replacements at these latter two positions . For Arg38 and Asn52, overlapping interactions lead to a higher reduction potential than expected from a strict additive effect of substitutions at these residues . This appears to arise from interaction of these two amino acids with a common heme element, namely, the heme propionate A group . The present results underscore the difficulty of predicting synergistic effects of multiple mutations within a protein.

FEBS Lett, 1995 Apr 17, 363(1-2), 53 - 6
Oligomannose-coated liposomes as an adjuvant for the induction of cell-mediated immunity; Sugimoto M et al.; The effect of the coating of ovalbumin-reconstituted liposomes with various oligosaccharides on their immunogenicity was investigated in mice . The coating of liposomes with oligomannose or yeast mannan drastically enhanced their ability to induce an ovalbumin-specific delayed-type footpad swelling response with a peak at 24 to 48 h post-challenge . Among various oligosaccharides tested, only those with mannose residue at the nonreducing termini manifested the activity when applied to liposomes . Since such oligosaccharides are ubiquitously found in the body, these results suggested the usefulness of oligomannose-coated liposomes as a safe adjuvant for the induction of cell-mediated immunity.

Genes Dev, 1995 Apr 15, 9(8), 1009 - 19
Tissue-specific regulation of the insulin gene by a novel basic helix-loop-helix transcription factor; Naya FJ et al.; The insulin gene is one of the best paradigms of tissue-specific gene expression . It is developmentally regulated and is expressed exclusively in the pancreatic beta-cell . This restricted expression is directed by a tissue-specific enhancer, within the promoter, which contains an E-box sequence . The insulin E-box binds an islet-specific protein complex, termed 3a1 . E-boxes bind proteins belonging to the basic helix-loop-helix (bHLH) family of transcription factors . The bHLH proteins function as potent transcriptional activators of tissue-specific genes by forming heterodimers between ubiquitous and cell-restricted family members . In addition, the cell-restricted bHLH members play an important role in specifying cell fate . To isolate the tissue-specific bHLH factor controlling insulin gene expression and study its role in islet cell differentiation, a modified yeast two-hybrid system was utilized to clone a novel bHLH factor, BETA2 (beta-cell E-box trans-activator 2), from a hamster insulin tumor (HIT) cell cDNA library . Northern analysis demonstrates that high-level expression of the BETA2 gene is restricted to pancreatic alpha- and beta-cell lines . As expected of tissue-specific bHLH members, BETA2 binds to the insulin E-box sequence with high affinity as a heterodimer with the ubiquitous bHLH factor E47 . More importantly, antibody supershift experiments clearly show that BETA2 is a component of the native insulin E-box-binding complex . Transient transfection assays demonstrate that the BETA2/E47 heterodimer synergistically interacts with a neighboring beta-cell-specific complex to activate an insulin enhancer . In contrast, other bHLH factors such as MyoD and E47, which can bind to the insulin E-box with high affinity, fail to do so . Thus, a unique, cooperative interaction is the basis by which the insulin E-box enhancer discriminates between various bHLH factors to achieve tissue-specific activation of the insulin gene.

Cancer Res, 1995 Apr 15, 55(8), 1752 - 7
Detection and cloning of a common region of loss of heterozygosity at chromosome 1p in breast cancer; Nagai H et al.; The short arm of chromosome 1 is frequently affected by rearrangements in a variety of human malignancies . Genetic alterations, predominantly deletions, which are indicative of the presence of a putative tumor suppressor gene at chromosome 1p, are observed in breast cancer . In order to define the altered locus, eleven highly polymorphic microsatellite markers on chromosome 1p were used to detect loss of heterozygosity . We analyzed 52 cases of breast cancer and found 4 common deleted regions at chromosome 1p . Twenty-two of 52 (42%) informative patients showed at least 1 affected locus . The region most frequently exhibiting loss of heterozygosity was 1p31 (11/39; 28%); the other three common deleted regions were 1p36 (10/44; 23%), 1p35-36 (5/40; 13%), and 1p13 (8/39; 21%) . These data suggest that one or more putative tumor suppressor genes may reside on chromosome 1p . We have cloned the entire region of interest at 1p31 in yeast artificial chromosomes . This yeast artificial chromosome contig can be used for fine mapping of the region and cloning of the candidate tumor suppressor gene.

J Biol Chem, 1995 Apr 14, 270(15), 8506 - 13
The protooncogene c-jun contains an unusual estrogen-inducible enhancer within the coding sequence; Hyder SM et al.; Estrogens have previously been shown to induce c-jun mRNA levels in target cells during hormone induced proliferation, and this appears to be a primary hormonal response involving transcriptional activation . In this report we have now identified an estrogen dependent enhancer within the coding sequence of c-jun . This element has the sequence GCAGAnnnTGACC which is identical to the consensus estrogen response element GGTCAnnnTGACC in the second half site, but varies considerably in the first half site . Synthetic oligodeoxynucleotides containing this jun sequence bind the estrogen receptor in cell-free studies using a competitive band shift assay with the consensus element . The jun element also confers hormone inducibility to reporter plasmids in yeast and mammalian based transcriptional systems . Structure-function studies illustrate that the TGACC half-site and its immediate flanking dinucleotides, but not the GCAGA half-site, are required for estrogen receptor binding . In contrast, both the GCAGA and TGACC half-sites are obligatory for hormone-inducible transcriptional activation . These results suggest a model in which the estrogen receptor functions as a heterodimer to regulate transcription of the c-jun protooncogene . Coupled with reports of estrogen response elements in c-fos and estrogenic induction of c-fos and c-jun in vivo, these findings also support a role for AP-1 components as early response genes in estrogen-induced proliferation.

Science, 1995 Apr 14, 268(5208), 286 - 90
Identification of a dual specificity kinase that activates the Jun kinases and p38-Mpk2; Lin A et al.; One Ras-dependent protein kinase cascade leading from growth factor receptors to the ERK (extracellular signal-regulated kinases) subgroup of mitogen-activated protein kinases (MAPKs) is dependent on the protein kinase Raf-1, which activates the MEK (MAPK or ERK kinase) dual specificity kinases . A second protein kinase cascade leading to activation of the Jun kinases (JNKs) is dependent on MEKK (MEK kinase) . A dual-specificity kinase that activates JNK, named JNKK, was identified that functions between MEKK and JNK . JNKK activated the JNKs but did not activate the ERKs and was unresponsive to Raf-1 in transfected HeLa cells . JNKK also activated another MAPK, p38 (Mpk2; the mammalian homolog of HOG1 from yeast), whose activity is regulated similarly to that of the JNKs.

Biochemistry, 1995 Apr 11, 34(14), 4696 - 701
Identification of sequences mediating guanylyl cyclase dimerization; Wilson EM et al.; Deletion mutagenesis was used to identify sequences required for dimerization and enzymatic activity of the intracellular domain of the membrane guanylyl cyclase, GC-A . The intracellular domain of GC-A contains a protein kinase-like domain near its amino terminus, a guanylyl cyclase catalytic domain near its carboxyl terminus, and, between these domains, a region of unknown function predicted to form an amphipathic alpha-helix . Gel filtration analysis of deletion mutants of the GC-A intracellular domain suggested that a 43 amino acid sequence within the interdomain region was both necessary and sufficient for dimerization and was required for guanylyl cyclase catalytic activity . The ability of this sequence to mediate protein dimerization was confirmed in the yeast two-hybrid system, in which its fusion to the lexA DNA-binding domain and to the VP16 transcriptional activation domain led to their dimerization and consequent activation of a lexA-HIS3 gene . Thus, we have identified sequences responsible for dimerization of the intracellular domain of a guanylyl cyclase and shown that they are required for enzyme activity . Modulation of their interaction may be important in guanylyl cyclase activation.

Genomics, 1995 Apr 10, 26(3), 615 - 8
The human collagenase-3 (CLG3) gene is located on chromosome 11q22.3 clustered to other members of the matrix metalloproteinase gene family; Pendas AM et al.; The gene coding for human collagenase-3 (CLG3), a recently described matrix metalloproteinase produced by breast carcinomas, has been localized by fluorescence in situ hybridization on chromosome 11q22.3 . Physical mapping of an isolated YAC clone containing CLG3 has revealed that this gene is tightly linked to those encoding other matrix metalloproteinases, including fibroblast collagenase (CLG1), stromelysin-1 (STMY1), and stromelysin-2 (STMY2) . Further mapping of this region using pulsed-field gel electrophoresis has shown that the CLG3 gene is localized to the telomeric side of the matrix metalloproteinase cluster, the relative order of the loci being centromere-STMY2-CLG1-STMY1-CLG3-telomere.

Genomics, 1995 Apr 10, 26(3), 543 - 9
A YAC contig containing the reeler locus with preliminary characterization of candidate gene fragments; Bar I et al.; The reeler mutation in the mouse maps to proximal chromosome 5 and defines a key gene involved in brain development and evolution . No gene product is known, and the locus is currently being characterized by positional cloning . YAC clones corresponding to the closest markers D5Mit61 and D5Mit72 have been isolated . Cloned extremities of the YAC inserts were used to construct a 1.1-Mb contig, a 700-kb fragment of which was shown to contain the reeler locus . The integrity of the contig was verified by physical mapping on genomic DNA . The classical allele of the reeler mutation was associated with a 150-kb deletion between D5Mit61 and D5Mit72, while no gross chromosomal anomaly was found in the Orleans allele . Candidate coding sequences were isolated to construct a preliminary transcriptional map of the reeler region . Cosmid clones mapping within the rl deletion revealed a large transcript of more than 11 kb, which was present in normal embryonic brain but barely detectable in homozygous rlOrl/rlOrl embryonic brain, suggesting strongly that it corresponds to the reeler transcript.

Genomics, 1995 Apr 10, 26(3), 502 - 9
YAC contigs mapping the human COL4A5 and COL4A6 genes and DXS118 within Xq21.3-q22; Srivastava AK et al.; Sequence-tagged sites (STSs) were developed for three loci of uncertain X chromosomal localization (DXS122, DXS137, and DXS174) and were used to seed YAC contigs . Two contigs now total about 3.3 Mb formatted with 34 STSs . One contains DXS122 and DXS174 within 250 kb on single YACs; it is placed in Xq21.3-q22.1 by FISH analysis, which is consistent with somatic cell hybrid panel analyses and with the inclusion of a probe that detects polymorphism at the DXS118 locus already assigned to that general region . The other contig, which contains DXS137, is in Xq22.2 by FISH, consistent with cell hybrid analyses and with the finding that it covers the human COL4A5 and COL4A6 genes known to be in that vicinity . In addition to extending the cloned coverage of this portion of the X chromosome, these materials should aid, for example, in the further analysis of Alport syndrome.

Genomics, 1995 Apr 10, 26(3), 489 - 501
High-resolution physical mapping of a 250-kb region of human chromosome 11q24 by genomic sequence sampling (GSS); Selleri L et al.; A physical map of the region of human chromosome 11q24 containing the FLI1 gene, disrupted by the t(11;22) translocation in Ewing sarcoma and primitive neuroectodermal tumors, was analyzed by genomic sequence sampling . Using a 4- to 5-fold coverage chromosome 11-specific library, 22 region-specific cosmid clones were identified by phenol emulsion reassociation hybridization, with a 245-kb yeast artificial chromosome clone containing the FLI1 gene, and by directed "walking" techniques . Cosmid contigs were constructed by individual clone fingerprinting using restriction enzyme digestion and assembly with the Genome Reconstruction and AsseMbly (GRAM) computer algorithm . The relative orientation and spacing of cosmid contigs with respect to the chromosome was determined by the structural analysis of cosmid clones and by direct visual in situ hybridization mapping . Each cosmid clone in the contig was subjected to "one-pass" end sequencing, and the resulting ordered sequence fragments represent approximately 5% of the complete DNA sequence, making the entire region accessible by PCR amplification . The sequence samples were analyzed for putative exons, repetitive DNAs, and simple sequence repeats using a variety of computer algorithms . Based upon the computer predictions, Southern and Northern blot experiments led to the independent identification and localization of the FLI1 gene as well as a previously unknown gene located in this region of chromosome 11q24 . This approach to high-resolution physical analysis of human chromosomes allows the assembly of detailed sequence-based maps and provides a tool for further structural and functional analysis of the genome.

Genomics, 1995 Apr 10, 26(3), 451 - 60
Refined physical map of the spinal muscular atrophy gene (SMA) region at 5q13 based on YAC and cosmid contiguous arrays; Roy N et al.; The gene for the autosomal recessive neurodegenerative disorder spinal muscular atrophy has been mapped to a region of 5q13 flanked proximally by CMS-1 and distally by D5S557 . We present a 2-Mb yeast artificial chromosome (YAC) contig constructed from three libraries encompassing the D5S435/D5S629/CMS-1-SMA-D5S557/D5S112 interval . The D5S629/CMS-1-SMA-D5S557 interval is unusual insofar as chromosome 5-specific repetitive sequences are present and many of the simple tandem repeats (STR) are located at multiple loci that are unstable in our YAC clones . A long-range restriction map that demonstrates the SMA-containing interval to be 550 kb is presented . Moreover, a 210-kb cosmid array from both a YAC-specific and a chromosome 5-specific cosmid library encompassing the multilocus STRs CATT-1, CMS-1, D5F149, D5F150, and D5F153 has been assembled . We have recently reported strong linkage disequilibrium with Type I SMA for two of these STRs, indicating that the gene is located in close proximity to or within our cosmid clone array.

J Biol Chem, 1995 Apr 7, 270(14), 8201 - 8
Purification and characteristics of the candidate prohormone processing proteases PC2 and PC1/3 from bovine adrenal medulla chromaffin granules; Azaryan AV et al.; The prohormone-processing proteases PC1/3 and PC2 belong to the family of mammalian subtilisin-related proprotein convertases (PC) possessing homology to the yeast Kex2 protease . The presence of PC1/3 and PC2 in secretory vesicles of bovine adrenal medulla (chromaffin granules) implicates their role in the processing the precursors of enkephalin, neuropeptide Y, somatostatin, and other neuropeptides that are present in chromaffin granules . In this study, PC1/3 and PC2 were purified to apparent homogeneity from the soluble fraction of chromaffin granules by chromatography on concanavalin A-Sepharose, Sephacryl S-200, pepstatin A-agarose, and anti-PC1/3 or anti-PC2 immunoaffinity resins . PC1/3 and PC2 were monitored during purification by measuring proteolytic activities with 35S-enkephalin precursor and Boc-Arg-Val-Arg-Arg-methylcoumarin amide (MCA) substrates and by following PC1/3 and PC2 immunoreactivity with specific anti-PC1/3 and anti-PC2 sera generated in this study . Purified PC1/3 and PC2 on SDS-polyacrylamide gels each show a molecular mass of 66 kDa . PC2 in the soluble fraction of chromaffin granules was present at 5- and 10-fold higher enzyme protein and activity, respectively, compared with that of PC1/3 . PC1/3 and PC2 cleaved paired basic and monobasic sites within peptide-MCA substrates, with Boc-Arg-Val-Arg-Arg-MCA and pGlu-Arg-Thr-Lys-Arg-MCA as the most effectively cleaved peptides tested . PC1/3 and PC2 showed pH optima of 6.5 and 7.0, respectively . Kinetic studies indicated apparent Km values for hydrolysis of Boc-Arg-Val-Arg-Arg-MCA as 66 and 40 microM, with Vmax values of 255 and 353 nmol/h/mg for PC1/3 and PC2, respectively . Specificity of the PC enzymes for dibasic sites was confirmed by potent inhibition by the active site-directed peptide inhibitors (D-Tyr)-Glu-Phe-Lys-Arg-CH2Cl and Ac-Arg-Arg-CH2Cl . Inhibition by EGTA and activation by Ca2+ indicated PC1/3 and PC2 as Ca(2+)-dependent proteases . In addition, PC enzymes were activated by dithiothreitol and inhibited by thiol-blocking reagents, p-hydroxymercuribenzoate and mercuric chloride . These results illustrate the properties of endogenous PC1/3 and PC2 as prohormone-processing enzymes.

J Biol Chem, 1995 Apr 7, 270(14), 7988 - 92
A conserved region in the amino terminus of DNA polymerase delta is involved in proliferating cell nuclear antigen binding; Zhang SJ et al.; Synthetic peptides to selected sequences in human DNA polymerase delta (pol delta) were used to identify the region involved in the interaction of pol delta to proliferating cell nuclear antigen . Peptides corresponding to sequences in five regions in the amino terminus of human pol delta and three in the carboxyl terminus, which are conserved with the yeast homologs of pol delta, were tested . These studies showed that the peptide corresponding to the N2 region (residues 129-149) selectively and specifically inhibited the PCNA stimulation of pol delta . This inhibition was relieved by titration with excess PCNA . The identification of the N-2 region as being involved in PCNA binding was supported by studies that demonstrated that the N2 peptide could bind PCNA . Deletion mutants of pol delta expressed in Sf9 cells provided evidence that the binding region for PCNA was located in the first 182 residues of the amino terminus . These studies provide reasonable evidence that residues within the region 129-149 of pol delta are involved in the binding site for PCNA.

Biochim Biophys Acta, 1995 Apr 6, 1266(1), 83 - 90
Modulation of the receptor binding affinity of amphiregulin by modification of its carboxyl terminal tail; Adam R et al.; Amphiregulin (AR), a heparin-binding, epidermal growth factor (EGF) receptor ligand has homology with EGF but exhibits a lower affinity for the EGF receptor than EGF . As the mature form of AR is truncated at the C terminus and lacks a conserved leucine residue known to be essential for high affinity binding of EGF to the EGF receptor, wild-type AR (AR1-84), a C-terminally extended AR construct incorporating six residues from the predicted coding sequence of AR (AR1-90) and a similarly extended construct with a Met86 to Leu substitution (AR1-90(leu86)) were expressed as recombinant proteins in yeast, purified by heparin affinity and C18 reverse phase chromatography and their relative biological activities determined . The growth factors were tested in mitogenesis and EGF receptor autophosphorylation assays and their relative order of potencies was found to be leu86 > met86 > wt . The AR1-90(leu86) construct was found to be 50- to 100-fold more active than wild type AR1-84 consistent with previously reported studies of the role of the equivalent C-terminal leucine in EGF or TGF alpha . Significantly, the C-terminally extended form of AR, AR1-90, which utilized six residues from the predicted coding sequence, was 10-times more active than wild type AR1-84 . This difference in activity of the C-terminally extended form of AR may be of biological significance since differential proteolytic processing of the AR precursor in vivo could result in production of multiple forms of the growth factor with differing affinities for the EGF receptor and hence differing biological potencies.

EMBO J, 1995 Apr 3, 14(7), 1520 - 31
Cloning and characterization of hTAFII18, hTAFII20 and hTAFII28: three subunits of the human transcription factor TFIID; Mengus G et al.; We have cloned cDNAs encoding three novel TAFIIs {TATA-binding protein (TBP)-associated factors} from the human (h) HeLa cell TFIID complexes hTAFII28, hTAFII20 and hTAFII18 . hTAFII28 is a core hTAFII present in both of the previously described hTFIID species which either lack or contain hTAFII30 (hTFIID alpha and hTFIID beta respectively), and is the homologue of Drosophila (d)TAFII30 beta . hTAFII18 is a novel hTAFII which shows homology to the N-terminal region of the yeast TAFIISPT3, but has no known Drosophila counterpart . In contrast to hTAFII28, hTAFII18 is a TFIID beta-specific hTAFII . hTAFII20 is the homologue of p22, an alternatively spliced form of dTAFII30 alpha (p32) . Using a combination of protein affinity chromatography and cotransfection and immunoprecipitation assays, we have identified a series of in vitro and intracellular interactions among the novel hTAFIIs and between the novel hTAFIIs and hTAFII30 or TBP . We show that hTAFII28 interacts with hTAFII18 both in vitro and intracellularly; in contrast to its Drosophila homologue, hTAFII28 also interacts directly with TBP . Deletion analysis indicates that TBP and hTAFII18 bind to distinct domains of hTAFII28 . hTAFII18 also interacts with TBP, but it interacts more strongly with hTAFII28 and hTAFII30 . The binding of hTAFII28 and hTAFII30 requires distinct domains of hTAFII18 . As observed with the homologous Drosophila proteins, hTAFII20 interacts directly with TBP; however, additional interactions between hTAFII20 and hTAFII28 or hTAFII30 were detected . These results reveal differences not only in subunit composition, but also in the organization of dTFIID and hTFIID complexes.

Gene, 1995 Apr 3, 155(2), 299 - 304
Human isoleucyl-tRNA synthetase: sequence of the cDNA, alternative mRNA splicing, and the characteristics of an unusually long C-terminal extension; Nichols RC et al.; The human isoleucyl-tRNA synthetase (IRS)-encoding cDNA, whose primary structure we report here, has an open reading frame (ORF) which encodes a protein of 1262 amino acids (aa) with strong homology to IRS from yeast (53.5%) and Tetrahymena (51.0%) and contains all the major consensus motifs of class-I hydrophobic amino-acyl-tRNA synthetases (aaRS; MRS, LRS, VRS, IRS) . However, the human enzyme has an unusually long C-terminal extension composed, in part, of a twice-repeated motif which shows no homology to any reported protein . We also report the presence of a coiled-coil-like motif in the C-terminal half of the protein . The mRNA has an additional exon in the 5'-untranslated region (UTR) which is alternatively spliced, giving rise to two types of mRNA, both of which are expressed in several human tissues . The longer of the two transcripts contains predicted secondary structure in the 5'-UTR which may reduce the translational efficiency of this mRNA . Two possible regulatory elements in the 5'-UTR, an interferon-stimulated response element (ISRE)-like sequence and a short ORF, have been identified . Because human IRS has previously been shown to be the target of antibodies in autoimmune disease, we discuss the role of protein structural features in the development of an autoimmune response to IRS.

J Bacteriol, 1995 Apr, 177(7), 1864 - 71
Purification and characterization of an endo-beta-1,6-glucanase from Trichoderma harzianum that is related to its mycoparasitism; de la Cruz J et al.; The enzymes from Trichoderma species that degrade fungal cell walls have been suggested to play an important role in mycoparasitic action against fungal plant pathogens . The mycoparasite Trichoderma harzianum produces at least two extracellular beta-1,6-glucanases, among other hydrolases, when it is grown on chitin as the sole carbon source . One of these extracellular enzymes was purified to homogeneity after adsorption to its substrate, pustulan, chromatofocusing, and, finally, gel filtration . The apparent molecular mass was 43,000, and the isoelectric point was 5.8 . The first 15 amino acids from the N terminus of the purified protein have been sequenced . The enzyme was specific for beta-1,6 linkages and showed an endolytic mode of action on pustulan . Further characterization indicated that the enzyme by itself releases soluble sugars and produces hydrolytic halli on yeast cell walls . When combined with other T . harzianum cell wall-degrading enzymes such as beta-1,3-glucanases and chitinases, it hydrolyzes filamentous fungal cell walls . The enzyme acts cooperatively with the latter enzymes, inhibiting the growth of the fungi tested . Antibodies against the purified protein also indicated that the two identified beta-1,6-glucanases are not immunologically related and are probably encoded by two different genes.

Mol Cell Biol, 1995 Apr, 15(4), 2117 - 24
Separate domains of the Ran GTPase interact with different factors to regulate nuclear protein import and RNA processing; Ren M et al.; The small Ras-related GTP binding and hydrolyzing protein Ran has been implicated in a variety of processes, including cell cycle progression, DNA synthesis, RNA processing, and nuclear-cytosolic trafficking of both RNA and proteins . Like other small GTPases, Ran appears to function as a switch: Ran-GTP and Ran-GDP levels are regulated both by guanine nucleotide exchange factors and GTPase activating proteins, and Ran-GTP and Ran-GDP interact differentially with one or more effectors . One such putative effector, Ran-binding protein 1 (RanBP1), interacts selectively with Ran-GTP . Ran proteins contain a diagnostic short, acidic, carboxyl-terminal domain, DEDDDL, which, at least in the case of human Ran, is required for its role in cell cycle regulation . We show here that this domain is required for the interaction between Ran and RanBP1 but not for the interaction between Ran and a Ran guanine nucleotide exchange factor or between Ran and a Ran GTPase activating protein . In addition, Ran lacking this carboxyl-terminal domain functions normally in an in vitro nuclear protein import assay . We also show that RanBP1 interacts with the mammalian homolog of yeast protein RNA1, a protein involved in RNA transport and processing . These results are consistent with the hypothesis that Ran functions directly in at least two pathways, one, dependent on RanBP1, that affects cell cycle progression and RNA export, and another, independent of RanBP1, that affects nuclear protein import.

Mol Cell Biol, 1995 Apr, 15(4), 1942 - 52
The Ras-related protein Cdc42Hs and bradykinin promote formation of peripheral actin microspikes and filopodia in Swiss 3T3 fibroblasts; Kozma R et al.; The Ras-related protein Cdc42 plays a role in yeast cell budding and polarity . Two related proteins, Rac1 and RhoA, promote formation in mammalian cells of membrane ruffles and stress fibers, respectively, which contain actin microfilaments . We now show that microinjection of the related human Cdc42Hs into Swiss 3T3 fibroblasts induced the formation of peripheral actin microspikes, determined by staining with phalloidin . A proportion of these microspikes was found to be components of filopodia, as analyzed by time-lapse phase-contrast microscopy . The formation of filopodia was also found to be promoted by Cdc42Hs microinjection . This was followed by activation of Rac1-mediated membrane ruffling . Treatment with bradykinin also promoted formation of microspikes and filopodia as well as subsequent effects similar to that seen upon Cdc42Hs microinjection . These effects of bradykinin were specifically inhibited by prior microinjection of dominant negative Cdc42HsT17N, suggesting that bradykinin acts by activating cellular Cdc42Hs . Since filopodia have been ascribed an important sensory function in fibroblasts and are required for guidance of neuronal growth cones, these results indicate that Cdc42Hs plays an important role in determining mammalian cell morphology.

Infect Immun, 1995 Apr, 63(4), 1468 - 72
Impaired responsiveness to gamma interferon of macrophages infected with lymphocytic choriomeningitis virus clone 13: susceptibility to histoplasmosis; Villarete L et al.; Lymphocytic choriomeningitis virus clone 13 (LCMV clone 13), a variant isolated from the spleens of neonatally infected mice, causes persistent infections in mice infected as adults . Such persistently infected mice succumb to a normally sublethal dose of Histoplasma capsulatum, and their macrophages contain overwhelming numbers of yeast cells of the fungus . Both LCMV clone 13 and H . capsulatum yeast cells target and replicate in macrophages of the host . We sought to study the effects of LCMV clone 13 on the ability of macrophages to control growth of H . capsulatum in vitro . We show that the growth of H . capsulatum within macrophages was not directly affected by the presence of LCMV clone 13 . However, macrophages containing LCMV clone 13 did not respond fully to gamma interferon (IFN-gamma) stimulation . Such unresponsiveness resulted in proliferation of the fungus within macrophages cultured in the presence of IFN-gamma . The addition of anti-IFN-alpha/beta antibodies to LCMV clone 13-infected macrophage cultures restored macrophage responsiveness to IFN-gamma . These results indicate that production of IFN-alpha/beta by LCMV clone 13-infected macrophages antagonizes their responsiveness to IFN-gamma . Such antagonism may be one of the mechanisms by means of which certain viruses cause immune suppression and susceptibility to opportunistic infections.

Nat Genet, 1995 Apr, 9(4), 424 - 31
The apolipoprotein(a) gene is regulated by sex hormones and acute-phase inducers in YAC transgenic mice; Frazer KA et al.; High plasma concentrations of apolipoprotein (a) (apo(a)) have been implicated as a major independent risk factor for atherosclerosis in humans . Apo(a) is a large, evolutionarily new gene (present primarily in primates) for which considerable controversy exists concerning the factors that regulate its expression . To investigate the in vivo regulation of apo(a), we have created several lines of YAC transgenic mice containing a 110-kb human apo(a) gene surrounded by greater than 60 kb of 5' and 3' flanking DNA . Studies in humans have suggested that acute-phase inducers increase and sex steroids decrease apo(a) concentrations, but these results are controversial . Analysis of the YAC transgenic mice conclusively supports the hypothesized role of sex steroids and refutes the suggested role of acute-phase inducers in regulating the apo(a) gene.

Clin Infect Dis, 1995 Apr, 20(4), 913 - 6
Bloodstream infection due to Trichosporon beigelii in a burn patient: case report and review of therapy; Hajjeh RA et al.; Trichosporon beigelii is a yeast that has recently been increasingly associated with systemic infections in immunocompromised patients . Few cases have been reported in nonleukopenic patients . We describe what we believe to be the first report of a bloodstream infection due to T . beigelii in a burn patient . Our patient was successfully treated with a combination of amphotericin B and flucytosine . Antifungal susceptibility testing of the T . beigelii isolate showed that the organism was inhibited but not killed by amphotericin B . Burn patients are known to have a transient defect in neutrophil function that can predispose them to some infections . We review the English-language literature of recently reported cases of trichosporonosis and review the various therapies for T . beigelii infection.

J Pathol, 1995 Apr, 175(4), 391 - 6
Interphase cytogenetic analysis of distinct X-chromosomal translocation breakpoints in synovial sarcoma; Janz M et al.; Synovial sarcomas show a specific translocation involving chromosomes X and 18, t(X;18)(p11.2;q11.2) . Two distinct X-chromosomal breakpoints occur in different synovial sarcoma tumour samples . These breakpoints are located within two related genomic regions containing ornithine aminotransferase-like sequences, termed OATL1 and OATL2 . Preliminary observations indicated the potential correlation of OATL1-associated breakpoints with biphasic tumours and OATL2-associated breakpoints with monophasic fibrous tumours . The present study uses interphase cytogenetics to investigate the nature of chromosomal aberrations in frozen synovial sarcoma tissue samples . Two-colour fluorescence in situ hybridization (FISH) was performed using probes specific for the centromeres of chromosome X or 18, along with yeast artificial chromosome probes corresponding to the distinct breakpoint regions on Xp . One monophasic epithelial and two monophasic fibrous synovial sarcomas showed an OATL2-associated breakpoint, while a biphasic tumour revealed a hybridization pattern indicating a breakpoint within the OATL1 region . These results confirm our previous suggestion of a relationship between alternative breakpoints in Xp11.2 and different histological phenotypes observed in synovial sarcomas . They also demonstrate the utility of the two-colour hybridization approach for the identification of chromosomal changes in interphase nuclei isolated from frozen tissues.

Biophys J, 1995 Apr, 68(4 Suppl), 50S - 54S
Actin's view of actomyosin interface; Miller CJ et al.; Actomyosin interactions were examined by using yeast actin mutants with alanines replacing charged amino acid pairs D24/D25, E99/E100, D80/D81, and E83/K84 . In the in vitro motility experiments, actin filaments of D24A/D25A or E99A/E100A mutants moved in the presence of 0.7% methylcellulose at the velocities of wild-type actin . Without methylcellulose, these mutant filaments, but not the D80/D81 or E83/K84 filaments, dissociated from the assay surface upon addition of ATP . Measurements of myosin subfragment-1 (S1) binding to D24A/D25A- and E99A/E100A-polymerized actins in the presence of ATP revealed a three- and twofold decrease in their binding constant, respectively, compared with wild-type actin . In contrast to this, all monomeric actins had the same binding affinity for S1 . The rates and extents of polymerization of D24A/D25A and E99A/E100A actins by S1 were reduced in comparison to wild-type actin . The local structure of subdomain-2 on actin, as probed by subtilisin cleavage, was not altered for either mutant . A twofold decrease in nucleotide exchange was detected for the D24A/D25A mutant actin . These results demonstrate the involvement of the D24/D25 and E99/E100 residues in the weak binding of myosin to actin and reveal that residues D80/D81 and E83/K84 do not modulate actomyosin interactions.

J Antibiot (Tokyo), 1995 Apr, 48(4), 317 - 20
Action of cytogenin on lymphoid cells and their cytokine production; Kumagai H et al.; Action of cytogenin on macrophages and T cells was investigated . Phagocytosis of yeast and production of PMA-elicited superoxide anion by macrophages taken from mice given cytogenin po were augmented . Cytogenin enhanced productions of IL-1 alpha by macrophages and IFN gamma and GM-CSF by spleen cells although it did not enhanced production of TNF alpha by macrophages and IL-6 by macrophages and spleen cells . Macrophages stimulated with cytogenin caused to stimulate proliferation of purified T cells in Intercell cultures in which each cell population was cultured without contact . Results suggest that cytogenin primarily activates macrophages to produce monokines such as IL-1 alpha and it causes to stimulate proliferation and differentiation of T cells resulting in production of lymphokines such as IFN gamma and GM-CSF.

Biosci Biotechnol Biochem, 1995 Apr, 59(4), 749 - 50
Extraction of extracellular L-asparaginase from Candida utilis; Kil JO et al.; L-Asparaginase was extracted from Candida utilis cells using various reducing agents, 2-mercaptoethanol, dithiothreitol, or cysteine . The extraction of the enzyme depended upon the kind and concentration of reducing agents, temperature, time of incubation, and pH of buffer used . The enzyme was typically extracted by incubating the cells at 50 degrees C for 4 h in extraction solution containing 20 mM 2-mercaptoethanol in 20 mM potassium phosphate buffer (pH 7.0) . The enzyme can be extracted from either cell precipitate or cell culture broth . The yeast cells were viable after extraction of L-asparaginase.

Biosci Biotechnol Biochem, 1995 Apr, 59(4), 576 - 81
Oxidation of ethylene glycol and glycolic acid by glycerol oxidase; Isobe K; A glycerol oxidase from Aspergillus japonicus oxidized ethylene glycol to glyoxal by the same reaction pathway as alcohol oxidases from methanol yeast . The optimum pH and temperature for the oxidation of ethylene glycol were around 7.0 and 40 degrees C, respectively . Those of glycolaldehyde were similar to those of ethylene glycol . The apparent Kms for ethylene glycol and glycolaldehyde were 195 and 48.8 mM, respectively . The maximum velocities for ethylene glycol and glycolaldehyde were 89.1 and 62.2 mumol/min/mg of protein, respectively . Glycerol oxidase also oxidized glycolic acid, which is not oxidized by the alcohol oxidases, to glyoxylic acid like glycolate oxidases from green plants, and the apparent Km and Vmax for glycolic acid were 114 mM and 2.68 mumol/min/mg of protein, respectively . The glycerol oxidase was applicable to the production of glyoxal and glyoxylic acid.

Plant Physiol, 1995 Apr, 107(4), 1055 - 8
Three classes of nuclear import signals bind to plant nuclei; Hicks GR et al.; Three nuclear localization signals (NLS), including an unusual Mat alpha 2-like NLS from maize (Zea mays) R, were found to compete for binding to plant nuclei . In addition, the authentic yeast Mat alpha 2 NLS, which does not function in mammals, was shown to function in plants in vivo . Our results indicate that plants possess a site at the nuclear pore complex that recognizes the three known classes of NLSs.

Plant Cell Physiol, 1995 Apr, 36(3), 511 - 6
Spectrophotometric and molecular properties of mutated rice phytochrome A; Tomizawa K et al.; A cDNA (PHYA) for the phytochrome A apoprotein (PHYA) of rice and three mutated sequences (phyA S/A, the first ten serine residues in the N-terminal domain of PHYA were changed to alanine residues; phyA ND, the first 80 N-terminal amino acids were deleted; phyA CD, the amino acids of the C-terminal domain from 689 to 1,128 were deleted) were expressed in yeast, and the wild-type and mutant apophytochromes were allowed to combine in vitro with the chromophore phycocyanobilin (PCB) . The PCB-attached product of phyA S/A gave very similar spectrophotometric peaks to the PhAr and PhyAfr forms of wild-type product . By contrast, the peak of the product of phyA CD in the Pfr form was significantly shifted towards a shorter wavelength, an indication that, whereas the C-terminal domain is not crucial for the PCB attachment, it greatly influences the absorption maximum of PhyAfr . The rate of 50% reversion from PhyAfr to PhAr in darkness was 3 h at 27 degrees C with all of the samples, showing that the S/A and CD mutations did not affect this property . No photoreversibility was detected with the product of phyA ND . Gel-filtration analysis of the wild-type PHYA and the product of phyA S/A showed that the apparent molecular mass of each was 330 kDa, suggesting that both exists as dimers in solution.

Development, 1995 Apr, 121(4), 1099 - 110
Scleraxis: a basic helix-loop-helix protein that prefigures skeletal formation during mouse embryogenesis; Cserjesi P et al.; Members of the basic helix-loop-helix (bHLH) family of transcription factors have been shown to regulate growth and differentiation of numerous cell types . Cell-type-specific bHLH proteins typically form heterodimers with ubiquitous bHLH proteins, such as E12, and bind a DNA consensus sequence known as an E-box . We used the yeast two-hybrid system to screen mouse embryo cDNA libraries for cDNAs encoding novel cell-type-specific bHLH proteins that dimerize with E12 . One of the cDNAs isolated encoded a novel bHLH protein, called scleraxis . During mouse embryogenesis, scleraxis transcripts were first detected between day 9.5 and 10.5 post coitum (p.c.) in the sclerotome of the somites and in mesenchymal cells in the body wall and limb buds . Subsequently, scleraxis was expressed at high levels within mesenchymal precursors of the axial and appendicular skeleton and in cranial mesenchyme in advance of chondrogenesis; its expression pattern in these cell types foreshadowed the developing skeleton . Prior to formation of the embryonic cartilaginous skeleton, scleraxis expression declined to low levels . As development proceeded, high levels of scleraxis expression became restricted to regions where cartilage and connective tissue formation take place . Scleraxis bound the E-box consensus sequence as a heterodimer with E12 and activated transcription of a reporter gene linked to its DNA-binding site . The expression pattern, DNA-binding properties and transcriptional activity of scleraxis suggest that it is a regulator of gene expression within mesenchymal cell lineages that give rise to cartilage and connective tissue.

Development, 1995 Apr, 121(4), 1053 - 63
Identifying tumor suppressors in genetic mosaics: the Drosophila lats gene encodes a putative protein kinase; Xu T et al.; We have identified recessive overproliferation mutations by screening and examining clones of mutant cells in genetic mosaics of the fruitfly Drosophila melanogaster . This type of screen provides a powerful approach for identifying and studying potential tumor suppressors . One of the identified genes, lats, has been cloned and encodes a putative protein kinase that shares high levels of sequence similarity with three proteins in budding yeast and Neurospora that are involved in regulation of the cell cycle and growth . Mutations in lats cause dramatic overproliferation phenotypes and various developmental defects in both mosaic animals and homozygous mutants.

Am J Physiol, 1995 Apr, 268(4 Pt 1), E572 - 9
Sulfation pathway of thyroid hormone metabolism in selenium-deficient male rats; Wu SY et al.; Male Sprague-Dawley rats were fed a selenium-deficient yeast-based laboratory diet or a control diet for 6 wk . The tissue type I 5'-monodeiodinase (5'-MDI) activity and the immunoassayable 5'-MDI were significantly (P < 0.05) reduced in the liver and the kidney but not in the thyroid of selenium-deficient rats . The mean serum concentrations of thyroxine sulfate (T4S), 3,3',5'-triiodothyronine sulfate (T3S), and reverse T3 sulfate (rT3S) (ng/dl) were significantly increased in selenium-deficient rats (15.7, 59.4, and 22.8, respectively, n = 12) compared with control rats (< 1.0, 18.5, and 9.1, respectively, n = 12, P < 0.01) . Kinetic studies were carried out during a constant infusion of unlabeled sulfated iodothyronines (T4S, T3S, or rT3S, n = 5-6/group) at a rate of 1 microgram/h by Alzet minipump for 48 h . The data showed that elevated serum concentrations of T4S or T3S in the selenium-deficient rat are due both to reduced metabolic clearance rate (MCR, mean, l.kg-1.day-1, 7.4 for T4S and 4.5 for T3S in selenium deficiency vs . 12 and 9.2, respectively in controls, P < 0.05) and increased production rate (mean, microgram.kg-1.day-1, 1.2 for T4S, and 2.7 for T3S in selenium deficiency vs . 0.12 and 1.7, respectively, in the controls, P < 0.05) . However, the increased serum rT3S concentration in selenium-deficient rats is due mainly to reduced MCR (mean, l.kg-1.day-1, 34 vs . 67 in controls, P < 0.05) and its daily production rate remained unchanged in selenium deficiency (mean, microgram.kg-1.day-1, 7.6 vs . 6.1 in the control group, P > 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Hum Genet, 1995 Apr, 95(4), 467 - 8
Dinucleotide polymorphism at the DXS1178 locus is tightly linked to PGK1 at Xq13; Fujita R et al.; A polymorphic CA repeat (locus name DXS1178) was isolated from a 1-megabase YAC (OTCC) containing the OTC gene, located at Xp21.1 . However, amplification in human-rodent hybrid cells and segregation analysis in three CEPH families mapped the DXS1178 locus at Xq13 . The mapping ambiguity is apparently caused by the chimeric nature of the OTCC YAC clone.

Hum Genet, 1995 Apr, 95(4), 429 - 34
New polymorphisms and markers in the HLA class I region: relevance to hereditary hemochromatosis (HFE); Totaro A et al.; Hereditary hemochromatosis (HFE) is an inherited disorder whose gene lies in the proximity of the histocompatibility antigen (HLA) class I region, on 6p21.3 . Despite efforts in refining the HFE region, a number of informative DNA markers, linked to the disease locus and amenable to use in an assay based on the polymerase chain reaction (PCR) is available . The gene content of this region is high, and the HFE gene has not so far been identified . We have used a strategy based on PCR protocols potentially able to detect both polymorphisms and expressed sequences . This approach has been applied to a 700-kb stretch (approximately) of DNA corresponding to the insert of a Centre d'Etude du Polymorphisme Humain yeast artificial chromosome (225 B1) of the possible candidate region . Five new polymorphisms have been detected among 20 specific fragments isolated . Four of them are tightly linked to the HFE locus . Because of the strong linkage disequilibrium with the disease demonstrated by these markers, they could represent starting points for the identification and characterization of the HFE gene . The remaining non-polymorphic fragments, being amplifiable and in most cases linked to NotI sites, may be useful starting points for the generation of a genomic contig of band 6p21.3 and for gene identification.

Genes Dev, 1995 Apr 1, 9(7), 843 - 54
A novel U2-U6 snRNA structure is necessary for mammalian mRNA splicing; Sun JS et al.; Splicing of mRNA precursors requires a complex and dynamic set of RNA-RNA base-pairing interactions in which the U2 and U6 snRNAs play central roles . Using a genetic suppression assay, we refine and extend a U2-U6 snRNA structure that may comprise the catalytic center of the spliceosome . We first show that a critical U2-U6 helix proven in yeast, helix Ia, is also essential for mammalian splicing . Mutations in the adjacent helix Ib, however, cannot be similarly suppressed, and relevant residues in both U2 and U6 are shown to participate in intramolecular, rather than intermolecular, base-pairing . We next demonstrate the requirement for a novel U2-U6 helix, helix III, which involves bases extending 3' from the branch site recognition sequence in U2 and 5' from an evolutionarily invariant sequence in U6 implicated previously in 5' splice site recognition . This configuration suggests that helix III may help juxtapose the pre-mRNA 5' splice site and branch site . We provide evidence for this by demonstrating that a branch site mutation can be suppressed by a mutation in the 5' splice site, provided that compensatory changes are made in the appropriate bases in U2 and U6 . Our results provide new insights into how U2 and U6 snRNAs interact with each other and with the pre-mRNA to initiate the first catalytic step in splicing.

J Cell Biol, 1995 Apr, 129(1), 105 - 20
rbSec1A and B colocalize with syntaxin 1 and SNAP-25 throughout the axon, but are not in a stable complex with syntaxin; Garcia EP et al.; rbSec1 is a mammalian neuronal protein homologous to the yeast SEC1 gene product which is required for exocytosis . Mutations in Sec1 homologues in the nervous systems of C . elegans and D . melanogaster lead to defective neurotransmitter secretion . Biochemical studies have shown that recombinant rbSec1 binds syntaxin 1 but not SNAP-25 or synaptobrevin/VAMP, the two proteins which together with syntaxin 1 form the synaptic SNARE complex . In this study we have examined the subcellular localization of rbSec1 and the degree of interaction between rbSec1 and syntaxin 1 in situ . rbSec1, which we show here to be represented by two alternatively spliced isoforms, rbSec1A and B, has a widespread distribution in the axon and is not restricted to the nerve terminal . This distribution parallels the localization of syntaxin 1 and SNAP-25 along the entire axonal plasmalemma . rbSec1 is found in a soluble and a membrane-associated form . Although a pool of rbSec1 is present on the plasmalemma, the majority of membrane-bound rbSec1 is not associated with syntaxin 1 . We also show that rbSec1 is not part of the synaptic SNARE complex or of the syntaxin 1/SNAP-25 complex we show to be present in non-synaptic regions of the axon . Thus, in spite of biochemical studies demonstrating the high affinity interaction of rbSec1 and syntaxin 1, our results indicate that rbSec1 and syntaxin 1 are not stably associated . They also suggest that the function of rbSec1, syntaxin 1, and SNAP-25 is not restricted to synaptic vesicle exocytosis at the synapse.

Vet Immunol Immunopathol, 1995 Apr, 45(3-4), 361 - 7
Effect on humoral tolerance (IgG and IgE) in dogs by oral administration of ovalbumin and Der pI; Deplazes P et al.; The effect of oral administration of ovalbumin (OVA) or recombinant house dust mite allergen (Der p I) to dogs upon specific IgG and IgE reactions to subcutaneous immunization with these antigens was studied . Daily feeding of 10 x 10 g of OVA resulted in a non-responsiveness to subsequent parenteral immunization with OVA in two young dogs . The same two dogs were also immunized parenterally with Der p I and showed a pronounced IgG response against native Der p I, confirming that the non-responsiveness to OVA was antigen-specific . Thus, it has been demonstrated that it is possible to induce oral tolerance in dogs . Two other dogs of the same litter that received 2 x 10 mg of recombinant Der p I in a crude yeast lysate per os reacted to immunization with OVA with pronounced IgG and IgE production against OVA, further confirming the antigen-specificity of the OVA tolerance . However, tolerance to Der p I was not induced, as evidenced by a strong IgG response to immunization after per os application of the antigen, possibly because the oral dose was too small.

Cytokine, 1995 Apr, 7(3), 232 - 6
Cloning, sequencing and expression of the ovine interleukin 6 gene; Ebrahimi B et al.; Gene amplification by reverse transcriptase PCR with heterologous primers has been used to obtain a cDNA clone encoding the structural sequences of ovine interleukin 6 from alveolar macrophages . This cDNA encodes a protein of M(r) = 23,429, which is 53% homologous in amino acid sequence to human IL = 6 . The clone hybridizes to an RNA of size 1260 nt in alveolar macrophages, expression of which is potentiated by LPS . The ovine IL-6 structural gene has been cloned into the yeast expression vector pOGS40, and used to produce a recombinant protein . This protein is capable of causing increased immunoglobulin production in pokeweed mitogen stimulated ovine peripheral blood mononuclear cells at concentrations of 10-100 ng/ml, but it only causes very limited replication of B9 cells, a murine IL-6 dependent cell line . This is in contrast to recombinant human IL-6, which is capable of stimulating B9 cell proliferation, but not immunoglobulin production by ovine PBMC.

Hum Mol Genet, 1995 Apr, 4(4), 731 - 9
Three genes that escape X chromosome inactivation are clustered within a 6 Mb YAC contig and STS map in Xp11.21-p11.22; Miller AP et al.; In order to study the distribution of genes that escape X chromosome inactivation, a high density yeast artificial chromosome (YAC) contig and STS map spanning approximately 6 Mb has been constructed in Xp11.21-p11.22 . The contig contains 113 YACs mapped with 53 markers, including 10 genes . Four genes have been assayed for their expression status on both the active and inactive human X chromosomes, and these data have been combined with previous results on two other genes in the contig . Three of these genes escape X inactivation and have been localized to a single YAC clone of approximately 1075 kb . The other three genes are subject to inactivation, with two of them lying among the genes that escape inactivation . These results suggest that there are both regional control signals as well as gene-specific elements that determine the X inactivation status of genes on the proximal short arm of the human X chromosome.

Hum Mol Genet, 1995 Apr, 4(4), 717 - 25
A primary expression map of the chromosome 15q15 region containing the recessive form of limb-girdle muscular dystrophy (LGMD2A) gene; Chiannilkulchai N et al.; Previous genetic and physical studies of LGMD2A, an autosomal recessive form of limb-girdle muscular dystrophy, have led to the establishment of a 10-12 Mb YAC contig encompassing the morbid locus . In order to progress toward the identification of the gene involved in LGMD2A, a primary transcription map of this genomic region was generated . The direct cDNA selection strategy was used with three YACs covering the candidate region and two different muscle cDNA libraries . Seventeen transcription units were identified among 171 cDNA fragments analysed . Five sequences corresponded to known genes, and twelve to new ones . They were characterized for their sequences, physical positions within the YAC contig, and expression patterns . Among those specifically transcribed in muscle, the calpain gene is a good positional and functional candidate for LGMD2A.

AIDS Res Hum Retroviruses, 1995 Apr, 11(4), 433 - 42
Phagocytosis of individual CD4+ T cells by HIV-induced T cell syncytia; Murphy S et al.; Transmission electron microscopic analysis of HIV-induced syncytia of the CD4+ SupT1 cell line has revealed profiles of whole T cells in the syncytium cytoplasm . Serial sections demonstrate that these T cells are completely enveloped by a second membrane in the syncytium cytoplasm and represent phagosomes . Pycnosis of engulfed T cell nuclei, vacuolation of the cytoplasm of engulfed T cells, and the association of engulfed T cells with dense vesicular clusters in the syncytium cytoplasm support the conclusion that they represent phagosomes . In addition, transmission electron micrographs of the syncytium surface reveal giant pseudopodial extensions wrapping around T cells, in a fashion similar to bacterial and yeast phagocytosis by professional phagocytes . These results suggest that phagocytosis is a characteristic acquired during HIV-induced syncytium formation, and that it may represent an avenue of T cell death in addition to fusion in HIV-infected SupT1 cell cultures.

Curr Biol, 1995 Apr 1, 5(4), 383 - 92
Two different subunits of importin cooperate to recognize nuclear localization signals and bind them to the nuclear envelope; Gorlich D et al.; BACKGROUND: Selective protein import into the cell nucleus occurs in two steps: binding to the nuclear envelope, followed by energy-dependent transit through the nuclear pore complex . A 60 kD protein, importin, is essential for the first nuclear import step, and the small G protein Ran/TC4 is essential for the second . We have previously purified the 60kD importin protein (importin 60) as a single polypeptide . RESULTS: We have identified importin 90, a 90 kD second subunit that dissociates from importin 60 during affinity chromatography on nickel (II)-nitrolotriacetic acid-Sepharose, a technique that was originally used to purify importin 60 . Partial amino-acid sequencing of Xenopus importin 90 allowed us to clone and sequence its human homologue; the amino-acid sequence of importin 90 is strikingly conserved between the two species . We have also identified a homologous budding yeast sequence from a database entry . Importin 90 potentiates the effects of importin 60 on nuclear protein import, indicating that the importin complex is the physiological unit responsible for import . To assess whether nuclear localization sequences are recognized by cytosolic receptor proteins, a biotin-tagged conjugate of nuclear localization signals linked to bovine serum albumin was allowed to form complexes with cytosolic proteins in Xenopus egg extracts; the complexes were then retrieved with streptavidin-agarose . The pattern of bound proteins was surprisingly simple and showed only two predominant bands: those of the importin complex . We also expressed the human homologue of importin 60, Rch1p, and found that it was able to replace its Xenopus counterpart in a functional assay . We discuss the relationship of importin 60 and importin 90 to other nuclear import factors . CONCLUSIONS: Importin consists of a 60 and a 90 kD subunit . Together, they constitute a cytosolic receptor for nuclear localization signals that enables import substrates to bind to the nuclear envelope.

Biochem Mol Biol Int, 1995 Apr, 35(4), 875 - 80
High membrane fluidity is related to NaCl stress in Candida membranefaciens; Khaware RK et al.; The effect of hypersaline stress on the lipid composition of the salt-tolerant yeast Candida membranefaciens was studied . Fatty acid analyses of the plasma membrane showed a growth phase- and dose-dependent increase in the level of linolenic acid (C18:3) in 1.35 M NaCl-stressed cells . Palmitoleic acid (C16:1) was completely undetectable at all phases of the life cycle . Changes in the levels of other fatty acids were insignificant . The degree of unsaturation of fatty acids in the plasma membranes was higher in presence of 1.35 M NaCl . The fluorescence polarisation value of DPH (1,6-diphenyl- 1,3,5-hexatriene) in the spheroplasts of the stressed cells was lower as compared to the control cells, indicating the fact that a higher membrane fluidity favours osmotic adaptation against NaCl stress . Among different phospholipids, levels of Phosphatidylinositol and Phosphatidylethanolamine were elevated in the salt-adapted cells as compared to their controls . The levels of Phosphatidylcholine and cardiolipin did not change significantly in response to hypersaline stress . The study points out that hypersalinity signals affect the lipid composition which in turn affects the membrane fluidity of C . membranefaciens.

J Reprod Med, 1995 Apr, 40(4), 323 - 6
Histiocytosis X of the vulva with a confusing clinical and pathologic presentation . A case report; Savell V et al.; Histiocytosis X (HX) is a rare disorder of Langerhans cells and most commonly occurs in children . We report a case of HX of the vulva in a 76-year-old woman that clinically simulated a yeast infection of the vulva but histologically resembled an amelanotic melanoma . We briefly report the clinical and pathologic features of this case, which responded to vincristine followed by vinblastine and was in complete remission after nine months.

Mol Biotechnol, 1995 Apr, 3(2), 85 - 92
Rapid identification of polymorphic CA-repeats in YAC clones; Rotman G et al.; Positional cloning of rare disease genes depends on the availability of highly polymorphic markers near the disease loci . The most abundant class of polymorphic markers in the human genome is CA-repeats . We have developed a strategy for the rapid isolation of highly polymorphic CA-repeats from YAC clones . Total DNA of yeast clones containing partly overlapping YACs is digested with frequent cutter restriction enzymes, blotted and hybridized with a poly(CA/GT) probe under high stringency conditions that enable preferential detection of long CA-repeats . The repeats detected in this way are isolated by PCR using vectorette linkers, sequenced, and appropriate flanking markers are constructed for genotyping . All of the CA-repeats identified using this approach were highly polymorphic . This simple and rapid approach should allow the development of highly polymorphic markers at any genomic region cloned in YACs.

J Cell Biochem, 1995 Apr, 57(4), 630 - 40
Activation of the c-fos promoter by increased internal pH; Murguia JR et al.; Changes in intracellular pH (pHin) take part in the mitogenic response . Their importance has been stressed by the finding that mouse fibroblasts expressing a yeast proton pumping ATPase (PMA1) exhibit a transformed phenotype and are tumorigenic . These cells do maintain a higher pHin, supporting the idea that elevated pHin may act as a proliferative trigger . Here we show that cells constitutively expressing PMA1 have higher levels of the AP-1 transcription factor . The use of stable transfectants and transient transfection assays show that PMA1 activity induces transactivation of the c-fos promoter . The activation of the promoter is mediated throughout the serum response element (SRE) . The use of protein kinase C inhibitors suggests that AP-1 activation is achieved through a pathway independent of protein kinase C.

J Small Anim Pract, 1995 Apr, 36(4), 147 - 50
Population sizes and frequency of Malassezia pachydermatis at skin and mucosal sites on healthy dogs; Bond R et al.; Skin and mucosal carriage of Malassezia pachydermatis was studied in 20 healthy pet dogs of various breeds and in 20 kennelled beagles . Using swabs, anal carriage was detected in 10 pet dogs and 11 beagles and the nose, mouth, prepuce and vulva were shown to be infrequently colonised . M pachydermatis was isolated from the external ear canal of 11 beagles and two pet dogs; both the population sizes and frequency of isolation were significantly (P < 0.05) greater in the beagles . The yeast was infrequently isolated from the axilla and groin in low numbers using contact plates and detergent scrub samples but was often cultured from the lower lip and the dorsal interdigital spaces; isolation frequencies and population sizes in the two groups of dogs were not significantly different . These results demonstrate that the anus, external ear canal and lip and interdigital skin of healthy dogs are frequently colonised by M pachydermatis.

Turk J Pediatr, 1995 Apr-Jun, 37(2), 93 - 102
Results of vaccinated infants born to HBsAg-positive mothers with different hepatitis B vaccines and doses; Kuru U et al.; Seventy-eight infants born to HBsAg-positive women were randomly assigned to receive either the plasma-derived vaccine or 0.5 ml (10 micrograms HBsAg) yeast-derived recombinant hepatitis B vaccine within 24 hours of birth, simultaneously with hepatitis B hyperimmunoglobulin . In 67 infants who received the plasma-derived vaccine, one of the doses of 0.5 ml (25 micrograms HBsAg) was used randomly . In all of the infants, the second and third doses of both vaccines were given at one and two months of age, respectively . The booster doses were given at 12 month of age in all of the infants . These vaccinated infants were followed up until 13 months of age . There were differences in the seroconversion rates with different vaccines and doses . The recipients of the half-dose of plasma-derived vaccine showed lower seroconversion rates than the others, and the newborns in this group showed more seronegativity (13.2%) than the others (p < 0.05) . The lowest anti-HBs geometric mean titers (GMTs) were obtained in newborns vaccinated with Hevac B 0.5 ml . Sixty percent of the anti-HBs GMTs in this group were under 100 mlU/ml . There were statistically significant differences between males and females in anti-HBs seronegativity rates, with males having lower anti-HBs GMTs than females . The difference was particularly significant among male newborns vaccinated with a half-dose of plasma-derived vaccine.

Curr Genet, 1995 Apr, 27(5), 451 - 9
Heterozygosity at the b mating-type locus attenuates fusion in Ustilago maydis; Laity C et al.; Mating and pathogenesis of the corn smut fungus, Ustilago maydis, are controlled by two unlinked mating-type loci, a and b . Yeast-like haploids that differ at both loci are compatible and fuse to establish a pathogenic dikaryon . Mating is assayed in vitro by co-inoculation on culture medium containing activated charcoal; compatible combinations have a characteristic "fuzzy" appearance caused by the growth of aerial hyphae . In general, this test has not been useful for assaying the mating ability of strains that are already mycelial (e.g., those heterozygous at b or at both mating-type loci) . Using an assay for cytoduction involving transfer of a mitochondrial marker during transient cell fusion, and engineered strains with defined genotypes, we examined the mating abilities of strains heterozygous or hemizygous at the mating-type loci . The data (which have not been available from conventional pathogenicity or plate mating tests) show that heterozygosity at b attenuates fusion in haploid and diploid strains, whereas strains heterozygous at a retain the ability to fuse with a compatible haploid partner . It appears, therefore, that subsequent fusion events are attenuated once fusion has occurred to establish the U . maydis dikaryon.

RNA, 1995 Apr, 1(2), 219 - 22
GNRA tetraloops make a U-turn; Jucker FM et al.; The U-turn (uridine turn) is an RNA structural motif that contains a change in backbone direction stabilized by specific interactions across the bend . It was first identified in the anticodon loop and the T-loop of yeast tRNA(Phe) (Quigley & Rich, 1976, Science 194:796-806) and has recently also been found in the crystal structure of the hammerhead ribozyme (Pley HW, Flaherty KM, McKay DB, 1994a, Nature 372:68-74) . These U-turn motifs follow a UNR consensus sequence (where N is any nucleotide and R is G or A) . Here we report that the frequently occurring GNRA tetraloops also contain a U-turn motif, and we discuss the role of U-turns as abundant tertiary structural motifs in RNA.

Curr Opin Genet Dev, 1995 Apr, 5(2), 229 - 33
The ins and outs of RNA nucleocytoplasmic transport; Zapp ML; Nucleocytoplasmic transport of RNA is an obligatory step in gene expression and may also be a target for regulation . The cellular machinery has the capacity to export a myriad of RNA transcripts that differ significantly in sequence and structure . The molecular mechanisms of RNA transport are (as yet) largely unknown . Thus, biochemical and genetic approaches are being used to identify cellular factors that mediate this process . Major advances over the past year include the cloning of genes for nuclear pore complex components and isolation of yeast mutants that harbor specific defects in RNA export.

Biotechniques, 1995 Apr, 18(4), 688 - 97
Primer design for automated DNA sequencing utilizing T7 DNA polymerase and internal labeling with fluorescein-15-dATP; Wiemann S et al.; We have identified additional criteria for the walking primer design that improve the success rate of automated fluorescent DNA sequencing using the internal labeling technique and T7 DNA polymerase . These criteria resulted from the evaluation of over 220 sequences generated with walking primers and fluorescein-15-dATP as internal label in the course of the European Community (EC) yeast genome sequencing project . In this project primers were designed using standard commercial software . Intensities of sequencing signals varied over a broad range from very strong to very weak, depending on the primers used . This led us to evaluate primer performance relative to (i) the template sequence immediately downstream of the primer binding site and (ii) the primer sequence itself . Our experiments show that the position of the first labeled dATP to be incorporated downstream of the primer into the growing strand is substantial for the signal intensity of the sequence . The closer to the primer that the first 'A' is incorporated, the stronger the peak intensities are . An additional feature of sequencing with native T7 DNA polymerase is its ability to remove a 3'-terminal 'A' of the primer by the 3'-->5' exonuclease activity and to exchange the nucleotide with a labeled dATP by the polymerase activity.

Genes Chromosomes Cancer, 1995 Apr, 12(4), 283 - 7
Localization of the 8;13 translocation breakpoint associated with myeloproliferative disease to a 1.5 Mbp region of chromosome 13; Kempski H et al.; There are five reported cases of an atypical myeloproliferative disorder in which the leukemia cells have a consistent t(8;13)(p11;q12) translocation . We analyzed the breakpoint in metaphases from two of these patients by fluorescence in situ hybridization using a series of yeast artificial chromosomes (YACs) derived from the 13q12 region . We found that a YAC containing the FLT1 and FLT3 oncogenes was localized distal to the 13q12 breakpoint and was not rearranged . YAC66, a YAC that lies immediately adjacent to the chromosome 13 centromere, was localized proximal to the 13q12 breakpoint and was not rearranged . A third YAC, which is located between FLT1 and YAC66, was unrearranged in normal metaphase chromosomes, but showed hybridization signals on both derivative chromosomes in both cases . Thus, the breakpoints in these two cases are localized to the same 1.5 Mbp region of 13q12 . This may be the site of an unidentified gene involved in the pathogenesis of some types of leukemia.

J Cell Biol, 1995 Apr, 129(1), 157 - 67
KIF2 is a new microtubule-based anterograde motor that transports membranous organelles distinct from those carried by kinesin heavy chain or KIF3A/B; Noda Y et al.; Kinesin is known as a representative cytoskeletal motor protein that is engaged in cell division and axonal transport . In addition to the mutant assay, recent advances using the PCR cloning technique have elucidated the existence of many kinds of kinesin-related proteins in yeast, Drosophila, and mice . We previously cloned five different members of kinesin superfamily proteins (KIFs) in mouse brain (Aizawa, H., Y . Sekine, R . Takemura, Z . Zhang, M . Nangaku, and N . Hirokawa . 1992 . J . Cell Biol . 119:1287-1296) and demonstrated that one of them, KIF3A, is an anterograde motor (Kondo, S., R . Sato-Yashitake, Y . Noda, H . Aizawa, T . Nakata, Y . Matsuura, and N . Hirokawa . J . Cell Biol . 1994 . 125:1095-1107) . We have now characterized another axonal transport motor, KIF2 . Different from other KIFs, KIF2 is a central type motor, since its motor domain is located in the center of the molecule . Recombinant KIF2 exists as a dimer with a bigger head and plus-end directionally moves microtubules at a velocity of 0.47 +/- 0.11 microns/s, which is two thirds that of kinesin's . Immunocytological examination showed that native KIF2 is abundant in developing axons and that it accumulates in the proximal region of the ligated nerves after a 20-h ligation . Soluble KIF2 exists without a light chain, and KIF2's associated-vesicles, immunoprecipitated by anti-KIF2 antibody, are different from those carried by existing motors such as kinesin and KIF3A . They are also distinct from synaptic vesicles, although KIF2 is accumulated in so-called synaptic vesicle fractions and embryonal growth cone particles . Our results strongly suggest that KIF2 functions as a new anterograde motor, being specialized for a particular group of membranous organelles involved in fast axonal transport.

J Mol Biol, 1995 Mar 31, 247(3), 377 - 89
Exclusive expression of C . elegans osm-3 kinesin gene in chemosensory neurons open to the external environment; Tabish M et al.; In Caenorhabditis elegans three genetic loci osm-3, unc-104 and unc-116 have been identified, which encode anterograde motor kinesin . Here we show that osm-3 encodes a 672 amino acid long kinesin-like protein (KLP) that contains all three functional domains similar to the kinesin heavy chain, including a globular motor region, an alpha-helical coiled-coil rod, and a globular tail region . OSM-3 shows homology in both the motor and rod domains with kinesins from divergent species such as mouse KIF3, and sea urchin KRP95, and also with the rod domains of several non-kinesin proteins, such as myosin, ezrin, outer membrane proteins alpha precursor OMPA, yeast intracellular protein transport USO1, and the rat neurofilament NF-H . Temporal and spatial expression of the osm-3::lacZ fusion gene during development is limited to an exclusive set of 26 chemosensory neurons whose dendritic endings are exposed to the external environment, including six IL2 neurons of the inner labial sensilla, eight pairs of amphid neurons (ADF, ADL, ASE, ASG, ASH, ASI, ASJ, ASK) in the head, and two pairs of phasmid neurons (PHA and PHB) in the tail . Our data are consistent with the known structural defects in the amphid and phasmid sensilla in osm-3 mutants and also show the expression of the gene in IL2 neurons . Temporally, the gene is differentially expressed in all three types of chemosensory sensilla . Further work on osm-3, unc-104 and unc-116 mutants should give insight into the in vivo functions of the kinesin family during C . elegans neurogenesis.

J Biol Chem, 1995 Mar 31, 270(13), 7219 - 26
Interaction of the COOH-terminal transactivation domain of p65 NF-kappa B with TATA-binding protein, transcription factor IIB, and coactivators; Schmitz ML et al.; We show that the transactivating COOH terminus of the p65 subunit of human transcription factor NF-kappa B directly binds the general transcription factors TFIIB and TATA-binding protein (TBP) in vitro . Interaction of p65 with TFIIB required the most COOH-terminal sequence repeat within TFIIB . A functional interaction of TFIIB with p65 was evident from assays in yeast cells . Cotransfection experiments in COS cells revealed that only overexpression of TBP was able to further stimulate p65-dependent transactivation of a reporter gene . The coexpression of neither TBP nor TFIIB was able to relieve squelching, indicating the involvement of additional factors in transactivation by p65 . A cell-free assay using highly purified factors revealed a specific transcriptional stimulation through the COOH-terminal activation domain of NF-kappa B by at least one cofactor, PC1, isolated from HeLa cells . These data show that the potent acidic transactivation domains in the COOH terminus of p65 are able to functionally recruit various components of the basic transcription machinery as well as coactivators.

Biochem Pharmacol, 1995 Mar 30, 49(7), 971 - 7
In vitro inhibition of glutathione reductase by arsenotriglutathione; Styblo M et al.; Arsenotriglutathione, a product of the reaction of arsenate or arsenite with glutathione, is a mixed-type inhibitor (Ki = 0.34 mM) of the in vitro reduction of glutathione disulfide by purified yeast glutathione reductase . Notably, arsenotriglutathione was a 10-fold more potent inhibitor than either arsenite or glutathione . The inhibition of glutathione reductase by arsenotriglutathione was partly reversed by the addition of meso-2,3-dimercaptosuccinic acid (DMSA) . However, high concentrations of DMSA also inhibited the reduction of glutathione disulfide by the yeast enzyme (IC50 of 7 mM with 0.1 mM glutathione disulfide) . Ultrafiltration of the enzyme-arsenotriglutathione complex recovered about 74% of the original (non-inhibited) activity, suggesting that the inhibition of glutathione reductase by arsenotriglutathione had both reversible and irreversible components . The relatively high potency of arsenotriglutathione as an inhibitor of glutathione reductase may alter the reduction of glutathione disulfide and affect the availability of glutathione that is required for the reduction of arsenate to arsenite and for the formation of the arsenotriglutathione complex.

Proc Natl Acad Sci U S A, 1995 Mar 28, 92(7), 2539 - 43
Aceruloplasminemia: molecular characterization of this disorder of iron metabolism; Harris ZL et al.; Ceruloplasmin is an abundant alpha 2-serum glycoprotein that contains 95% of the copper found in the plasma of vertebrate species . We report here on the identification of a genetic defect in the ceruloplasmin gene in a patient previously noted to have a total absence of circulating serum ceruloplasmin in association with late-onset retinal and basal ganglia degeneration . In this patient T2 (transverse relaxation time)-weighted magnetic resonance imaging of the brain revealed basal ganglia densities consistent with iron deposition, and liver biopsy confirmed the presence of excess iron . Although Southern blot analysis of the patient's DNA was normal, PCR amplification of 18 of the 19 exons composing the human ceruloplasmin gene revealed a distinct size difference in exon 7 . DNA sequence analysis of this exon revealed a 5-bp insertion at amino acid 410, resulting in a frame-shift mutation and a truncated open reading frame . The validity of this mutation was confirmed by analysis of DNA from the patient's daughter, which revealed heterozygosity for this same 5-bp insertion . The presence of this mutation in conjunction with the clinical and pathologic findings demonstrates an essential role for ceruloplasmin in human biology and identifies aceruloplasminemia as an autosomal recessive disorder of iron metabolism . These findings support previous studies that identified ceruloplasmin as a ferroxidase and are remarkably consistent with recent studies on the essential role of a homologous copper oxidase in iron metabolism in yeast . The clinical and laboratory findings suggest that additional patients with movement disorders and nonclassical Wilson disease should be examined for ceruloplasmin gene mutations.

Biochemistry, 1995 Mar 21, 34(11), 3605 - 13
Mitochondrial presequence inserts differently into membranes containing cardiolipin and phosphatidylglycerol; Snel MM et al.; The interaction of the 25-residue presequence of yeast cytochrome oxidase subunit IV with lipid bilayers composed of phosphatidylglycerol, cardiolipin, or their (1:4) mixtures with phosphatidylcholine has been studied by spin-label ESR spectroscopy . Binding of the presequence progressively broadens the gel-to-fluid phase transition of dimyristoylphosphatidylglycerol bilayers, leading to abolition of the transition at a peptide/lipid ratio of > or = 1:5 mol/mol . The mobility of phosphatidylglycerol spin-labeled at the 5-position of the sn-2 chain is decreased in both gel and fluid phases on binding the presequence, with a progressively increasing ESR spectral anisotropy in the fluid phase . The ESR spectra of phosphatidylglycerol spin-labeled at the 14-position of the sn-2 chain contain a second motionally restricted component, in addition to the fluid bilayer spectral component, that arises from direct interaction of the bound presequence with the lipid chains . The proportion of this motionally restricted component is greater for dioleoylphosphatidylglycerol bilayers (corresponding to 2-3 lipids per peptide) than for cardiolipin bilayers (1-2 lipids/peptide), and this component is present also in the mixed bilayers containing 80% phosphatidylcholine . The ESR spectra of the presequence spin-labeled with a maleimide derivative at cysteine-19 evidence high mobility in solution and a very strong reduction in mobility on binding to bilayers containing negatively charged lipids . At low peptide to lipid ratios, the ESR spectra of the spin-labeled presequence sense the phase transition of dimyristoylphosphatidylglycerol.(ABSTRACT TRUNCATED AT 250 WORDS)

Mol Gen Genet, 1995 Mar 20, 246(6), 761 - 6
Genetic and physical mapping of the lateral suppressor (ls) locus in tomato; Schumacher K et al.; Tomato plants homozygous for the recessive lateral suppressor (ls) mutation show a number of phenotypic abnormalities among which the lack of lateral meristem initiation during vegetative growth and the absence of petals on the flower are the most prominent . As a first step towards the isolation of the Ls gene by means of map-based cloning, we have determined its position on the restriction fragment length polymorphism (RFLP) map of tomato . RFLP analysis of 527 F2 plants segregating for the ls allele allowed us to define an interval of 0.8 cM in which the Ls gene is located . Analysis of the physical distance between the two flanking RFLP markers by pulsed field gel electrophoresis revealed that they lie no further than 375 kb apart . Knowledge of the physical distance together with the availability of a tomato yeast artificial chromosome (YAC) library, makes it feasible to isolate the Ls gene by a map-based cloning approach.

Genomics, 1995 Mar 20, 26(2), 379 - 81
Thioredoxin, a mediator of growth inhibition, maps to 9q31; Heppell-Parton A et al.; The human thioredoxin gene has been provisionally mapped to 3p11-p12 . Recently thioredoxin cDNA has been isolated in a procedure that detects transcripts coding for growth-suppressing proteins, and thus the chromosomal location of the gene is of particular interest . Chromosome 3 is believed to harbor several tumor suppressor genes important in the development of lung and other common epithelial tumors . To establish more firmly the chromosomal location of the human thioredoxin gene, a somatic hybrid panel was used; it identified chromosome 9 as the location of the transcribed thioredoxin gene . Fluorescence in situ hybridization of a YAC encoding the transcribed thioredoxin gene refined the localization to 9q31.

Genomics, 1995 Mar 20, 26(2), 265 - 71
Identification of a YAC spanning the translocation breakpoints in uterine leiomyomata, pulmonary chondroid hamartoma, and lipoma: physical mapping of the 12q14-q15 breakpoint region in uterine leiomyomata; Fejzo MS et al.; Uterine leiomyomata are the most common tumors in women and can cause abnormal uterine bleeding, pelvic pain, and infertility . Approximately 200,000 hysterectomies are performed annually in the U.S . to relieve patients of the medical sequelae of these benign neoplasms . Our efforts have focused on cloning the t(12;14)(q14-q15;q23-q24) breakpoint in uterine leiomyoma to further our understanding of the biology of these tumors . Thirty-nine YACs and six cosmids mapping to 12q14-q15 have been mapped by fluorescence in situ hybridization to tumor metaphase chromosomes containing a t(12;14) . One YAC spanned the translocation breakpoint and was mapped to tumor metaphases from a pulmonary chondroid hamartoma containing a t(12;14)(q14-q15;q23-q24) and a lipoma containing a t(12;15)(q15;q24); this YAC also spanned the breakpoint in these two tumors, suggesting that the same gene on chromosome 12 may be involved in the pathobiology of these distinct benign neoplasms.

Genomics, 1995 Mar 20, 26(2), 258 - 64
The gamma-aminobutyric acid receptor gamma 3 subunit gene (GABRG3) is tightly linked to the alpha 5 subunit gene (GABRA5) on human chromosome 15q11-q13 and is transcribed in the same orientation; Greger V et al.; GABAA receptors are heterooligomeric ligand-gated ion channels that mediate the effect of the inhibitory neurotransmitter gamma-aminobutyric acid . The GABAA receptors consist of at least 15 different receptor subunits that can be classified into 5 subfamilies (alpha, beta, gamma, delta, rho) on the basis of sequence similarity . Chromosomal mapping studies have revealed that several of the GABAA receptor subunit genes appear to be organized as clusters . One such cluster, which consists of the GABAA receptor beta 3 (GABRB3) and alpha 5 (GABRA5) subunit genes, is located in chromosome 15q11-q13 . It is shown here that the GABAA receptor gamma 3 subunit gene (GABRG3) also maps to this region . Lambda and P1 phage clones surrounding both ends of GABRG3 were isolated; the clones derived from the 5' end of GABRG3 were linked to an existing phage contig spanning the 3' end of GABRA5 . The two genes are located within 35 kb of each other and are transcribed in the same orientation.

Genomics, 1995 Mar 20, 26(2), 239 - 44
Cloning of the human heparan sulfate-N-deacetylase/N-sulfotransferase gene from the Treacher Collins syndrome candidate region at 5q32-q33.1; Dixon J et al.; Treacher Collins syndrome is an autosomal dominant disorder of craniofacial development, the features of which include conductive hearing loss and cleft palate . Previous studies have shown that the Treacher Collins syndrome locus is flanked by D5S519 and SPARC, and a yeast artificial chromosome contig encompassing this "critical region" has been completed . In the current investigation a cosmid containing D5S519 has been used to screen a human placental cDNA library . This has resulted in the cloning of the human heparan sulfate-N-deacetylase/N-sulfotransferase gene . Two different mRNA species that have identical protein coding sequences but that differ in the size and sequence of the 3' untranslated regions (3' UTR) have been identified . The smaller species has a 3' UTR of 1035 bp, whereas that of the larger is 4878 bp.

Genomics, 1995 Mar 20, 26(2), 229 - 38
High-density physical mapping of a 3-Mb region in Xp22.3 and refined localization of the gene for X-linked recessive chondrodysplasia punctata (CDPX1); Wang I et al.; The study of patients with chromosomal rearrangements has led to the mapping of the gene responsible for X-linked recessive chondrodysplasia punctata (CDPX1; MIM 302950) to the distal part of the Xp22.3 region, between the loci PABX and DXS31 . To refine this mapping, a yeast artificial chromosome (YAC) contig map spanning this region has been constructed . Together with the YAC contig of the pseudo-autosomal region that we previously established, this map covers the terminal 6 Mb of Xp, with an average density of 1 probe every 100 kb . Newly isolated probes that detect segmental X-Y homologies on Yp and Yq suggest multiple complex rearrangements of the ancestral pseudoautosomal region during evolution . Compilation of the data obtained from the study of individuals carrying various Xp22.3 deletions led us to conclude that the CDPX disease displays incomplete penetrance and, consequently, to refine the localization of CDPX1 to a 600-kb interval immediately adjacent to the pseudoautosomal boundary . This interval, in which 12 probes are ordered, provides the starting point for the isolation of CDPX1.

Genomics, 1995 Mar 20, 26(2), 192 - 8
A YAC contig map of Plasmodium falciparum chromosome 4: characterization of a DNA amplification between two recently separated isolates; Rubio JP et al.; We have generated a physical map of Plasmodium falciparum chromosome 4 using yeast artificial chromosomes (YACs) . The map is defined by a YAC contig spanning approximately 1.05 Mb, which has been restriction mapped to a resolution of 30 kb and is punctuated by 22 sequence-tagged sites . The physical information obtained has enabled us to compare and contrast the structure of chromosome 4 in detail between FCR3 and B8, two recently separated isolates of P . falciparum, leading to characterization of a novel chromosome polymorphism occurring in a subtelomeric region . Comparison of chromosomes 4 from 10 different isolates has shown that chromosome size polymorphisms are restricted to both subtelomeric regions . These analyses provide a high-resolution physical map that will be important to complement genetic analysis of this human pathogen.

J Biol Chem, 1995 Mar 17, 270(11), 5687 - 90
Structure of a conformationally constrained Arg-Gly-Asp sequence inserted into human lysozyme; Yamada T et al.; To examine the effect of a conformational constraint introduced into the Arg-Gly-Asp (RGD) sequence on cell adhesion activity, we constructed a mutant protein by inserting an RGD-containing sequence flanked by two Cys residues between Val74 and Asn75 of human lysozyme . The CRGDSC-inserted lysozyme was expressed in yeast, purified, and designated as Cys-RGD4 . Using baby hamster kidney cells, Cys-RGD4 was shown to possess even higher cell adhesion activity than that of the RGDS-inserted lysozyme, RGD4 . The Cys-RGD4 protein was co-crystallized with a lysozyme inhibitor, tri-N-acetylchitotriose, and the three-dimensional structure was determined at 1.6-A resolution by x-ray crystallography . In contrast to RGD4, the inserted RGD-containing region of Cys-RGD4 was well defined . The structural analysis revealed that the two inserted Cys residues form a new disulfide bond in Cys-RGD4, as expected, and that the RGD region assumes a type II' beta-turn conformation of Gly-Asp with a hydrogen bond between the C = O of Arg and the H-N of Ser . In addition, it was confirmed that two more hydrogen bonds are present in the RGD region of the Cys-RGD4 lysozyme . These results suggest that the conformation of the RGD-containing region is rigid and stable in the Cys-RGD4 molecule and that the type II' beta-turn structure of RGD is essential for binding to integrins with high affinity.

Nature, 1995 Mar 16, 374(6519), 280 - 2
Association of Cdk-activating kinase subunits with transcription factor TFIIH; Serizawa H et al.; The RNA polymerase II large subunit contains an essential carboxy-terminal domain (CTD) believed to be involved in the response to regulators during transcription initiation . The CTD is phosphorylated on a portion of RNA polymerase II molecules in vivo and it can be phosphorylated by the general transcription factor TFIIH in vitro . A highly purified TFIIH from rat liver has been described; this, like human and yeast TFIIH, contains associated CTD kinase and helicase activities . We report here that two polypeptides of the purified mammalian TFIIH are the MO15/Cdk7 kinase and cyclin H subunits of the Cdk-activating kinase Cak, previously identified as a positive regulator of Cdc2 and Cdk2 . TFIIH and Cak preparations are each capable of phosphorylating recombinant CTD and recombinant Cdk2 proteins . The presence of Cak in TFIIH indicates that Cak may have roles in transcriptional regulation and in cell-cycle control.

Oncogene, 1995 Mar 16, 10(6), 1095 - 101
A reciprocal translocation (1;15) (36.2;q24) in a neuroblastoma cell line is accompanied by DNA duplication and may signal the site of a putative tumor suppressor-gene; Amler LC et al.; Cytogenetic analyses and molecular deletion studies of human neuroblastomas have indicated the chromosomal bands 1p36.1-1p36.2 as a location of genetic information which may be involved in tumorigenesis . To define this putative neuroblastoma locus in more detail we have analysed cell lines with alterations of distal 1p . Here we show, by fluorescence in situ hybridization (FISH), that cell line NGP has a reciprocal 1;15 translocation . Loci D1S214/D1S96 could be shown to map telomeric/distal, D1S228 centromeric/proximal to the break . We have identified yeast artificial chromosomes (YACs) that cover the break and map to D1S160 and D1S244 . This chromosomal position is within the smallest region of overlap (SRO) found in neuroblastoma tumors (Weith et al., 1989; Caron et al., 1993; Schleiermacher et al., 1994) and within the region of a constitutional interstitial deletion of a neuroblastoma patient (Biegel et al., 1993) . Mapping studies with FISH revealed that the translocation is associated with duplication of DNA . It appears, as if the subchromosomal region we describe here is a good candidate for harboring the postulated neuroblastoma suppressor-gene.

EMBO J, 1995 Mar 15, 14(6), 1248 - 56
Different domains of the murine RNA polymerase I-specific termination factor mTTF-I serve distinct functions in transcription termination; Evers R et al.; Termination of mouse ribosomal gene transcription by RNA polymerase I (Pol I) requires the specific interaction of a DNA binding protein, mTTF-I, with an 18 bp sequence element located downstream of the rRNA coding region . Here we describe the molecular cloning and functional characterization of the cDNA encoding this transcription termination factor . Recombinant mTTF-I binds specifically to the murine terminator elements and terminates Pol I transcription in a reconstituted in vitro system . Deletion analysis has defined a modular structure of mTTF-I comprising a dispensable N-terminal half, a large C-terminal DNA binding region and an internal domain which is required for transcription termination . Significantly, the C-terminal region of mTTF-I reveals striking homology to the DNA binding domains of the proto-oncogene c-Myb and the yeast transcription factor Reb1p . Site-directed mutagenesis of one of the tryptophan residues that is conserved in the homology region of c-Myb, Reb1p and mTTF-I abolishes specific DNA binding, a finding which underscores the functional relevance of these residues in DNA-protein interactions.

EMBO J, 1995 Mar 15, 14(6), 1099 - 108
'Sheltered disruption' of Neurospora crassa MOM22, an essential component of the mitochondrial protein import complex; Nargang FE et al.; MOM22 is a component of the protein import complex of the mitochondrial outer membrane of Neurospora crassa . Using the newly developed procedure of 'sheltered disruption', we created a heterokaryotic strain harboring two nuclei, one with a null allele of the mom-22 gene and the other with a wild-type allele . Homokaryons bearing the mom-22 disruption could not be isolated, suggesting that mom-22 is an essential gene . The mutant nucleus can be forced to predominate in the heterokaryon through the use of specific nutritional and inhibitor resistance markers . Cultivation of the heterokaryon under conditions favoring the mutant nucleus resulted in selective depletion of MOM22 . MOM22-depleted cells did not grow and contained mitochondria with an altered morphology and protein composition . Protein import into isolated, MOM22-depleted mitochondria was abolished for most precursor proteins destined for all subcompartments . In contrast, precursors of MOM19, MOM22 and MOM72 became inserted normally into the outer membrane, defining a novel MOM22-independent import pathway which remained intact in mutant mitochondria . Furthermore, the specific binding of the ADP/ATP carrier to the outer membrane was unaffected, but subsequent transport across the outer membrane did not occur . Our data show that MOM22 is an essential component of Neurospora cells specifically required for the biogenesis of mitochondria.

Biochem J, 1995 Mar 15, 306 ( Pt 3), 643 - 50
Lack of glycosyl-phosphatidylinositol anchoring leads to precursor retention by a unique mechanism in Dictyostelium discoideum; Pauly PC et al.; Gp80, a cell-adhesion molecule in Dictyostelium discoideum, is modified by N- and O-linked oligosaccharides, and a glycosylphosphatidylinositol (GPI) anchor . To identify sequences important for the addition of these modifications to gp80, we created a hybrid protein in which the C-terminal 136 amino acids of yeast invertase were replaced by the C-terminal 110 amino acids of gp80 . When expressed in D . discoideum, this protein (Inv-gp80) was not GPI-anchored and was retained in a pre-Golgi compartment . Inv-gp80 did, however, display characteristics of a transmembrane protein, suggesting a novel mechanism for its retention . We also expressed a truncated version of the hybrid protein in which the C-terminal 22 amino acids of the Inv-gp80 were deleted . The truncated protein (Inv-gp80stop) was O-glycosylated and secreted . These observations indicate that the hybrid protein is not abnormally folded and demonstrate the importance of the C-terminal 22 amino acids in the retention of Inv-gp80 . Together, the data suggest that oligomerization of the protein blocks its GPI anchoring.

Nucleic Acids Res, 1995 Mar 11, 23(5), 725 - 8
Multiple RNA binding domains (RBDs) just don't add up; Shamoo Y et al.; One of the most common motifs for binding RNA in eukaryotes is the RNA binding domain (RBD) or RNA Recognition Motif (RRM) . One of the more intriguing aspects of these proteins is their modular nature . Proteins have been found containing from one to four RRMs . In most instances, these domains have some basal level of non-sequence specific RNA binding affinity . In addition, many also have a higher affinity for a specific structure or sequence of RNA . In the cases of heterogenous nuclear ribonucleoprotein A1 (hnRNP A1), yeast poly-A binding protein and splicing factor U2AF65, the individual free energy of binding of the RBDs for RNA are not strictly additive . By invoking a model in which the amino acids connecting adjoining RBDs are considered to be flexible linkers with an interresidue spacing of about 3.5 A, it is possible to predict the apparent association constants for at least some multi-RBD proteins to single-stranded RNA . We have surveyed the literature and found that individual RBDs are separated by 'linker' sequences of highly variable length . These linkers provide a critical determinant of binding affinity and may modulate cis versus trans binding . A clearer understanding of multi-RBD binding is essential to critically evaluating the role of these proteins in RNA splicing, packaging and transport.

Gene, 1995 Mar 10, 154(2), 271 - 5
Cloning and characterisation of multiple acetyl-CoA carboxylase transcripts in ovine adipose tissue; Barber MC et al.; A full-length ovine acetyl-CoA carboxylase-encoding cDNA (ACC) has been cloned from adipose tissue and completely sequenced . The open reading frame of 7041 nucleotides (nt) is highly homologous to the previously cloned human, rat, chicken, yeast and algal ACC (85, 89, 82, 54 and 54% identity, respectively) . Transcript heterogeneity was found in the 5' and 3' untranslated regions (UTR) resulting in ACC transcripts in the range of 9.0 kb to 9.4 kb . Heterogeneity at the 5' end was generated by the insertion of a 47-nt sequence, resulting in transcripts with either 272 or 319 nt in the 5'-UTR . Heterogeneity at the 3' end was the result of the use of different polyadenylation signals . RNase protection analysis demonstrated that shorter transcripts containing 1635 nt predominated over longer transcripts of 2065 nt in the 3'-UTR.

Gene, 1995 Mar 10, 154(2), 199 - 203
Xenopus laevis ribosomal protein L22: full-length cDNA sequence and expression analysis; Rapanotti MC et al.; A cDNA clone was isolated from a Xenopus laevis embryo library and sequenced . Primer extension experiments indicated the full-length nature of the insert and the encoded product was identified on a two dimensional gel as ribosomal protein (r-protein) L22 . The 510-bp L22 cDNA sequence presents short untranslated regions and a 5'-end polypyrimidine tract found in all other vertebrate r-protein mRNA (rp mRNA) so far analyzed . Both the nucleotide (nt) and the deduced amino acid (aa) sequences have been compared with the homologous sequences from other species . The L22 nt sequence is about 70% similar to the mammalian L27a rp mRNA and about 60% homologous to the Drosophila, Tetrahymena and yeast corresponding mRNAs . The 148-aa sequence presents a higher conservation, being 90% similar to the mammalian sequence and more than 70% to the other species . Expression analysis showed that, both during X . laevis embryogenesis and in X . laevis cultured cells during growth-rate changes, L22 synthesis is translationally regulated . Therefore X . laevis L22 mRNA is a new example of the correlation between the polypyrimidine terminal tract and the translational regulation observed in other rp mRNAs.

Cell, 1995 Mar 10, 80(5), 813 - 23
Defective DNA-dependent protein kinase activity is linked to V(D)J recombination and DNA repair defects associated with the murine scid mutation; Blunt T et al.; Murine cells homozygous for the severe combined immune deficiency mutation (scid) and V3 mutant hamster cells fall into the same complementation group and show similar defects in V(D)J recombination and DNA double-stranded break repair . Here we show that both cell types lack DNA-dependent protein kinase (DNA-PK) activity owing to defects in DNA-PKcs, the catalytic subunit of this enzyme . Furthermore, we demonstrate that yeast artificial chromosomes containing the DNA-PKcs gene complement both the DNA repair and recombination deficiencies of V3 cells, and we conclude that DNA-PKcs is encoded by the XRCC7 gene . As DNA-PK binds to DNA ends and is activated by these structures, our findings provide novel insights into V(D)J recombination and DNA repair processes.

Science, 1995 Mar 10, 267(5203), 1494 - 8
Involvement of CRAF1, a relative of TRAF, in CD40 signaling; Cheng G et al.; CD40 is a receptor on the surface of B lymphocytes, the activation of which leads to B cell survival, growth, and differentiation . A yeast two-hybrid screen identified a gene, CRAF1, encoding a protein that interacts directly with the CD40 cytoplasmic tail through a region of similarity to the tumor necrosis factor-alpha (TNF-alpha) receptor-associated factors . Overexpression of a truncated CRAF1 gene inhibited CD40-mediated up-regulation of CD23 . A region of CRAF1 was similar to the TNF-alpha receptor-associated factors TRAF1 and TRAF2 and so defined a shared TRAF-C domain that was necessary and sufficient for CD40 binding and homodimerization . The CRAF1 sequence also predicted a long amphipathic helix, a pattern of five zinc fingers, and a zinc ring finger . It is likely that other members of the TNF receptor superfamily use CRAF-related proteins in their signal transduction processes.

J Biol Chem, 1995 Mar 3, 270(9), 4748 - 52
The vitamin D receptor interacts with general transcription factor IIB; MacDonald PN et al.; The vitamin D receptor (VDR) heterodimerizes with retinoid X receptors (RXR) on many vitamin D-responsive promoter elements, suggesting that this complex is the active factor in vitamin D-mediated transcription . However, the mechanism of transcriptional regulation following VDR-RXR binding to DNA is not well characterized . Using a yeast two-hybrid protein interaction assay, we demonstrate that VDR forms specific protein: protein contacts with the basal transcription factor TFIIB . Deletion analysis indicated that the carboxyl-terminal ligand binding domain of VDR interacted with a 43-residue amino-terminal domain in TFIIB . The interaction with TFIIB showed selectivity for the ligand binding domain of VDR as similar regions of RXR alpha or of retinoic acid receptor alpha did not couple with TFIIB . Binding assays with purified proteins showed a direct interaction between VDR and TFIIB in vitro . These data suggest a mechanism for VDR-dependent transcription in which protein contacts between VDR and TFIIB may impart regulatory information to the transcription preinitiation complex.

J Biol Chem, 1995 Mar 3, 270(9), 4632 - 9
Molecular cloning of a human genomic region containing the H blood group alpha(1,2)fucosyltransferase gene and two H locus-related DNA restriction fragments . Isolation of a candidate for the human Secretor blood group locus; Rouquier S et al.; We have used the human H blood group alpha(1,2)fucosyltransferase (FUT1) cDNA to screen chromosome 19 cosmid libraries in a search for the human Secretor (Se) blood group gene (FUT2) . One cosmid has been isolated that contains two distinct segments that cross-hybridize with FUT1 . We have assembled a 100-kilobase (kb) cosmid contig, localized to 19q13.3, encompassing FUT1 and the two FUT1-related sequences, termed Sec1 and Sec2, for Secretor candidate 1 and 2 . Sec1 and Sec2 are separated by 12 kb and are 65.5 kb and 35 kb apart, respectively, from the FUT1 gene . We used a cosmid-dependent direct cDNA selection method to clone a cDNA corresponding to a transcript that emanates from Sec2 . This cDNA detects a 3.35-kb transcript in human tissues known to express the Se locus . Together with sequence and expression data reported in the accompanying article (Kelly, R . J., Rouquier, S., Giorgi, D., Lennon, G . G., and Lowe, J . B . (1995) J . Biol . Chem . 270, 4640-4649), these data demonstrate that Sec2 corresponds to the human Se blood group locus (FUT2) . Our results furthermore define the physical relationship between the H and Se loci and confirm a hypothesis that these two loci represent distinct but closely linked alpha(1,2)fucosyltransferase genes.

J Biol Chem, 1995 Mar 3, 270(9), 4575 - 87
Cloning of an SNF2/SWI2-related protein that binds specifically to the SPH motifs of the SV40 enhancer and to the HIV-1 promoter; Sheridan PL et al.; We have isolated a human cDNA clone encoding HIP116, a protein that binds to the SPH repeats of the SV40 enhancer and to the TATA/inhibitor region of the human immunodeficiency virus (HIV)-1 promoter . The predicted HIP116 protein is related to the yeast SNF2/SWI2 transcription factor and to other members of this extended family and contains seven domains similar to those found in the vaccinia NTP1 ATPase . Interestingly, HIP116 also contains a C3HC4 zinc-binding motif (RING finger) interspersed between the ATPase motifs in an arrangement similar to that found in the yeast RAD5 and RAD16 proteins . The HIP116 amino terminus is unique among the members of this family, and houses a specific DNA-binding domain . Antiserum raised against HIP116 recognizes a 116-kDa nuclear protein in Western blots and specifically supershifts SV40 and HIV-1 protein-DNA complexes in gel shift experiments . The binding site for HIP116 on the SV40 enhancer directly overlaps the site for TEF-1, and like TEF-1, binding of HIP116 to the SV40 enhancer is destroyed by mutations that inhibit SPH enhancer activity in vivo . Purified fractions of HIP116 display strong ATPase activity that is preferentially stimulated by SPH DNA and can be inhibited specifically by antibodies to HIP116 . These findings suggest that HIP116 might affect transcription, directly or indirectly, by acting as a DNA binding site-specific ATPase.

Oncogene, 1995 Mar 2, 10(5), 995 - 1001
Characterization of the region of the short arm of chromosome 8 amplified in breast carcinoma; Dib A et al.; Chromosomal region 8p11.2-p12 is consistently amplified in human breast cancer . We have constructed a 2.8 Mb YAC contig of this region, centered on the human Fibroblast Growth Factor Receptor 1 (FGFR1) locus and encompassing the Adrenergic beta 3 Receptor (ADRB3) locus . A smaller centromeric YAC contig spanning 1.4 Mb was also assembled, and included the Ankyrin 1 (ANK1) and Tissue-type Plasminogen Activator (PLAT) genes . Results from mapping of the contigs showed physical linkage of the ADRB3 and FGFR1 genes, which were colocalized within the same YAC clone and separated by about 900 kb, FGFR1 being in centromeric position . It also showed physical linkage of ANK1 and PLAT genes, which appear to be separated by a maximum of 700 kb . In parallel, several loci were mapped according to their amplification status in a large panel of breast tumor samples . The overall amplification pattern suggested a continuous amplicon with a core around FGFR1 . Data from both the detailed physical map and the amplification status allowed to establish the following gene order, from telomere to centromere: ADRB3-D8S105-FGFR1-ANK1-PLAT-POLB . The precise localization and YAC cloning of the core of the amplicon will allow to isolate a putative oncogene involved in mammary carcinogenesis.

Nature, 1995 Mar 2, 374(6517), 62 - 4
A type VII myosin encoded by the mouse deafness gene shaker-1; Gibson F et al.; Genetic deafness is common, affecting about 1 in 2,000 births . Many of these show primary abnormalities of the sensory neuroepithelia of the inner ear, as do several hearing-impaired mouse mutants, suggesting that genes involved in sensory transduction could be affected . Here we report the identification of one such gene, the mouse shaker-1 (sh1) gene . Shaker-1 homozygotes show hyperactivity, head-tossing and circling due to vestibular dysfunction, together with typical neuroepithelial-type cochlear defects involving dysfunction and progressive degeneration of the organ of Corti . The sh1 gene encodes an unconventional myosin molecule of the type VII family . Three mutations are described, two mis-sense mutations and a splice acceptor site mutation, all in the region encoding the myosin head . The myosin type VII molecule encoded by sh1 is the first molecule to be identified that is known, by virtue of its mutations, to be involved in auditory transduction.

Exp Gerontol, 1995 Mar-Apr, 30(2), 99 - 124
Prohibitin: potential role in senescence, development, and tumor suppression; McClung JK et al.; Prohibitin is an evolutionarily conserved gene with homologues found in organisms ranging from yeast to man . In man the gene is located on chromosome 17 at q21 . The deduced amino acid sequences of the protein products from mouse and rat are identical; and these differ from the human protein sequence by a single conserved amino acid . Prohibitin has antiproliferative activity and available data suggest a role in such diverse processes as normal cell cycle regulation, replicative senescence, cellular immortalization, and the development of sporadic breast tumors . Although its functional activity is presently unknown, the 30,000-Da protein has been located in the inner membrane of mitochondria, where it is postsynthetically modified, as well as on the plasma membrane of B cells, where it is associated with the IgM receptor . Prohibitin's evolutionary conservation and ubiquitous expression indicate that it is a fundamentally important gene; and current data suggest a functional role in such dissimilar processes as development, senescence, and tumor suppression.

Int J Radiat Biol, 1995 Mar, 67(3), 303 - 13
Linear dose-response relationship and no inverse dose-rate effect observed for low X-ray dose-induced mitotic recombination in Drosophila melanogaster; Schweizer PM; Mitotic recombination has emerged lately as a surprisingly common cause of recessive functional gene loss in mammalian cells and has been implicated in tumour suppressor gene loss in human neoplasms . In an assay, primarily monitoring mitotic recombination in Drosophila melanogaster, the ability of low dose acute- and chronic X-ray irradiation to induce clonal expression of recessive mutations of formally heterozygous loci was investigated . Mosaic spots of recessive wing-hair misshape mutations (mwh and flr) and of hair-into-bristles transforming mutation (zw3tic) were enhanced by a factor of two over control level following irradiation of heterozygous larvae to doses as low as 0.01, 0.03 or 0.1 Gy X-rays . The frequencies of mosaic spots induced with eight doses in the interval 0.01-2.0 Gy was linearly related to the dose . The regression lines show no significant intercept at zero dose . During the entire larval developmental period exposure of the exponentially growing target cell population to conditions of chronic irradiation at dose-rate of 15.7 x 10(-5) Gy/min provided no evidence of an inverse dose-rate effect as reported in yeast . In Drosophila, the probability of mitotic recombination per induced DNA double-strand break appears to be at least one order of magnitude higher than in man.

Hum Genet, 1995 Mar, 95(3), 342 - 6
Refinement of the X-linked cleft palate and ankyloglossia (CPX) localisation by genetic mapping in an Icelandic kindred; Forbes SA et al.; The gene responsible for X-linked cleft palate and ankyloglossia (CPX) has previously been localized to the proximal region of the q arm of the X chromosome in both Icelandic and North American Indian kindreds . In this study, further linkage analysis has been performed on the Icelandic family and has resulted in a significant reduction in the size of the interval containing the mutated gene . A new polymorphism at DXS95, together with DXS1002 and DXS349, defines the proximal boundary of the CPX interval, whereas DXYS1X defines the distal boundary . Multipoint analysis supports this localisation with a peak lod score of 12.7, more than 2 lod score units higher than the next most likely position . In order to assess the physical size of the CPX interval prior to initiating yeast artificial chromosome cloning, metaphase fluorescence in situ hybridisation analysis was performed with the closest flanking markers . The size of the interval between DXS95 and DXYS1X was estimated to be approximately 2-3 Mb.

Hum Genet, 1995 Mar, 95(3), 303 - 7
The breakpoint on 7p in a patient with t(6;7) and craniosynostosis is spanned by a YAC clone containing the D7S503 locus; Tsuji K et al.; We previously reported a patient with an apparently balanced t(6;7) translocation and craniosynostosis . We now demonstrate, by fluorescence in situ hybridization, that the yeast artificial chromosome clone 933-e-1 from the Centre d'Etude du Polymorphisme Humain library harbouring the D7S503 locus spans the breakpoint on distal 7p . Recent reports have defined a candidate region for a Saethre-Chotzen craniosynostosis locus between the loci D7S513 and D7S516, a region that includes the D7S503 locus . Since the translocation carrier shows only some of the symptoms characteristic for the Saethre-Chotzen syndrome, it remains unresolved whether the gene disrupted by the translocation event is the only one causing craniosynostosis in this chromosomal region.

Hum Genet, 1995 Mar, 95(3), 287 - 92
Analysis of pericentromeric chromosome 21 specific YAC clones by FISH: identification of new markers for molecular-cytogenetic application; Yurov YB et al.; Fluorescence in situ hybridization (FISH) of chromosome 21 specific yeast artificial chromosome (YAC) clones after Alu-PCR (polymerase chain reaction) amplification has been used to find new region-specific DNA probes for the heterochromatic region of chromosome 21 . Six overlapping YAC clones from a pericentromeric contig map (region 21cen-21q11) were analyzed . Four YAC clones were characterized as hybridizing to several chromosomal locations . They are, therefore, either chimeric or shared by different chromosomes . Two of them containing alphoid satellite DNA, are localized at the centromeric regions of chromosomes 13 and 21 (clone 243A11), and on 13cen, 21cen and 1q3 (clone 781G5); the two others are localized at both 21q11 and 13q2 (clone 759D3), and at 18p (clone 770B3) . Two YACs were strongly specific for chromosome 21q11 only (clones 124A7 and 881D2) . These YACs were used effectively as probes for identifications of chromosome 21 during metaphase and interphase analysis of 12 individuals, including three families with Down syndrome offspring, and 6 aminocyte samples . The location of YAC clones on 21q11 close to the centromeric region allows the application of these clones as molecular probes for the analysis of marker chromosomes with partial deletions of the long arm as well as for pre- and postnatal diagnosis of trisomy 21 when alphoid or more distal region-specific DNA probes are uninformative . Overlapping YAC clones covering human chromosome 21q may be systematically used to detect a set of band-specific DNA probes for molecular-cytogenetic application.

Cell Immunol, 1995 Mar, 161(1), 61 - 71
Different subcellular localization of cytochrome b and the dormant NADPH-oxidase in neutrophils and macrophages: effect on the production of reactive oxygen species during phagocytosis; Johansson A et al.; When neutrophils and macrophages phagocytose a prey, e.g., complement (C3b)-opsonized yeast particles, the oxygen radical generating NADPH-oxidase is activated . In neutrophils, most of the production of oxygen metabolites occurred in an intracellular compartment, possibly in the phagolysosome . In contrast, no intracellular production could be detected in human macrophages . In these cells, the subcellular localization of the superoxide-generating NADPH-oxidase and associated cytochrome b was assessed in intact cells with indirect immunofluorescence and confocal laser scanning microscopy, and with subcellular fractionation, using centrifugation on Percoll density gradients . A dual localization of the cytochrome b as well as the dormant NADPH-oxidase activity in neutrophils was in agreement with earlier immunocytochemical, biochemical, and subcellular fractionation studies . Furthermore, most of the activity was recovered from the specific granules, whereas only a small fraction was retained in the plasma membrane . In contrast, the cytochrome b/NADPH-oxidase activity in macrophages localized primarily in the plasma membrane fraction . We suggest that the macrophages are incapable of producing reactive oxygen species intraphagosomally, due to an absence of a granule-localized pool of the membrane components of the NADPH-oxidase.

Cancer Res, 1995 Mar 1, 55(5), 984 - 8
Detection of CDKN2 deletions in tumor cell lines and primary glioma by interphase fluorescence in situ hybridization; Dreyling MH et al.; Deletions of chromosomal band 9p21 have been detected in various tumor types including melanoma, glioma, lung cancer, mesothelioma, and bladder cancer . Recently, the CDKN2 gene (p16INK4A, MTS I, CDK41) has been proposed as a candidate tumor suppressor gene because it is frequently deleted in cell lines derived from multiple tumor types . We performed fluorescence in situ hybridization (FISH) with interphase cells using yeast artificial chromosome clones and a cosmid contig of the CDKN2 region . In 10 cell lines (4 glioma, 2 melanoma, 2 non-small cell lung cancer, 2 bladder cancer) with 9p alterations detected by molecular or cytogenetic analysis, interphase FISH with the CDKN2 cosmid contig detected all 9p deletions previously identified by molecular analysis . Using this probe, FISH analysis of primary glioblastoma tumors revealed homozygous deletions of the CDKN2 region in 6 of 9 tumors (67%) whereas a yeast artificial chromosome probe containing the interferon type I (IFN) gene cluster was deleted in only 4 cases (44%) . Thus, it is likely that the CDKN2 region is the target of 9p deletions in gliomas . Interphase FISH will play an important role in defining the clinical significance of 9p deletions in primary tumors because it is especially applicable to clinical samples which may be contaminated by normal cells.

Hum Mol Genet, 1995 Mar, 4(3), 415 - 22
Aniridia-associated cytogenetic rearrangements suggest that a position effect may cause the mutant phenotype; Fantes J et al.; Current evidence suggests that aniridia (absence of iris) is caused by loss of function of one copy of the PAX6 gene, which maps to 11p13 . We present the further characterisation of two aniridia pedigrees in which the disease segregates with chromosomal rearrangements which involve 11p13 but do not disrupt the PAX6 gene . We have isolated three human YAC clones which encompass the PAX6 locus and we have used these to show that in both cases the chromosomal breakpoint is at least 85 kb distal of the 3' end of PAX6 . In addition, the open reading frame of PAX6 is apparently free of mutations . We propose that the PAX6 gene on the rearranged chromosome 11 is in an inappropriate chromatin environment for normal expression and therefore that a 'position effect' is the underlying mechanism of disease in these families.

Protein Sci, 1995 Mar, 4(3), 521 - 33
Transmembrane helices predicted at 95% accuracy; Rost B et al.; We describe a neural network system that predicts the locations of transmembrane helices in integral membrane proteins . By using evolutionary information as input to the network system, the method significantly improved on a previously published neural network prediction method that had been based on single sequence information . The input data were derived from multiple alignments for each position in a window of 13 adjacent residues: amino acid frequency, conservation weights, number of insertions and deletions, and position of the window with respect to the ends of the protein chain . Additional input was the amino acid composition and length of the whole protein . A rigorous cross-validation test on 69 proteins with experimentally determined locations of transmembrane segments yielded an overall two-state per-residue accuracy of 95% . About 94% of all segments were predicted correctly . When applied to known globular proteins as a negative control, the network system incorrectly predicted fewer than 5% of globular proteins as having transmembrane helices . The method was applied to all 269 open reading frames from the complete yeast VIII chromosome . For 59 of these, at least two transmembrane helices were predicted . Thus, the prediction is that about one-fourth of all proteins from yeast VIII contain one transmembrane helix, and some 20%, more than one.

Ger J Ophthalmol, 1995 Mar, 4(2), 107 - 15
Involvement of anterior chamber angle structures in disseminated histoplasmosis: report of three cases; Font RL et al.; This study describes the involvement of anterior chamber (AC) angle structures in patients with disseminated histoplasmosis . The postmortem eyes from three patients (aged 33, 41, and 42 years, respectively) with disseminated histoplasmosis, two of whom had acquired immunodeficiency syndrome, were examined by light microscopy using hematoxylin-eosin, periodic acid-Schiff (PAS), and Gomori's methenamide silver (GMS) stains . Electron microscopy studies of the choroid were performed in one eye . Significant numbers of budding yeast forms of Histoplasma capsulatum measuring 2-5 microns in diameter were observed within the trabecular meshwork, Schlemm's canal and in the deep intrascleral plexuses . All eyes showed massive involvement of the choroidal vasculature, including the choriocapillaris . The organisms were observed freely as well as in small clusters within the cytoplasm of circulating monocytes . The vessels of the limbal conjunctiva (two eyes) and ciliary body (three eyes) contained many Histoplasma organisms . In one eye, several budding yeast were noted in an iris vessel and in occasional histiocytes within the ciliary muscle . Blood smears containing Histoplasma organisms were observed in two cases . None of the patients had an ophthalmologic examination prior to death . Involvement of the intravascular structures of the eye as well as the AC angle was observed in three patients with disseminated histoplasmosis . The fungus most likely reached the AC angle structures by direct hematogenous dissemination or via the aqueous humor by migration from vessels in the ciliary body and iris . An abnormal retrograde blood flow into the AC angle structures may have also played an important role . We suggested that the intraocular pressure be monitored in cases of suspected disseminated histoplasmosis to detect functional alterations indicative of a blockage in the outflow channels.

Biomed Chromatogr, 1995 Mar-Apr, 9(2), 80 - 4
Dye-ligand affinity chromatography on continuous beds; Mohammad J et al.; Continuous beds derivatized with three triazine dyes (Cibacron Blue 3G-A, Pricon Red HE-3B and Pricon Red H-3B) were used for chromatographic purification of dehydrogenases from yeast enzyme concentrate . All three columns, which were prepared by a very simple and cost-effective method, provided strong binding of glucose-6-phosphate dehydrogenase and lactate dehydrogenase . The Cibacron Blue 3G-A column showed high affinity for alcohol dehydrogenase . Under the same conditions, the Pricon Red HE-3B column showed a lower affinity and the Pricon Red H-3B column showed none . The adsorbed dehydrogenases were eluted specifically from the columns in high yields (71-113% by desorption with the coenzymes NADP, NADH and NAD respectively) . Non-specific binding of human serum albumin and transferrin to these columns was also investigated . Enzyme assays and analyses by capillary electrophoresis showed that the continuous beds derivatized with triazine dyes gave a high degree of purification.

Lipids, 1995 Mar, 30(3), 235 - 46
2,3-Oxidosqualene cyclase: from azasqualenes to new site-directed inhibitors; Cattel L et al.; 2,3-Oxidosqualene cyclases (OSC) are enzymes which convert 2,3-oxidosqualene (OS) into polycyclic triterpenoids such as lanosterol, cycloartenol, and alpha- and beta-amyrin . Our interest in the study of OSC is the development of new OSC inhibitors for potential use as hypocholesterolemic, antifungal, or phytotoxic drugs . In particular, we describe the biological activity and the mechanism of a series of acyclic azasqualene derivatives mimicking the C-2, C-8, and C-20 carbonium ions formed during OS cyclization . Some of these carbonium ion analogues are very promising as specific hypocholesterolemic agents . The toxicity, the biodistribution, and the pharmacokinetics of different azasqualene derivatives in mice are also presented . In order to obtain new, site-directed irreversible inhibitors of OSC, a series of squalene derivatives containing functional groups that can link covalently to an active-site thiol group was designed . Among these compounds, squalene maleimide was the most active toward mammalian OSC, whereas squalene Ellman behaved as an irreversible inhibitor of OSC from yeast.

J Med Genet, 1995 Mar, 32(3), 231 - 3
Assignment of microsatellite sequences to the region duplicated in CMT1A (17p12): a useful tool for diagnosis; Cudrey C et al.; Charcot-Marie-Tooth disease type 1A (CMT1A), the most prevalent form of the peripheral hereditary neuropathies, has been associated with a duplication of a genomic segment of 1.5 Mb, located in 17p11.2 . Recently, the same segment has been found to be deleted in patients with another peripheral neuropathy, hereditary neuropathy with liability to pressure palsies (HNPP) . Highly polymorphic markers are rare in this area, rendering the diagnosis highly dependent either on invasive examinations (like nerve biopsy) or not totally reliable (like gene dosage) . Thus, we used a contig of YACs, including the whole region duplicated in CMT1A, to map highly polymorphic microsatellite loci, designed in Genethon . We showed that four of these loci are located in the duplicated region, allowing us to propose them as diagnostic markers for CMT1A and HNPP.

Genomics, 1995 Mar 1, 26(1), 9 - 20
Localization of new genes and markers to the distal part of the human major histocompatibility complex (MHC) region and comparison with the mouse: new insights into the evolution of mammalian genomes; Amadou C et al.; We have refined and extended the map of the distal half of the human major histocompatibility complex . The map is continuous from HLA-E to 1000 kb telomeric of HLA-F and includes six new markers and genes . In addition, the corresponding sequences that were not previously mapped in the mouse genome have been located . The human and the mouse organizations have therefore been compared . This comparison allows us to demonstrate that the structure of the distal part of the MHC is similar in the two species . In addition, this comparison shows the presence of a breakpoint of synteny telomeric of the distal part of the H-2 region . Indeed, the region telomeric of HLA in human is found on a chromosome different from that carrying H-2 in mouse . The mapping analysis of paralogous genes (structurally related genes) around the breakpoint shows that the human organization probably represents the putative human/mouse ancestral one . This evolutionary breakpoint was precisely mapped in human, and the surrounding region was cloned into yeast artificial chromosomes . Finally, we show that the region found around the breakpoint was involved several times in chromosome recombinations in the mouse lineage, as it seems to correspond also to the t-complex distal inversion point.

Genomics, 1995 Mar 1, 26(1), 31 - 8
Structure of the terminal 300 kb of DNA from human chromosome 21q; Reston JT et al.; The most distal 300 kb of human chromosome 21q was cloned and mapped using telomeric yeast artificial chromosomes (YACs) . The region contains low-copy subtelomeric repeats at the telomeric end, chromosome 21-specific sequences more centromerically, and the S100B gene at a distance of 100-140 kb from the chromosome terminus . RecA-assisted restriction endonuclease cleavage of genomic DNA showed that the cloned fragments correspond to telomere-terminal genomic DNA, and restriction enzyme mapping of the YACs shows that the smaller clone (175 kb) corresponds exactly to the telomeric end of the larger one (300 kb) . PCR assays for 21q-specific markers were used to show that COL6A1, COL6A2, and LA161 were all outside of the subtelomeric region spanned by the YACs and thus at least 300 kb from the 21q terminus . The molecular probes provide telomeric closure for existing 21q maps.

Genomics, 1995 Mar 1, 26(1), 21 - 30
Efficient pooling designs for library screening; Bruno WJ et al.; We describe efficient methods for screening clone libraries, based on pooling schemes that we call "random k-sets designs." In these designs, the pools in which any clone occurs are equally likely to be any possible selection of k from the v pools . The values of k and v can be chosen to optimize desirable properties . Random k-sets designs have substantial advantages over alternative pooling schemes: they are efficient, flexible, and easy to specify, require fewer pools, and have error-correcting and error-detecting capabilities . In addition, screening can often be achieved in only one pass, thus facilitating automation . For design comparison, we assume a binomial distribution for the number of "positive" clones, with parameters n, the number of clones, and c, the coverage . We propose the expected number of resolved positive clones--clones that are definitely positive based upon the pool assays--as a criterion for the efficiency of a pooling design . We determine the value of k that is optimal, with respect to this criterion, as a function of v, n, and c . We also describe superior k-sets designs called k-sets packing designs . As an illustration, we discuss a robotically implemented design for a 2.5-fold-coverage, human chromosome 16 YAC library of n = 1298 clones . We also estimate the probability that each clone is positive, given the pool-assay data and a model for experimental errors.

Genomics, 1995 Mar 1, 26(1), 115 - 22
Construction of a YAC contig spanning the Xq13.3 subband; Villard L et al.; The loci involved in several X-linked mental retardation syndromes have been linked to the pericentromeric region of the X chromosome long arm (Xq12-q21) . To isolate candidate genes for these diseases, we set up the construction of YAC contigs spanning this region . Two of these syndromes (the Juberg-Marsidi syndrome and the alpha-thalessemia mental retardation syndrome) have been recently linked, with high lod scores, to polymorphic probes previously assigned to Xq13.3 . We therefore constructed a first YAC contig, encompassing this band, from DXS441 to PGK1 . The physical map, deduced from the isolated clones, extends over 2.1 Mb of genomic DNA . Restriction analysis of the YAC contig allowed us to map precisely the loci previously assigned to that chromosomal region and to define their relative order . The validity of this physical map has been checked by comparing Sfi I digests of the YACs to genomic fragments obtained with the same enzyme . A cDNA selection approach, already performed with a previous partial contig, has been extended to cover the whole region.

Plant Mol Biol, 1995 Mar, 27(6), 1221 - 6
Isolation and characterization of gmsti, a stress-inducible gene from soybean (Glycine max) coding for a protein belonging to the TPR (tetratricopeptide repeats) family; Hernandez Torres J et al.; In close vicinity of two fus nuclear genes (chloroplast-specific translation elongation factor cEF-G) of soybean (Glycine max) we localized a split nuclear gene coding for a protein with tetratricopeptide repeats (TPR) . A full-length cDNA was sequenced (1871 nucleotides) . It encodes a protein (569 amino acids) with high sequence identity to the yeast STI1 stress-inducible and the human transformation-sensitive IEF SSP 3521 protein which both carry TPR elements . The soybean gene is heat-inducible . This is the first evidence for the existence of plant genes coding for proteins which belong to the TPR family . We call the gene gmsti and the protein GMSTI in analogy to the yeast counterpart.

Res Vet Sci, 1995 Mar, 58(2), 133 - 7
Evaluation of a detergent scrub technique for the quantitative culture of Malassezia pachydermatis from canine skin; Bond R et al.; A detergent scrub technique using wash fluid consisting of 0.075 M phosphate-buffered saline, pH 7.9 containing 0.1 per cent Triton X-100 was evaluated for the quantitative culture of Malassezia pachydermatis from canine skin . Preliminary studies showed that the detergents Triton X-100, Tween 40 and Tween 80 were equally able to disperse suspensions of pure cultures of M pachydermatis, but that the yeast counts were significantly reduced (P < 0.001) after suspension in saline, Triton X-100 or Tween 40 for two hours . The counts in skin washings were also reduced (P < 0.001) after suspension for three hours in 0.1 and 0.05 per cent solutions of Triton X-100 . Vortexing, or manual or mechanical shaking of the samples yielded comparable counts . The correlation between the counts on diseased skin measured by using detergent scrubs and a contact plate technique was highly significant (P < 0.001) . The detergent scrub technique was suitable for the quantitation of M pachydermatis on canine skin provided that the samples were processed without delay.

Mol Plant Microbe Interact, 1995 Mar-Apr, 8(2), 200 - 6
High-resolution mapping of the physical location of the tomato Cf-2 gene; Dixon MS et al.; To isolate the tomato Cf-2 resistance gene by map-based cloning, plants recombinant for RFLP markers close to Cf-2 were selected by exploiting the flanking morphological markers yv (yellow virescent) and tl (thiaminless) . Using these recombinants, a high-resolution linkage map of the region encompassing the Cf-2 gene has been generated containing several new RFLP markers . Mapping of two YAC clones carrying Lycopersicon esculentum and L . peruvianum DNA, indicates that in both genotypes the physical distance between the two closet flanking markers is less than 40 kb . This study also positions Cf-2 relative to the Mi gene, which confers resistance to root-knot nematodes.

Clin Infect Dis, 1995 Mar, 20(3), 692 - 5
Clinical evidence of spinal and cerebral histoplasmosis twenty years after renal transplantation; Livas IC et al.; Disseminated infection with Histoplasma capsulatum frequently involves the nervous system, but the CNS process is generally not clinically apparent . We report an unusual case of a renal transplant recipient with long-standing immunosuppression who presented with clinical evidence of mass lesions in both his cerebral cortex and his spinal cord . Findings of CSF examination were normal, but stereotaxic biopsies of his cortical lesions demonstrated yeast forms and cultures of biopsy specimens yielded H . capsulatum . Clinical defects referable to both the cortical and spinal lesions decreased in severity after the patient received antifungal therapy . Our case illustrates that disseminated histoplasmosis can present in myriad ways and that widespread disease in the CNS can be occult in immunocompromised patients.

Chromosome Res, 1995 Mar, 3(2), 137 - 8
Establishment of the marker order pter-NRAS-NGFB-D1S189-D1S252-D1S440-D1S453-D1S514-CEN-D1S442-D1S498-qte r in relation to the centromere on human chromosome 1; Hoggard N et al.; Recent developments in genetic linkage mapping of the human genome have generated a large number of short tandem repeat polymorphic markers (Weissenbach et al . 1992, Gyapay et al . 1994), and eventual integration of these markers into a physical map is a logical progression . A number of Genethon microsatellite (CA repeat) markers have been provisionally localized to 1p13, but their exact position with respect to other sequences is unknown . In order to confirm the order of these markers and their position with respect to known genes within 1p13 and the centromere, we have isolated yeast artificial chromosomes (YACs) corresponding to the markers and have carried out double and triple fluorescence in situ hybridization (FISH) studies . Knowledge of both the order of microsatellite markers and their integration with a physical map of known genes can be an essential component in analysis of disease loci such as human cancer, where regions of chromosomes showing high levels of loss of heterozygosity need to be mapped in detail.

Development, 1995 Mar, 121(3), 903 - 14
A dominant inhibitory version of the small GTP-binding protein Rac disrupts cytoskeletal structures and inhibits developmental cell shape changes in Drosophila; Harden N et al.; The Rho subfamily of Ras-related small GTP-binding proteins is involved in regulation of the cytoskeleton . The cytoskeletal changes induced by two members of this subfamily, Rho and Rac, in response to growth factor stimulation, have dramatic effects on cell morphology . We are interested in using Drosophila as a system for studying how such effects participate in development . We have identified two Drosophila genes, DRacA and DRacB, encoding proteins with homology to mammalian Rac1 and Rac2 . We have made transgenic flies bearing dominant inhibitory (N17DRacA), and wild-type versions of the DRacA cDNA under control of an Hsp70 promoter . Expression of the N17DRacA transgene during embryonic development causes a high frequency of defects in dorsal closure which are due to disruption of cell shape changes in the lateral epidermis . Embryonic expression of N17DRacA also affects germband retraction and head involution . The epidermal cell shape defects caused by expression of N17DRacA are accompanied by disruption of a localized accumulation of actin and myosin thought to be driving epidermal cell shape change . Thus the Rho subfamily may be generating localized changes in the cytoskeleton during Drosophila development in a similar fashion to that seen in mammalian and yeast cells . The Rho subfamily is likely to be participating in a wide range of developmental processes in Drosophila through its regulation of the cytoskeleton.

Plant Physiol, 1995 Mar, 107(3), 905 - 13
Cloning and expression analysis of the cytosolic NADP(+)-dependent isocitrate dehydrogenase from potato . Implications for nitrogen metabolism; Fieuw S et al.; A full-length cDNA (icdh-1) encoding a cytosolic NADP(+)-dependent isocitrate dehydrogenase (ICDH-1) from potato (Solanum tuberosum L.) has been isolated . Analysis of the deduced protein sequence revealed considerable homologies with the corresponding proteins from other eukaryotes such as tobacco, alfalfa, soybean, cattle, pig, and yeast . The gene was transcribed in all tissues tested, with the highest amount of icdh-1 transcript being found in green tissues, in flowers, and in roots . In leaves, enzyme activities were dependent on the age, with fully mature leaves showing the highest level of RNA expression and enzyme activity . This observation may indicate that NADP(+)-dependent ICDH is not only involved in amino acid biosynthesis via the glutamine synthetase/glutamine oxoglutarate aminotransferase cycle but also in cycling, redistribution, and export of amino acids . The latter assumption has been strengthened by our finding of a preferential expression of NADP(+)-dependent ICDH in leaf veins . Under in vivo conditions, the expression pattern paralleled the enzyme activity, indicating coarse control on the RNA level . Experiments carried out with detached leaves revealed an influence of light, nitrate, and sucrose on icdh-1 transcript levels and in some cases also on NADP(+)-dependent ICDH activity . In darkness, nitrate or sucrose induced icdh-1 mRNA expression . Leaves kept under starvation conditions exhibited a decrease of their protein content, whereas icdh-1 expression and ICDH activity increased significantly.

Trends Biochem Sci, 1995 Mar, 20(3), 117 - 22
Parallel signal processing among mammalian MAPKs; Cano E et al.; The intracellular signalling field is dominated by the mitogen-activated protein kinase (MAPK) cascade and its control, which involves the small GTPase Ras and sequential kinase activation . Until recently, ERK1 and ERK2 were the only cloned and well-characterized mammalian MAPKs; diverse ligand-stimulated, proline-directed protein phosphorylation events were attributed to these kinases . The recent discovery of two other MAPK subtypes, the JNK/SAPK subfamily and p38/RK (mammalian equivalents of HOG1 in yeast), reveals extreme complexity within the family and, most intriguingly, the existence in mammalian cells of parallel MAPK cascades that can be activated simultaneously.

Eur J Biochem, 1995 Mar 1, 228(2), 456 - 62
Purification, cDNA cloning and characterization of proteinase B, an asparagine-specific endopeptidase from germinating vetch (Vicia sativa L.) seeds; Becker C et al.; Proteinase B, an asparagine-specific endopeptidase, has been purified from germinating vetch (Vicia sativa L.) seeds . The final preparation consists of two enzymically active proteins with molecular masses of approximately 39 kDa and 37 kDa . Synthetic substrates were used to confirm cleavage specificity of the proteinase B preparation . As expected, the enzyme cleaves the substrates at the C-terminal side of Asn residues . The octapeptide ETRNGVEE was digested most efficiently . When Gly was replaced by Ile or Glu, cleavage took place with lower efficiency . Polyclonal antibodies displayed both proteins in cotyledon extracts of germinated vetch seeds . In addition, a strong cross-reacting protein band was found in cotyledon extracts of developing seeds, indicating the presence of a very similar enzyme during seed development . cDNA clones encoding proteinase B precursor have been obtained on the basis of the N-terminal amino acid sequence DDDFEGTRWAILLAGS, by means of the polymerase chain reaction . The cDNA clones contain an open reading frame of 1479 bp encoding a polypeptide of 493 amino acids . The precursor displayed 59% sequence identity to the cDNA-derived amino acid sequence of a vacuolar Asn-specific enzyme from the developing castor beam endosperm which is thought to catalyze the post-translational processing of pro-proteins into the mature forms . Proteinase B is synthesized de novo during seed germination . The results of Southern-blot analyses suggested that there are at least two genes for proteinase B.

Eur J Biochem, 1995 Mar 1, 228(2), 431 - 8
Interactions of human nuclear proteins P1Mcm3 and P1Cdc46; Burkhart R et al.; Human nuclear proteins P1Mcm3 and P1Cdc46 have high sequence similarities with the corresponding yeast proteins known to be required for the initiation of genome replication . Nuclei of proliferating HeLa cells contain relatively high amounts of P1Mcm3 (about 10(6) molecules/nucleus) of which only a small fraction is bound to a nuclear structure, most probably chromatin . At 0.5 M NaCl, the structure-bound nuclear protein can be partially solubilized as a dimer composed of P1Mcm3 and the related protein P1Cdc46 . However, most protein P1Mcm3 is not bound to a nuclear structure and appears in the nucleoplasm . About 10% of protein P1Mcm3 in the soluble fraction is free and uncomplexed, and the remaining P1Mcm3 forms stable complexes with protein P1Cdc46 . These P1Mcm3/Cdc46 complexes occur as dimers and in high-molecular-mass complexes (approximately 500 kDa) . The high-molecular-mass complexes dissociate in 0.5 M NaCl and release P1Mcm3/Cdc46 dimers . It has frequently been proposed that the Mcm proteins may function as licensing factors for genome replication . Our data imply that the active form of an Mcm protein is not a monomer, but a protein complex that includes an Mcm3/Cdc46 dimer . DNA polymerase alpha is not a component of this complex.

Mutat Res, 1995 Mar, 346(3), 173 - 9
Genotoxic potential of psoralen cross-links versus monoadducts in normal human lymphoblasts; Laquerbe A et al.; Using the 4,5',8-trimethylpsoralen in combination with the reirradiation protocol, we show that, in normal human lymphoblasts, the cytotoxic potential of photoinduced cross-links (CL) is higher than that of monoadducts (MA) . In contrast to cytotoxicity, the significant increase in the proportion of CL, at a constant level of total adducts, had no effect on the induction of mutations at the HPRT locus . Comparison with the data obtained in yeast and rodent cells using the same double irradiation protocol shows that the mutagenic potential of CL versus MA varies between species . This suggests that the equilibrium between the excision, the recombinational and the mutagenic components of the repair pathways which probably determine the mutagenic efficiency of CL versus MA is likely to be species-dependent.

Genes Dev, 1995 Mar 1, 9(5), 534 - 46
The Drosophila tumor suppressor gene warts encodes a homolog of human myotonic dystrophy kinase and is required for the control of cell shape and proliferation; Justice RW et al.; Homozygous loss of the warts (wts) gene of Drosophila, caused by mitotic recombination in somatic cells, leads to the formation of cell clones that are fragmented, rounded, and greatly overgrown compared with normal controls . Therefore, the gene is required for the control of the amount and direction of cell proliferation as well as for normal morphogenesis . The absence of wts function also results in apical hypertrophy of imaginal disc epithelial cells . Secretion of cuticle over and between the domed apical surfaces of these cells leads to a honeycomb-like structure and gives the superficial wart-like phenotype of mitotic clones on the adult . One wts allele allows survival of homozygotes to the late larval stage, and these larvae show extensive imaginal disc overgrowth . Because of the excess growth and abnormalities of differentiation that follow homozygous loss, we consider wts to be a tumor suppressor gene . The wts gene is defined by the breakpoints of overlapping deficiencies in the right telomeric region of chromosome 3, region 100A, and by lethal P-element insertions and excisions . It encodes a protein kinase that is most similar to human myotonic dystrophy kinase, the Neurospora cot-1 protein kinase, two cell-cycle regulated kinases of yeast, and several putative kinases from plants . These proteins define a new subfamily of protein kinases that are closely related to but distinct from the cyclic AMP-dependent kinases . Although myotonic dystrophy is defined by a neuromuscular disorder, it is sometimes associated with multiple pilomatrixomas, which are otherwise rare epithelial tumors, and with other tumors including neurofibromas and parathyroid adenomas . Our results raise the possibility that homozygous loss of the myotonic dystrophy kinase may contribute to the development of these tumors.

Arerugi, 1995 Mar, 44(3 Pt 1), 128 - 33
{The correlation between the levels of anti-Malassezia furfur IgE antibodies and severities of face and neck dermatitis of patients with atopic dermatitis}; Kawano S et al.; It is well known that the seborrheic areas including the face and neck of patients with atopic dermatitis (AD) are frequently affected after adolescence . Malassezia furfur (MF), a lipophilic yeast and a normal habitat of seborrheic areas, has been thought to be one of the most important factors in provoking face and neck lesions of AD . In the present study, we assessed serum levels of anti-MF IgE antibodies by enzyme-linked immunosorbent assay (AlaSTAT method) in 123 adolescent and adult AD patients and evaluated the correlations between levels of anti-MF IgE and those of total IgE and some other allergen-specific IgE . The correlation between the levels of anti-MF IgE and severities of face and neck dermatitis was also assessed . Anti-MF IgE antibodies were detected in high frequency in AD patients (77%), while they were not found in controls . Significant correlations were noted between the levels of anti-MF IgE and those of total IgE and anti-Candida IgE . The levels of anti-MF IgE were well correlated with the severities of face and neck dermatitis . These result may indicate that MF is an important provocative factor for face and neck dermatitis in AD patients.

J Biochem (Tokyo), 1995 Mar, 117(3), 554 - 9
Chicken alpha-enolase but not beta-enolase has a Src-dependent tyrosine-phosphorylation site: cDNA cloning and nucleotide sequence analysis; Tanaka M et al.; Chicken alpha- and beta-enolase cDNAs have been cloned and analyzed to reveal that alpha- but not beta-enolase has a Src-dependent phosphorylation site . The deduced amino acid sequence of the chicken alpha-enolase showed more than 90% homologies with those of other vertebrate alpha-enolases including amphibian (Xenopus laevis) alpha-like enolase . The chicken beta-enolase, on the other hand, shares 84-85% amino acid sequence homology with mammalian beta-enolases . These chicken enolases also showed more than 70% sequence identity with an insect (Drosophila melanogaster) enolase and around 60% with two yeast enolases . The amino acid sequence between residues 33 and 50 in chicken alpha-enolase coincided with the reported tryptic peptide sequence of rabbit beta-enolase, the tyrosine residue in which was phosphorylated in vitro by Rous-sarcoma-virus tyrosine kinase . The present finding suggested that the tyrosine residue at position 44 in chicken alpha-enolase is the phosphorylation site by the tyrosine kinase . In chicken beta-enolase, on the other hand, the corresponding tyrosine residue was found to be replaced with a histidine residue, in accordance with the previous observation that chicken beta-enolase was not phosphorylated in vivo or in vitro . Northern blot analysis indicated that alpha-enolase mRNA can be expressed in a wide range of chicken tissues, and that the gene expression switch from alpha to beta-enolase occurs just after hatching in developing chicken muscle.

Curr Genet, 1995 Mar, 27(4), 390 - 2
Selection of specific gene probes by combined use of low-stringency PCR amplification and Southern-blot hybridization; Magdolen U et al.; We have developed a protocol to isolate a gene from which only limited (amino-acid) sequence information is available . It involves two PCR amplifications using one constant primer and a set of nested primers and subsequent crosswise Southern hybridization . The amplified DNA giving a signal in both lanes is further processed for use in gene bank screening by applying standard procedures . In this way the structural gene for a thiol protease, BLH1, the homologue of the bleomycin A (a cancerostatic drug) resistance gene of rabbit (and man), was isolated from yeast genomic DNA.

Acta Derm Venereol, 1995 Mar, 75(2), 105 - 9
Hydrolysis of fatty acid esters by Malassezia furfur: different utilization depending on alcohol moiety; Mayser P et al.; The lipophilic yeast Malassezia furfur belongs to the resident skin flora but has been implicated in various skin diseases . While topical oily preparations may support its growth, their formulation may be altered by yeast-dependent enzymatic degradation . Different synthetic fatty acid (mono-)esters used as refatting agents were mixed with 10(4), 10(5) and 5 x 10(5) yeasts/microliters, respectively, and incubated at 35 degrees C for a maximum of 48 h on selective agar for pathogenic fungi (Merck) . The amount and pattern of generated free acids were evaluated by densitometric and gas chromatographic analysis, while yeast density was determined in a Neubauer chamber . Depending on the inoculum, yeast-dependent hydrolysis occurred immediately and was best effected in ethyl esters, followed by isopropyl esters, whereas hydrolysis of decyl oleate was only limited . Of the fatty acids released, unsaturated fatty acids were more stimulative to growth than saturated fatty acids; no toxic effects were observed . In conclusion, yeast-dependent hydrolysis of these synthetic fatty acid (mono-)esters was critically dependent on alcohol moiety, while growth promotion was best effected by unsaturated fatty acids . These results may help to improve the compatibility of topical formulations, especially in seborrheic areas.

Biofizika, 1995 Mar-Apr, 40(2), 383 - 8
{Change in certain physical characteristics of cells at various stages of the cell cycle}; Samoilova OP et al.; Experiments on semisynchronized cultures of animal and plant cells, performed in our laboratory approximately 30 years ago, have demonstrated that there exist regular changes of certain physical parameters of the cells during the cell cycle . In particular on yeast cultures there has been observed the appearance of specific electron paramagnetic resonance (EPR) signals with simultaneous change of the static magnetic susceptibility of the cultures in the period immediately preceding the beginning of the intensive budding . On chlorella cultures grown at certain regimes of light and darkness it is possible to observe characteristic changes of the kinetics of the photoconduction signal at the frequency of 10 HHz (SHR-photoconduction) . It can be conjectured that these effects are of a similar nature linked to some changes of the intracellular structures at certain stages of the cell cycle . The work performed much later makes it possible to link these changes to the appearance of spin, glass-like structures in macromolecular intracellular bodies.

Somat Cell Mol Genet, 1995 Mar, 21(2), 133 - 7
YAC contig mapping of six expressed sequences encoded by human chromosome 21; Yu J et al.; Six cDNA clones from human chromosome 21 have been mapped in a set of complete YAC contig spanning the entire chromosome 21q . The mapping positions between two STSs on the YAC contig and the NotI coordinates starting from the telomere of 21q were determined for the cDNA clones . The YAC contig mapping positions agree well with those using a comprehensive somatic cell hybrid mapping panel.

Int J Immunopharmacol, 1995 Mar, 17(3), 183 - 8
Nitric oxide production by chicken macrophages activated by Acemannan, a complex carbohydrate extracted from Aloe vera; Karaca K et al.; Cultures of normal chicken spleen cells and HD11 line cells produce nitric oxide (NO) in response to Acemannan, a complex carbohydrate derived from the Aloe vera plant . Neither cell type produced detectable amounts of NO in response to similar concentrations of yeast mannan, another complex carbohydrate . Nitric oxide production was dose dependent and inhibitable by the nitric oxide synthase inhibitor NG-methyl-L-arginine . In addition, the production of NO was inhibited by preincubation of ACM with concanavalin A in a dose-dependent manner . These results suggest that ACM-induced NO synthesis may be mediated through macrophage mannose receptors, and macrophage activation may be accountable for some of the immunomodulatory effects of ACM in chickens.

J Membr Biol, 1995 Mar, 144(2), 121 - 9
Oriented channel insertion reveals the motion of a transmembrane beta strand during voltage gating of VDAC; Zizi M et al.; Yeast VDAC channels (isolated from the mitochondrial outer membrane) form large aqueous pores whose walls are believed to consist of 1 alpha helix and 12 beta strands . Each channel has two voltage-gating processes: one closes the channels at positive potentials, the other at negative . When VDAC is reconstituted into phospholipid (soybean) membranes, the two gating processes have virtually the same steepness of voltage dependence and the same midpoint voltage . Substituting lysine for glutamate at either end of one putative beta strand (E145K or E152K) made the channels behave asymmetrically, increasing the voltage dependence of one gating process but not the other . The asymmetry was the same whether 1 or 100 channels were in the membrane, indicating oriented channel insertion . However, the direction of insertion varied from membrane to membrane, indicating that the insertion of the first channel was random and subsequent insertions were directed by the previously inserted channel(s) . This raises the prospect of an auto-directed insertion with possible implications to protein targeting in cells . Each of the mutations affected a different gating process because the double mutant increased voltage dependence of both processes . Thus this strand may slide through the membrane in one direction or the other depending on the gating process . We propose that the model of folding for VDAC be altered to move this strand into the sensor region of the protein where it may act as a tether and guide/restrict the motion of the sensor.

Clin Exp Allergy, 1995 Mar, 25(3), 265 - 72
Purification and characterization of the serotype-specific polysaccharide antigen of Trichosporon cutaneum serotype II: a disease-related antigen of Japanese summer-type hypersensitivity pneumonitis; Mizobe T et al.; Summer-type hypersensitivity pneumonitis (SHP) is a unique type of hypersensitivity pneumonitis and the most prevalent in Japan . Our previous study clarified that the causative agent of the disease is Trichosporon cutaneum, and that the patients with SHP have high titres of antibodies against the serotype-specific antigen of polysaccharide nature which exist in the high molecular weight fraction of the culture supernatant of the yeast . In this study, we purified the serotype-specific antigen of serotype II T . cutaneum by gel filtration and affinity chromatography using a monoclonal antibody, D-8, specific for a high molecular weight antigen of serotype II T . cutaneum, and elucidated the structure of the antigen . This affinity-purified antigen was shown to be an essentially acidic polysaccharide comprising mannose, xylose, and glucuronic acid (6:44:4.7) . Chemical analysis showed that this polysaccharide antigen contains a (1-3)-linked mannan backbone attached with short side chains of (1-4)-linked mannose and a small proportion of (1-2)-linked xylose residues by substituting the 2- or 4-positions of the (1-3)-linked mannose residues of the main chain . Approximately one-fifth of the side chains were terminated with glucuronic acid residues . The antigenic epitope of the serotype-specific antigen was shown to involve the terminal glucoronic acid residues as revealed by immunodiffusion test and sandwich enzyme-linked immunosorbent assay using monoclonal antibody D-8.

Ecotoxicol Environ Saf, 1995 Mar, 30(2), 158 - 63
Does a heterogeneous distribution of food or pesticide affect the outcome of toxicity tests with Collembola?
Krogh PH.
The reproduction of two closely related soil microarthropods, Folsomia candida Willem and Folsomia fimetaria L . (Insecta: Collembola), was tested under the influence of the insecticide dimethoate . Dimethoate had an adverse effect on the survival of adults and their reproduction in concentrations of about the recommended field dose, with F . fimetaria being more sensitive than F . candida . The experimental conditions were altered to evaluate the realism in the basic single species/single chemical reproductive test system . The importance of the spatial distribution of dimethoate was studied with food applied to the surface (original procedure), mixed homogeneously in the whole soil profile or only in the top layer, or mixed heterogeneously into the soil preserving the small granula of the yeast originally in the commercial formulation . Toxicity decreased significantly when exposure could be avoided in an uncontaminated bottom layer and even more if food was available in this soil horizon . But the results indicate that Collembola were not able to completely avoid dimethoate when they had the choice . For extrapolation purposes a simple test system may be sufficient as EC50 was changed less than one order of magnitude with the different test designs . In terms of EC50 the outcome of a toxicity test with a heterogeneous distribution of food and dimethoate was changed only slightly but the effects to suboptimally fed populations should be considered because they may be more vulnerable.

Genes Chromosomes Cancer, 1995 Mar, 12(3), 224 - 8
Defining the position of the breakpoint of the constitutional t(3;6) occurring in a family with renal cell carcinoma; van den Berg A et al.; In a family with a constitutional translocation t(3;6), the oldest member carrying the translocation had developed multiple nonpapillary renal cell carcinomas (RCCs) . The translocation breakpoint was positioned between 3p13 and 3p14.1 . This is close to the region in which a t(3;8) breakpoint has been reported in a family with hereditary RCC . We defined the location of the t(3;6) and t(3;8) breakpoints by fluorescence in situ hybridization (FISH) analysis with yeast artificial chromosomes (YACs) from the 3p14-13 region . Both interphase nuclei and metaphase cells from translocation-carrying members of both families have been used, allowing the definition of flanking YACs for each breakpoint . We could thereby clearly confirm that the breakpoints are different, the t(3;8) breakpoint being most distal . In addition, we have shown that both translocation breakpoints are located distal to the homozygously deleted region in the U2020 lung cancer cell line.

Zh Evol Biokhim Fiziol, 1995 Mar-Apr, 31(2), 129 - 40
{A theoretical analysis of the secondary structure and topology of dolichol-coupled enzymes}; Shpakov AO; Hydropathic profile for several dolichol-coupled enzymes (mammal N-acetylglucosamine-1-phosphate transferases (GPT), yeast products of genes ALG7, ALG1, DMP1 and SEC59) taking part in the biosynthesis of complex oligosaccharide used for N-glycosylation of proteins in endoplasmic reticulum (ER) of eukaryotic cells has been calculated and constructed . On the basis of analysis of present research and available data sites of amino acid sequence (AAS) potentially capable to penetrate ER membrane were identified . A tendency of AAS segments of dolichol-coupled enzymes to form alpha-helices, beta-folded structures and beta-bends was calculated using correlational methods . For COOH-terminal part of ALG1 the prediction about the capability to form helix-helix structures was made . It was concluded that at least three types of topological models in ER membrane were present for the mentioned dolichol-coupled enzymes.

Rev Inst Med Trop Sao Paulo, 1995 Mar-Apr, 37(2), 99 - 102
Blood culture as a parameter of treatment effectiveness in experimental histoplasmosis of the hamster; Finquelievich J et al.; The aim of this study was to determine the value of blood culture as a parameter of treatment effectiveness in experimental histoplasmosis . A total of thirty five hamsters, weighing approximately 120g, were inoculated intracardially with 0.1 ml of a suspension containing 4 x 10(7) cells/ml of the yeast phase of H . capsulatum . Treatments were started one week after the infection and lasted for 3 weeks . The azoles, (itraconazole, saperconazole and fluconazole) were administered once a day by gavage, at a dose of 8 mg/kg; Amphotericin B was given intraperitoneally every other day at a dose of 6mg/kg . Blood samples (1 ml) were obtained by heart punction from the 4th day after infection and were seeded in Sabouraud honey-agar and BHI-agar . The hamsters that survived were killed one week after treatment completion and the following criteria were considered for treatment evaluation: 1) rate of spontaneous death, at the end of the experience; 2) microscopic examination of Giemsa smears from liver and spleen and 3) determination of CFU in spleen cultures . Amphotericin B was the most effective drug, with negative blood cultures at day 20, negative spleen cultures in all cases and all the animals survived until the end of the study . Fluconazole was the less effective drug, blood cultures were positive during the whole experience, spleen cultures showed a similar average of CFU when compared with the control animals and 42.8% of these animals died . Saperconazole and itraconazole showed a similar activity, with survival of all hamsters and negative blood cultures at 23 and 26 days respectively . Blood culture seems to be valuable parameter for treatments' evaluation in experimental histoplasmosis of the hamster.

Proc Natl Acad Sci U S A, 1995 Feb 28, 92(5), 1709 - 13
The murine N-ras gene is not essential for growth and development; Umanoff H et al.; The mammalian ras gene family encodes key cell-signaling, cell growth-related proteins that have been highly conserved in species from yeast to man . Specific point mutations in the ras genes are associated with various mammalian tumors . To understand the developmental role of the N-ras protooncogene in the mouse, we have disrupted its gene function by homologous recombination in embryonic stem cells . Mice derived from these cells that are homozygous for the N-ras mutation do not produce any detectable N-Ras protein and are morphologically and histologically indistinguishable from their heterozygous and wild-type siblings . Since N-ras is expressed at high levels in hematopoietic cells, we examined different populations of cells in peripheral blood and found no differences between mutant and normal animals . Our results show that N-ras gene function is dispensable for normal mouse development, growth, and fertility.

Gene, 1995 Feb 27, 154(1), 39 - 45
A calcineurin-B-encoding gene expressed during differentiation of the amoeboflagellate Naegleria gruberi contains two introns; Remillard SP et al.; One of two similar genes in the unicellular eukaryote Naegleria gruberi is shown to encode calcineurin B (CnB), the regulatory subunit of calcium-calmodulin-regulated protein phosphatase 2B . Over a span of 156 amino acids, excluding divergent N-termini, the encoded sequence shows 62% identity with vertebrate CnB, and also shows sequence elements specific, among calcium-binding proteins, to CnB . In contrast, the sequence shows only 23% identity with N . gruberi flagellar calmodulin . CNB mRNA is readily detected in amoebae; its abundance increases fourfold during differentiation to flagellates, reaches a peak at 50-70 min, when flagella are forming, and then declines . A genomic clone matches an expressed cDNA, except that it is interrupted by two phase I introns . The position of one intron, which separates the divergent N-terminal domain from the four calcium-binding domains (EF hands), is shared with a yeast CNB gene; the other is located in the central helix between the two pairs of calcium-binding loops; features that support an ancient origin . These introns, the first found in protein-coding genes of Naegleria, are flanked by characteristic splice junction sequences . N . gruberi CnB also shares similarities with recoverins . The finding in a protist of a CNB gene that contains two introns separating functional domains, shares similarities to recoverins and shows increased expression during differentiation is provocative . If the phylogeny of major groups derived from ribosomal RNA is accepted, Naegleria is among the earliest branching eukaryotes known to contain canonical pre-mRNA introns.

J Biol Chem, 1995 Feb 24, 270(8), 4152 - 7
DNA repair protein XPA binds replication protein A (RPA); Matsuda T et al.; XPA is a zinc finger DNA-binding protein, which is missing or altered in group A xeroderma pigmentosum cells and known to be involved in the damage-recognition step of the nucleotide excision repair (NER) processes . Using the yeast two-hybrid system to search for proteins that interact with XPA, we obtained the 34-kDa subunit of replication protein A (RPA, also known as HSSB and RFA) . RPA is a stable complex of three polypeptides of 70, 34, 11 kDa and has been shown to be essential in the early steps of NER as well as in replication and recombination . We also demonstrate here that the RPA complex associates with XPA . These results suggest that RPA may cooperate with XPA in the early steps of the NER processes.

J Biol Chem, 1995 Feb 24, 270(8), 4042 - 52
Role of partner homology in DNA recombination . Complementary base pairing orients the 5'-hydroxyl for strand joining during Flp site-specific recombination; Lee J et al.; Absolute homology between partner substrates within the strand exchange region is an essential requirement for recombination mediated by the yeast site-specific recombinase Flp . Using combinations of specially designed half- and full-site Flp substrates, we demonstrate that the strand joining step of recombination is exquisitely sensitive to spacer homology . At each exchange point, 2-3 spacer nucleotides adjacent to the nick within the cleaved strand of one substrate must base pair with the corresponding segment of the un-nicked strand from the second substrate for efficient strand joining in the recombinant mode . In accordance with the "cis-activation/trans-nucleophilic attack" model for each of the two transesterification steps of Flp recombination (strand cleavage and strand joining), we propose that the limited strand pairing orients the DNA-nucleophile (5'-hydroxyl) for attack on its target diester (3'-phosphotyrosyl-Flp) . During one round of recombination, 4-6 terminal base pairs of the spacer (2-3 base pairs at each spacer end) must unpair, following strand cleavage, within a DNA substrate and pair with the partner substrate prior to strand union . In this model, the extent of branch migration of the covalently closed Holliday intermediate is limited to the central core of the spacer . The templated positioning of reactive nucleic acid groups (which is central to the model) may be utilized by other recombination systems and by RNA splicing reactions.

J Biol Chem, 1995 Feb 24, 270(8), 3574 - 81
Identification of a protein with homology to hsp90 that binds the type 1 tumor necrosis factor receptor; Song HY et al.; The yeast-based two hybrid has been used to identify a novel protein that binds to the intracellular domain of the type 1 receptor for tumor necrosis factor (TNFR-1IC) . The TNF receptor-associated protein, TRAP-1, shows strong homology to members of the 90-kDa family of heat shock proteins . After in vitro transcription/translation and 35S labeling, TRAP-1 was precipitated using a fusion protein consisting of glutathione S-transferase and TNFR-1IC, showing that the two proteins directly interact . The ability of deletion mutants of TNFR-1 to interact with TRAP-1 was tested using the two hybrid system . This showed that the amino acid sequences that mediate binding are diffusely distributed outside of the domain in the C terminus of TNFR-1IC that signals cytotoxicity . The 2.4-kilobase TRAP-1 mRNA was variably expressed in skeletal muscle, liver, heart, brain, kidney, pancreas, lung, and placenta . TRAP-1 mRNA was also detected in each of eight different transformed cell lines . Identification of TRAP-1 may be an important step toward defining how TNFR-1, which does not contain protein tyrosine kinase activity, transmits its message to signal transduction pathways.

Eur J Biochem, 1995 Feb 15, 228(1), 96 - 101
Two distinct regions of Ras participate in functional interaction with GDP-GTP exchangers; Segal M et al.; We have previously implemented a combined genetic/biochemical approach, for analysis of insertion-deletion mutants, to identify sites of Harvey-Ras participating in the interaction with guanine nucleotide exchangers, using the yeast Cdc25 as a model exchanger . We showed that positions 101-106 may be required for catalyzed exchange . We here present a further improved strategy to define more precisely the residues on Ras participating in this interaction . Non-conservative replacements at positions 103 or 105 abolished response to Cdc25 while substitutions at positions 102 or 104 were partially affected . The same substitutions had no effect on coupling to adenylyl cyclase . Since the strategy enables us to assess Ras functional interaction with both the exchanger and effector simultaneously, we have also examined the effect of substitutions in the distal part of the switch II region (amino acids 69-78) . In contrast to other reports, substitutions at positions 69 or 73 prevented Cdc25 response while mutations at position 74 did not prevent this interaction . However, all these substitutions partly affected cyclase activation . These findings establish the crucial role of the 102-105 region in the catalyzed exchange reaction and suggest that the 69-74 area would be required for the functional interaction with both exchangers and effector molecules.

EMBO J, 1995 Feb 15, 14(4), 685 - 96
Regulation of Raf-1 kinase activity by the 14-3-3 family of proteins; Li S et al.; We have identified the beta (beta) isoform of the 14-3-3 family of proteins as an activator of the Raf-1 protein kinase . 14-3-3 was isolated in a yeast two-hybrid screen for Raf-1 kinase domain binding proteins . Purified bovine brain 14-3-3 interacted specifically with both c-Raf-1 and the isolated Raf-1 kinase domain . Association was sensitive to the activation status of Raf-1; 14-3-3 bound to unactivated Raf-1, but not Raf-1 activated by protein kinase C alpha or Ras and Lck . The significance of these interactions under physiological conditions was demonstrated by co-immunoprecipitation of Raf-1 and 14-3-3 from extracts of quiescent, but not mitogen-stimulated, NIH 3T3 cells . 14-3-3 was not a preferred Raf-1 substrate in vitro and did not significantly affect Raf-1 kinase activity in a purified system . However, in cell-free extracts 14-3-3 acted as a Ras-independent activator of both c-Raf-1 and the Raf-1 kinase domain . The same results were obtained in vivo using transfection assays; 14-3-3 enhanced both c-Raf-1- and Raf-1 kinase domain-stimulated expression of AP-1- and NF-kappa B-dependent reporter genes and accelerated Raf-1 kinase domain-triggered differentiation of PC12 cells . We conclude that 14-3-3 is a latent co-activator bound to unactivated Raf-1 in quiescent cells and mediates mitogen-triggered but Ras-independent regulatory effects aimed directly at the kinase domain.

EMBO J, 1995 Feb 15, 14(4), 639 - 49
Cloning and functional characterization of a mammalian zinc transporter that confers resistance to zinc; Palmiter RD et al.; A cDNA encoding a zinc transporter (ZnT-1) was isolated from a rat kidney cDNA expression library by complementation of a mutated, zinc-sensitive BHK cell line . This cDNA was used to isolate the homologous mouse ZnT-1 gene . The proteins predicted for these transporters contain six membrane-spanning domains, a large intracellular loop and a C-terminal tail . ZnT-1 is homologous to zinc and cobalt resistance genes of yeast . Immunocytochemistry with an antibody to a myc epitope added to the C-terminus of ZnT-1 revealed localization to the plasma membrane . Transformation of normal cells with a mutant ZnT-1 lacking the first membrane-spanning domain conferred zinc sensitivity on wild-type cells, suggesting that ZnT-1 functions as a multimer . Deletion of the first two membrane-spanning domains resulted in a non-functional molecule, whereas deletion of the C-terminal tail produced a toxic phenotype . Mutant cells have a slightly higher steady-state level of intracellular zinc and high basal expression of a zinc-dependent reporter gene compared with normal cells . Mutant cells have a lower turnover of 65Zn compared with normal cells or mutant cells transformed with ZnT-1 . We propose that ZnT-1 transports zinc out of cells and that its absence accounts for the increased sensitivity of mutant cells to zinc toxicity.

Gene, 1995 Feb 14, 153(2), 249 - 54
Isolation of a mouse cDNA encoding MTJ1, a new murine member of the DnaJ family of proteins; Brightman SE et al.; We report the isolation and sequencing of MTJ1, a 1792-bp cDNA from an M27 murine lung carcinoma cell line . The largest ORF within MTJ1 encodes a 63,869-Da protein, containing a 73-amino-acid (aa) sequence (the J domain) that is conserved in proteins of the DnaJ family of chaperonins . The J domain of MTJ1 is bracketed by potential transmembrane domains in a similar configuration to the J domain of the yeast DnaJ-like protein, SEC63 . Polyclonal antibodies raised against deduced aa sequences within MTJ1 recognized antigens of 62, 42 and 41 kDa that were enriched in the nuclear and heavy microsome subcellular fractions of murine tumor cells . Northern analysis detected a major 3.2-kb transcript that was present in all murine organs examined, but was relatively underexpressed in brain and heart.

Gene, 1995 Feb 14, 153(2), 171 - 4
A cDNA encoding Arabidopsis thaliana cytoplasmic ribosomal protein L18; Baima S et al.; A cDNA encoding ribosomal (r) protein L18 of Arabidopsis thaliana (At) was isolated and characterized . The nucleotide sequence contains a 563-bp open reading frame that encodes a 20.9-kDa basic protein . Amino-acid comparison indicated that the predicted L18 r-protein of At has a high degree of homology with L18 of distantly related organisms such as yeast, Xenopus laevis and rat . Genomic DNA analysis suggested that L18 is encoded by a single locus in At . An mRNA of approx . 0.9 kb is detected in all the tissues and developmental stages analyzed.

Cell, 1995 Feb 10, 80(3), 497 - 506
OBF-1, a novel B cell-specific coactivator that stimulates immunoglobulin promoter activity through association with octamer-binding proteins; Strubin M et al.; Recent biochemical and genetic studies indicate that in addition to the octamer-binding proteins Oct-1 and Oct-2, other B cell components are required for lymphoid-restricted, octamer site-mediated immunoglobulin gene promoter activity . Using a genetic screen in yeast, we have isolated B cell-derived cDNAs encoding Oct-binding factor 1 (OBF-1), a novel protein that specifically associates with Oct-1 and Oct-2 . Biochemical studies demonstrate that OBF-1 has no intrinsic DNA-binding activity and recognizes the POU domains of Oct-1 and Oct-2, but not those of Oct-4 and Oct-6 . The OBF-1 mRNA is expressed in a highly cell-specific manner, being most abundant in B cells and essentially absent in most of the other cells or tissues tested . Furthermore, expression of OBF-1 in HeLa cells selectively stimulates the activity of a natural immunoglobulin promoter in an octamer site-dependent manner . Thus, OBF-1 has all the properties expected for a B cell-specific transcriptional coactivator protein.

J Biol Chem, 1995 Feb 10, 270(6), 2466 - 72
Design of a ruthenium-cytochrome c derivative to measure electron transfer to the initial acceptor in cytochrome c oxidase; Geren LM et al.; A ruthenium-labeled cytochrome c derivative was prepared to meet two design criteria: the ruthenium group must transfer an electron rapidly to the heme group, but not alter the interaction with cytochrome c oxidase . Site-directed mutagenesis was used to replace His39 on the backside of yeast C102T iso-1-cytochrome c with a cysteine residue, and the single sulfhydryl group was labeled with (4-bromomethyl-4' methylbipyridine) (bis-bipyridine)ruthenium(II) to form Ru-39-cytochrome c (cyt c) . There is an efficient pathway for electron transfer from the ruthenium group to the heme group of Ru-39-cyt c comprising 13 covalent bonds and one hydrogen bond . Electron transfer from the excited state Ru(II*) to ferric heme c occurred with a rate constant of (6.0 +/- 2.0) x 10(5) s-1, followed by electron transfer from ferrous heme c to Ru(III) with a rate constant of (1.0 +/- 0.2) x 10(6) s-1 . Laser excitation of a complex between Ru-39-cyt c and beef cytochrome c oxidase in low ionic strength buffer (5 mM phosphate, pH7) resulted in electron transfer from photoreduced heme c to CuA with a rate constant of (6 +/- 2) x 10(4) s-1, followed by electron transfer from CuA to heme a with a rate constant of (1.8 +/- 0.3) x 10(4) s-1 . Increasing the ionic strength to 100 mM leads to bimolecular kinetics as the complex is dissociated . The second-order rate constant is (2.5 +/- 0.4) x 10(7) M-1s-1 at 230 mM ionic strength, nearly the same as that of wild-type iso-1-cytochrome c.

Genomics, 1995 Feb 10, 25(3), 691 - 700
A 6-Mb YAC contig in Xp22.1-p22.2 spanning the DXS69E, XE59, GLRA2, PIGA, GRPR, CALB3, and PHKA2 genes; Alitalo T et al.; We report the generation of an approximately 6-Mb contig of 70 overlapping yeast artificial chromosomes (YAC) covering the interval between DXS16 and DXS1229 in Xp22.1-p22.2 . Within this region lie the genes for calbindin (CALB3), gastrin-releasing peptide receptor (GRPR), phosphatidyl-inositol glycan-class A protein (PIGA), glycine receptor alpha-2 (GLRA2), phosphorylase kinase alpha (PHKA2), XE59 (a gene escaping X chromosome inactivation), and DXS69E (71-7A) . YACs were isolated initially from four libraries either by hybridization or using sequence tagged sites (STSs) for DXS16, DXS9, GLRA2, DXS207, DXS43, DXS1416, DXS1317, DXS1195, and DXS418 . Additional STSs were obtained from the end fragments of the original YACs studied, thus allowing us to cover the contig with a series of 73 STSs, approximately 1 per 100 kb . YAC contig construction allowed the following locus order to be established: Xpter-DXS16-DXS69E-DXS414-XE59 - DXS9 - (GLRA2, DXS987) - (PIGA, DXS207) - DXS1053-DXS197-(GRPR,DXS43)-CALB3-DXS14 16- DXS1317 - DXS1195 - DXS418 - DXS257 - (PHKA2, DXS999)-DXS443-DXS1229-Xcen . Restriction mapping of the DXS16-DXS43 interval predicted the existence of several CpG islands, suggesting the presence of other genes in the region . This work provides a starting point for further mapping and positional cloning of several X-linked disease genes.

Genomics, 1995 Feb 10, 25(3), 674 - 81
Thermal asymmetric interlaced PCR: automatable amplification and sequencing of insert end fragments from P1 and YAC clones for chromosome walking; Liu YG et al.; Isolation of DNA segments adjacent to known sequences is a tedious task in genome-related research . We have developed an efficient PCR strategy that overcomes the shortcomings of existing methods and can be automated . This strategy, thermal asymmetric interlaced (TAIL)-PCR, utilizes nested sequence-specific primers together with a shorter arbitrary degenerate primer so that the relative amplification efficiencies of specific and nonspecific products can be thermally controlled . One low-stringency PCR cycle is carried out to create annealing site(s) adapted for the arbitrary primer within the unknown target sequence bordering the known segment . This sequence is then preferentially and geometrically amplified over nontarget ones by interspersion of high-stringency PCR cycles with reduced-stringency PCR cycles . We have exploited the efficiency of this method to expedite amplification and sequencing of insert end segments from P1 and YAC clones for chromosome walking . In this study we present protocols that are amenable to automation of amplification and sequencing of insert end sequences directly from cells of P1 and YAC clones.

Genomics, 1995 Feb 10, 25(3), 644 - 9
A bidirectional YAC walk from the Norrie disease (NDP) locus; Black GC et al.; The region of Xp between DXS7 and the centromere contains the gene for Norrie disease in addition to the genes for several other ophthalmic disorders . A 650-kb YAC containing the loci MAOA, MAOB, and NDP has been used as the starting point for a bidirectional chromosomal walk . A contig of 16 YACs covering between 2 and 3 Mb has been developed in which the following markers/genes are located (in physical order): Xpter--DXS1201 (256ze5)--DXS6668--DXS228--DXS77--MAOA--++ +MAOB--FR12 (pseudogene)--NDP--DXS6670--RRM2P3--DXS6671--DXS742 --Xcen . Seven new STSs are described both for end clones and for internal Alu PCR products from the contig . The contig contains the breakpoint of the t75-2ma-1b (t75) translocation, close to the 5' end of the MAOB gene.

Genomics, 1995 Feb 10, 25(3), 638 - 43
Isolation of a YAC clone covering a cluster of nine S100 genes on human chromosome 1q21: rationale for a new nomenclature of the S100 calcium-binding protein family; Schafer BW et al.; S100 proteins are low-molecular-weight calcium-binding proteins of the EF-hand superfamily and appear to be involved in the regulation of a number of cellular processes such as cell cycle progression and differentiation . More than 10 members of the S100 protein family have been described from human sources so far . We have now isolated a YAC clone from human chromosome 1q21, on which 9 different genes coding for S100 calcium-binding proteins could be localized . Moreover, we have mapped the gene coding for S100P to human chromosome 4p16 and thereby completed the chromosomal assignments of all known human S100 genes . The clustered organization of S100 genes in the 1q21 region allows us to introduce a new logical nomenclature for these genes, which is based on the physical arrangement on the chromosome . The new nomenclature should facilitate and further the understanding of this protein family and be easily expandable to other species.

Mol Cell Biochem, 1995 Feb 9, 143(1), 67 - 71
Specific species and tissue differences for the gene expression of calcium-binding protein regucalcin; Shimokawa N et al.; The existence and expression of gene encoding the Ca(2+)-binding protein regucalcin in various species and tissues were investigated with Southern and Northern hybridization analyses using regucalcin cDNA (0.9 kb of open reading frame) . Genomic Southern hybridization analysis demonstrated that regucalcin gene was widely conserved among higher animals including human, monkey, rat, mouse, dog, bovine, rabbit and chicken . The gene was not found in yeast . The Northern blot analysis of poly (A) +RNAs extracted from the liver of various species showed that regucalcin mRNA was predominantly expressed in rat and mouse, although the expression was also seen in human, bovine and chicken . Furthermore, the enzyme-linked immunoadsorbent assay (ELISA) with rabbit-anti-regucalcin IgG indicated that hepatic regucalcin concentration was most pronounced in rat as compared with that of guinea pig, mouse and chicken . These observations show that the gene expression of regucalcin and its protein synthesis is unique in the liver of rats, suggesting the existence of a specific mechanism in demonstrating regucalcin synthesis from gene.

J Biol Chem, 1995 Feb 3, 270(5), 1971 - 4
The biochemistry of neurotransmitter secretion; Bajjalieh SM et al.; The progress that has resulted from the convergence of biochemistry with yeast genetics has accelerated the pace at which the molecular events of membrane transport are being elucidated . Future research will focus not only on testing the proposed sequence of protein-protein interactions but also on identifying how calcium regulation is imposed on this system . As our understanding of the basic mechanisms of neurosecretion increases, attention will undoubtedly shift to how the molecules of release are modified to produce changes in synaptic efficacy.

J Biol Chem, 1995 Feb 3, 270(5), 2145 - 51
Biochemical properties of the ligand-binding 20-kDa subunit of the beta-glucan receptors on human mononuclear phagocytes; Szabo T et al.; beta-Glucan receptors are present on mammalian leukocytes and initiate phagocytosis of particulate yeast beta-glucans, such as zymosan particles . Human monocytes and U937 cells express two membrane proteins of 180 and 160 kDa, each of which binds particulate yeast glucan through a 20-kDa polypeptide constituent . In this report, the structural composition of the two beta-glucan receptors and the biochemical properties of their polypeptide constituents were examined . The 180-kDa receptor was composed of three disulfide-linked polypeptides of 95, 60, and 20 kDa, whereas the 160-kDa receptor was a multimer of two polypeptides of 27 and 20 kDa . Unlike other receptor constituents, the 20-kDa polypeptide was nonglycosylated and focused at two distinct isoelectric points . Immunoblots of the focused polypeptides showed the two 20-kDa variants and the 95-kDa subunit to be constitutively tyrosine-phosphorylated, a feature not previously reported for receptors on human mononuclear phagocytes . Dephosphorylation of the receptor proteins resulted in the loss of antigenic phosphotyrosine without affecting the antigenicity of either 20-kDa variant for the anti-idiotypic antibody to beta-glucan receptors . Separate analysis of the 160-kDa receptor showed it contained both variants of the 20-kDa polypeptide . Thus, the 20-kDa subunit constituent of the two beta-glucan receptors is a functionally and chemically unique polypeptide with apparent microheterogeneity in its primary structure.

Int J Radiat Biol, 1995 Feb, 67(2), 161 - 8
Separation of DNA fragments induced by ionizing irradiation using a graded-field gel electrophoresis; Dahm-Daphi J et al.; A method is described that allows a separation of X-ray induced DNA fragments by graded-field gel electrophoresis . Synchronized G1 and asynchronous CHO cells were embedded in agarose and irradiated with X-ray doses ranging from 1 to 100 Gy . Following proteolysis by sarcosine and proteinase K, electrophoresis was run for 49 h using graded electric fields with stepwise increasing field strength (0.6, 1.5, 3 and 9 V/cm) . Since the molecular size of DNA able to migrate decreased with increasing voltage, each voltage step led to the generation of a distinct DNA band with the largest fragments in band 1, fragments of intermediate size in bands 2 and 3 and the smallest fragments in band 4 . Using yeast chromosomal DNA as a reference, the molecular weight of eluted fragments was calculated to range from 1 to 10 Mbp . It could be shown that the fragment size was not the only criterion that discriminates migrating from non-migrating DNA . DNA fragments were found to be retained in the well by an unknown factor presumably associated with DNA conformation . This retention factor increased with increasing fragment size . Graded-field gel electrophoresis also allowed the determination of the absolute number of double-stranded breaks (dsb) induced, which amounted to 11.5 x 10(-12) dsbs Gy-1 Da-1 corresponding to 37 dsbs/G1 cell.

Eur J Biochem, 1995 Feb 1, 227(3), 780 - 6
Identification by UV cross-linking of oligo(U)-binding proteins in mitochondria of the insect trypanosomatid Crithidia fasciculata; Leegwater P et al.; RNA editing in trypanosomes is the process of insertion and deletion of U residues at specific sites of mitochondrial transcripts mediated by short guide RNAs (gRNAs) that have a 3' oligo(U) extension . Here we describe the identification by UV cross-linking of proteins present in mitochondrial extracts from Crithidia fasciculata with a high affinity for gRNAs, and the characterization of the binding specificity . A 65-kDa protein binds to gRNAs provided they are equipped with a U tail, to post-transcriptionally labelled mitoribosomal 9S and 12S RNAs that also possess a 3' terminal stretch of U residues, and to free oligo(U) sequences with a minimal length of 23-29 nucleotides . It does not bind to a number of control RNAs, one of which has an internal U stretch of 13 residues . Poly(U), but not poly(C) or total yeast RNA, efficiently competes for binding to gRNA . Proteins of 88 kDa and 30 kDa also bind to gRNAs with a U tail, to mitochondrial ribosomal RNAs and to oligo(U) . These proteins, however, require longer oligo(U) for binding (> 39 nucleotides) and they also have an affinity for other U-rich RNAs and poly(C) . For comparison, part of the analysis was also carried out with a mitochondrial extract from Trypanosoma brucei . In this organism, gRNA-binding proteins of 83 kDa and 64 kDa were found with the same preference for 3'-terminal oligomeric U stretches as the C . fasciculata 65-kDa protein, whereas the binding specificity of a 26-kDa protein resembled that of the C . fasciculata 88-kDa and 30-kDa proteins . The possible involvement of the proteins in the editing process is discussed.

DNA Cell Biol, 1995 Feb, 14(2), 163 - 73
CYP52 (cytochrome P450alk) multigene family in Candida maltosa: identification and characterization of eight members; Ohkuma M et al.; Previously, we characterized three genes and presented evidence for an n-alkane-inducible cytochrome P450 (P450alk) multigene family in an n-alkane-assimilating and diploid-type yeast, Candida maltosa . In the present report, we isolated and characterized additional members of this gene family, including a total of thirteen P450alk-related sequences (eight genes and five of their alleles) . Two sets, each consisting of two genes, were tandemly arranged in the genome . A gene replacement experiment showed that at least one gene had only a single allele in the genome . The determined nucleotide and the deduced amino acid sequences indicated that all had a characteristic constituent for P450s and exhibited amino acid identities from 94% to 37% to each other . Six genes showed relatively higher similarities to each other than to the other two genes and were thus classified into a subfamily . All the members of this subfamily were assigned to the same single chromosome, showing a good correlation between sequence similarity and chromosomal linkage . Although all the genes except for one were induced by n-alkane, their inducibilities by some other aliphatic carbon sources showed variabilities.

Arch Biochem Biophys, 1995 Feb 1, 316(2), 821 - 6
Inhibitors of pig kidney trehalase; Kyosseva SV et al.; Trehazolin, a new trehalase inhibitor isolated from the culture broth of Micromonospora, was reported to be a highly specific inhibitor for porcine and silk worm trehalases with IC50 values of 5.5 x 10(-9) and 3.7 x 10(-9) M, respectively (O . Ando, H . Satake, K . Itoi, A . Sato, M . Nakajima, S . Takashi, H . Haruyama, Y . Ohkuma, T . Kinoshita, and R . Enokita (1991) J . Antibiot . 44, 1165-1168) . We also found that trehazolin is a very powerful and quite specific inhibitor against purified pig kidney trehalase, giving an IC50 value of 1.9 x 10(-8) M . Lineweaver-Burk plots showed that this compound was a competitive inhibitor of the trehalase . However, even at concentrations of 200 micrograms/ml, trehazolin did not inhibit the rat intestinal maltase or sucrase, yeast alpha-glucosidase or almond beta-glucosidase . Validoxylamine A and validamycin A, two other trehalase inhibitors, showed potent competitive inhibition against purified pig kidney trehalase, with IC50 values of 2.4 x 10(-9) and 2.5 x 10(-4) M, respectively . On the other hand, validoxylamine A was almost inactive against rat intestinal sucrase and maltase, with some inhibition being observed at millimolar concentration . A number of other glucosidase inhibitors, such as MDL 25637, castanospermine, and deoxynojirimycin were also tested against the purified trehalase and showed reasonable inhibitory activity.

Arch Biochem Biophys, 1995 Feb 1, 316(2), 659 - 64
Identification of a 42-kDa plant mitochondrial outer membrane protein, MOM42, involved in the import of precursor proteins into plant mitochondria; Perryman RA et al.; A 42-kDa plant outer mitochondrial membrane protein, MOM42, has been identified as an essential component of the plant mitochondrial precursor protein translocation apparatus . Immunological cross-reactivity has been detected between antibodies raised against both Neurospora and yeast mitochondrial outer membrane proteins and plant mitochondrial outer membrane proteins . Immunocompetition studies showed that import of precursors to Rieske FeS protein, ATPase su9-DHFR, and the adenine nucleotide transporter was inhibited in the presence of antibody to MOM42 . The inhibition of Rieske Fes and su9-DHFR import was greater than that of the adenine nucleotide transporter . The competition studies suggest that the MOM42 is involved in the translocation of bound precursor proteins . The import data and the Western blots suggest that components of the mitochondrial import system are highly conserved.

J Nutr, 1995 Feb, 125(2), 302 - 8
Selenium deficiency alters thyroid hormone metabolism in guinea pigs; Cammack PM et al.; In guinea pigs, activity of glutathione peroxidase in most organs is markedly lower than in organs of other rodents despite comparable dietary intakes and tissue levels of selenium . To determine if metabolism of selenium with respect to other selenoproteins also differs in guinea pigs, we measured the effects of selenium intake on thyroid hormone metabolism . Weanling male Hartley Albino guinea pigs were fed a selenium-deficient Torula yeast-based diet, or the same diet supplemented with 0.5 mg selenium/kg diet as sodium selenate for 72 d . Growth was impaired in guinea pigs fed the unsupplemented diet . Activity of glutathione peroxidase was higher in tissues and plasma of supplemented guinea pigs than in selenium-deficient animals . However, it was still far lower than reported values for other rodent species . In selenium deficiency, activity of type 1 5'-iodothyronine deiodinase was 60% less in liver and 45% less in kidney . Concentration of thyroxine was 68% lower in kidney of selenium-deficient animals, and levels of 3,3',5-triiodothyronine in kidney and plasma were 44 and 31% lower, respectively . Thus, with the exception of thyroxine concentrations, thyroid hormone metabolism responds to selenium deficiency in guinea pigs as it does in rats, although the magnitude of that response is not as great.

Hum Genet, 1995 Feb, 95(2), 197 - 200
Localization of the gene for a novel human adenylyl cyclase (ADCY7) to chromosome 16; Hellevuo K et al.; A novel form of human adenylyl cyclase (ADCY7) has been discovered in the human erythroleukemia cell line (HEL) . This cell line has been widely used as a model for studies of the characteristics of human platelets . Data from HEL cells suggests that ADCY7 may be the major AC form in human platelets . In the current study polymerase chain reaction (PCR) techniques coupled with use of human/rodent somatic hybrid panels and a yeast artificial chromosome (YAC) library were used to determine the chromosomal localization of the gene (adcy7) for ADCY7 enzyme . A 251-bp product from the 3' untranslated region of human adcy7 was amplified for PCR mapping and the results localize the adcy7 gene to region 16q12-16q13 of the human genome . The AC enzyme family is characterized by the presence of 12 membrane-spanning domains in its sequences, and this chromosomal region is known to contain other genes coding for proteins characterized by 12 membrane-spanning domains.

Am J Pathol, 1995 Feb, 146(2), 450 - 62
Regulatory roles of tumor necrosis factor-alpha and interleukin-1 beta in monocyte chemoattractant protein-1-mediated pulmonary granuloma formation in the rat; Flory CM et al.; Intravenous infusion of particulate yeast cell wall glucan into rats results in the synchronous development of angiocentric pulmonary granulomas that are composed almost entirely of monocytes and macrophages . Previous studies indicate that locally produced monocyte chemoattractant protein-1 (MCP-1) is required for full granuloma development . Because tumor necrosis factor-alpha (TNF-alpha) and interleukin 1 (IL-1) can induce MCP-1 production in a variety of cell types, we sought to determine their potential regulatory roles in this model . A single infusion of anti-TNF-alpha antibody at the time of glucan infusion (time 0) markedly reduced MCP-1 mRNA levels at 1 and 6 hours but not at later time points; there was no effect on granuloma size or number measured at 48 hours . When multiple infusions of anti-TNF-alpha antibody were administered over a 23-hour period (0 to 23 hours), MCP-1 mRNA was reduced through 24 hours, there was a significant reduction in peak bronchoalveolar lavage fluid MCP-1 activity at 48 hours, and there were marked reductions in granuloma size and number at 48 hours . Similar results were observed in animals that received infusions of anti-IL-1 beta . Infusion of anti-IL-1 beta at time 0 resulted in moderate reductions in MCP-1 mRNA at 1 and 6 hours and had no effect on granuloma size or number measured at 48 hours . When multiple infusions of anti-IL-1 beta were administered over a 23-hour period (0 to 23 hours), MCP-1 mRNA was reduced through 24 hours, there was a moderate reduction in peak bronchoalveolar lavage fluid MCP-1 activity at 48 hours, and there were marked reductions in granuloma size and number at 48 hours . A single infusion of anti-TNF-alpha and anti-IL-1 beta together at time 0 resulted in marked reductions in whole lung MCP-1 and mRNA at 1 and 6 hours, but not at 24 hours . Multiple combined infusions of anti-TNF-alpha and anti-IL-1 beta over a 23-hour period resulted in additive reductions in MCP-1 mRNA through 24 hours, bronchoalveolar lavage fluid MCP-1 activity at 48 hours, and granuloma size and number at 48 hours . These data suggest that locally produced TNF-alpha and IL-1 beta play regulatory roles in glucan-induced pulmonary granulomatous vasculitis through the modulation of local MCP-1 production.

J Pharmacol Exp Ther, 1995 Feb, 272(2), 939 - 44
Mepyramine, a histamine H1 receptor antagonist, inhibits the metabolic activity of rat and human P450 2D forms; Hiroi T et al.; The interaction of antihistaminics, including mepyramine, with rat hepatic cytochrome P450s (P450s) was investigated . We first investigated mepyramine binding to eight forms of rat hepatic P450s . Mepyramine bound specifically to P450 2D1, which suggests that it inhibits P450 2D activity . Histamine H1 receptor antagonists (mepyramine, diphenhydramine, chlorpheniramine and triprolidine) inhibited the lidocaine 3-hydroxylation activity catalyzed by P450 2D1 but did not inhibit the testosterone hydroxylation activities catalyzed by P450s other than P450 2D forms . The Ki values of these antagonists for the catalytic activity of P450 2D1 were low and were similar to those of quinine and quinidine, which are specific inhibitors of P450 2D forms . The Ki value of mepyramine was especially low, at 34 nM . Furthermore, the effects of mepyramine on human P450 2D6 were investigated . Among the ten forms of human P450 expressed in yeast, mepyramine bound specifically to P450 2D6 in a binding assay . In human hepatic microsomes, mepyramine inhibited the debrisoquine 4-hydroxylation activity catalyzed by P450 2D6 . These results indicate that histamine H1 receptor antagonists such as mepyramine are potent inhibitors of P450 2D forms because of their high affinity for these enzymes.

Am J Hum Genet, 1995 Feb, 56(2), 428 - 33
Haplotype analysis in Australian hemochromatosis patients: evidence for a predominant ancestral haplotype exclusively associated with hemochromatosis; Jazwinska EC et al.; Hemochromatosis (HC), an inherited disorder of iron metabolism, shows a very strong founder effect in Australia, with the majority of patients being of Celtic (Scots/Irish) origin . Australian HC patients thus provide an ideal group in which to examine HC-gene-region haplotypes, to analyze the extent of linkage disequilibrium and genetic heterogeneity in HC . We have analyzed chromosomes from 26 multiply affected HC pedigrees, and we were able to assign HC status unambiguously to 107 chromosomes--64 as affected and 43 as unaffected . The haplotypes examined comprise the following highly polymorphic markers: the serological marker HLA-A and the microsatellites D6S248, D6S265, HLA-F, and D6S105 . All show highly significant allelic association with HC and no evidence of separation from the disease locus by recombination . Analysis identified a predominant ancestral haplotype comprising alleles 5-1-3-2-8 (marker order: D6S248-D6S265-HLA-A-HLA-F-D6S105), present in 21 (33%) of 64 affected chromosomes, and exclusively associated with HC (haplotype relative risk 903) . No other common haplotype was significantly associated with HC . Haplotype analysis in Australian HC patients thus provides strong evidence for (a) the introduction of HC into this population on an ancestral haplotype, (b) a common mutation associated with HC in Australian patients, and (c) a candidate HC-gene region extending between and including D6S248 and D6S105.

J Cell Biol, 1995 Feb, 128(3), 307 - 19
The Hansenula polymorpha PER8 gene encodes a novel peroxisomal integral membrane protein involved in proliferation; Tan X et al.; We previously described the isolation of mutants of the methylotrophic yeast Hansenula polymorpha that are defective in peroxisome biogenesis . Here, we describe the characterization of one of these mutants, per8, and the cloning of the PER8 gene . In either methanol or methylamine medium, conditions that normally induce the organelles, per8 cells contain no peroxisome-like structures and peroxisomal enzymes are located in the cytosol . The sequence of PER8 predicts that its product (Per8p) is a novel polypeptide of 34 kD, and antibodies against Per8p recognize a protein of 31 kD . Analysis of the primary sequence of Per8p revealed a 39-amino-acid cysteine-rich segment with similarity to the C3HC4 family of zinc-finger motifs . Overexpression of PER8 results in a markedly enhanced increase in peroxisome numbers . We show that Per8p is an integral membrane protein of the peroxisome and that it is concentrated in the membranes of newly formed organelles . We propose that Per8p is a component of the molecular machinery that controls the proliferation of this organelle.

Blood, 1995 Feb 1, 85(3), 772 - 9
Molecular characterization of 16p deletions associated with inversion 16 defines the critical fusion for leukemogenesis; Marlton P et al.; The inversion of chromosome 16 {inv(16)} in acute myeloid leukemia (AML) is associated with a p-arm deletion in a subset of patients . The inversion results in two fusion genes: 5'-CBFB/MYH11-3' on 16p and 5'-MYH11/CBFB-3' on 16q . We have studied cells from 42 patients with inv(16) (38 patients) or t(16;16) (four patients) to define the frequency and characteristics of the deletion further . Using fluorescence in situ hybridization (FISH) with probes from cosmids, cosmid contigs, and yeast artificial chromosomes (YACs), we found that six patients with inv(16) had a deletion of between 150 and 350 kb centromeric to the p-arm inversion breakpoint cluster region (p-ibc) . This region was shown to contain the 5' portion of the myosin heavy chain (MYH11) gene . YACs containing the p-ibc, which had been useful as FISH probes in the diagnosis of inv(16), detected the inversion in deletion as well as nondeletion patient cells . Thus, the deleted region identified in patients is entirely contained within the human genomic content of the YACs . Southern blot experiments using probes flanking the p-ibc indicated that the deletion removes segments within 10 kb centromeric of the p-ibc . Reverse transcription-polymerase chain reaction (RT-PCR) using primers from the 5' region of CBFB and the 3' region of MYH11 (distal to the p-ibc) produced the 5'-CBFB/MYH11-3' chimeric transcript in inv(16)/del patients . These data confirm that the 5'-CBFB/MYH11-3' chimeric transcript, rather than the reciprocal 5'-MYH11/CBFB-3', is the critical product for chromosome 16-related leukemogenesis.

J Virol, 1995 Feb, 69(2), 728 - 33
The RNA polymerase PB2 subunit is not required for replication of the influenza virus genome but is involved in capped mRNA synthesis; Nakagawa Y et al.; An established cell line, clone 64, in which the expression of the RNA polymerase PB1 and PA subunit genes and the nucleoprotein (NP) gene but not the PB2 subunit gene of influenza virus can be induced by the addition of dexamethasone, was used to analyze the replication and transcription machineries of the influenza virus . Both NS-CATc and NS-CATv, the chimeric nonstructural protein chloramphenicol acetyltransferase (NS-CAT) RNAs in the sense and antisense orientations positioned between the 5'- and 3'-terminal sequences of influenza virus RNA segment 8 (the NS gene), respectively, can be transcribed into the corresponding complementary-strand RNA in clone 64 cells only when treated with dexamethasone . Although sense-strand poly(A)+ CAT RNA was detected in the dexamethasone-treated clone 64 cells transfected with NS-CATv RNA, CAT activity was not detected in these cells and the isolated poly(A)+ CAT RNA was inert in an in vitro translation system . However, when the poly(A)+ CAT RNA was capped by using a purified yeast mRNA capping enzyme (mRNA guanylyltransferase), the capped poly(A)+ CAT RNA became translatable in the in vitro translation system . These results indicated that PB1, PA, and NP can support the replication of the influenza virus genome as well as the transcription to yield uncapped poly(A)+ RNA and that PB2 is specifically required for the synthesis of capped RNA.

J Virol, 1995 Feb, 69(2), 1107 - 14
Hepatitis B virus X protein interacts with a probable cellular DNA repair protein; Lee TH et al.; The mechanism of action of hepatitis B virus (HBV) X protein in transcriptional transactivation and in tumorigenesis remains obscure . We have used the yeast two-hybrid system to identify a cellular protein that can interact with HBV X protein . This protein, designated X-associated protein 1 (XAP-1), is a human homolog of the UV-damaged DNA-binding protein (UV-DDB) recovered from a monkey cell cDNA library . UV-DDB is presumed to be involved in DNA repair . The interaction between X protein and XAP-1 protein was verified by immunoprecipitation of yeast cell lysates expressing both proteins and by in vitro mixing with X protein expressed as a glutathione S-transferase fusion protein and XAP-1 protein either in HeLa cell extracts or synthesized by in vitro translation . We speculate that the interaction of X protein with a DNA repair protein may recruit cellular proteins to repair the partially double-stranded HBV genome or may modify cellular transcription processes . An effect on the cellular DNA repair system may explain a cofactor role for HBV in liver cancer development.

Cell Struct Funct, 1995 Feb, 20(1), 33 - 9
Expression and phosphorylation of BiP/GRP78, a molecular chaperone in the endoplasmic reticulum, during the differentiation of a mouse myeloblastic cell line; Nakai A et al.; To determine the functional significance of endoplasmic reticulum chaperones in hematopoietic cells, we analyzed the expression and post-translational modification of BiP/GRP78 and GRP94 as well as the cytoplasmic chaperones HSP70 and HSC70 during the differentiation of a mouse myeloid leukemia cell line, M1 . The amounts of BiP/GRP78 and GRP94 increased several-fold when M1 cells were induced to differentiate into macrophage-like cells by treatment with interleukin-6 (IL-6) . Synthesis began to increase at 4 hr after IL-6 treatment . The phosphorylated form of BiP/GRP78 increased during the later stages of differentiation . These data suggested that the chaperone activity of BiP/GRP78 and GRP94 may be needed for differentiated macrophage-like cells or for the differentiation event itself, and that functionally different BiP/GRP78 accumulate during the differentiation of M1 cells.

Biol Chem Hoppe Seyler, 1995 Feb, 376(2), 111 - 7
Purification and characterisation of pyruvate decarboxylase from pea seeds (Pisum sativum cv . Miko); Mucke U et al.; Pyruvate decarboxylase (PDC) was purified from pea seeds . The catalytically active holoenzyme is an oligomer of two types of subunits with molecular masses of about 65 kDa and 68 kDa, respectively . The active enzyme is a mixture of tetramers, octamers and even higher oligomers . These differences in the quaternary structure compared with PDC from yeast (tetramer) do not result in a different kinetic behaviour . The activity of pea PDC as well as that of yeast PDC is regulated by its substrate pyruvate resulting in a sigmoid shape of the v/S-plot . At the optimum pH of 6.0 a S0.5-value of 1 mM pyruvate is found that increases with rising pH and increasing concentrations of phosphate . The substrate analogue activator pyruvamide activates the enzyme resulting in a hyperbolic v/S-plot . The stability of PDC from pea seeds in solution is about one order of magnitude higher than that of yeast PDC . Despite the described similarities of the two enzymes no significant cross reactivity of the anti-pea PDC antibody with the enzyme from yeast occurs.

Diagn Cytopathol, 1995 Feb, 12(1), 51 - 5
Mycoses of the breast: diagnosis by fine-needle aspiration; Farmer C et al.; Fungal infections of the breast are unusual and may clinically mimic carcinoma . When studied by fine-needle aspiration (FNA), such masses may yield necrosis, granulomatous inflammation, reactive histiocytes, and atypical epithelial cells . Cohesive groups of atypical epithelial cells featured nuclear enlargement and overlapping, as well as prominent nucleoli . The organisms may be widely scattered, so that careful evaluation was required for their identification . In concert with provocative clinical findings, these features may lead to an erroneous diagnosis of malignancy . We describe three women with mycotic masses of the breast initially studied by FNA . The first patient presented at age 31 with a large, firm breast mass, chest wall extension, and radiographic evidence of vertebral bone involvement . FNA was requested to confirm the clinical diagnosis of advanced breast carcinoma . In addition to the atypia described above, the smears showed yeast forms indicative of blastomycosis surrounded by neutrophils . She remains well, following antifungal treatment . The second case of Blastomycosis was diagnosed by FNA of a breast mass in a 64-yr-old woman, who also responded to treatment . The third patient's preoperative needle aspiration showed granulomas, but no organisms were identified, even with special stains; silver stains of surgically excised tissue showed histoplasmosis.

Mol Endocrinol, 1995 Feb, 9(2), 243 - 54
Two classes of proteins dependent on either the presence or absence of thyroid hormone for interaction with the thyroid hormone receptor; Lee JW et al.; The thyroid hormone (T3) receptors (TRs) are hormone-dependent transcription factors that regulate expression of a variety of specific target genes . To help elucidate the mechanisms that underlie this transcriptional regulation and other potential TR activities, we used the yeast interaction trap to isolate clones encoding proteins that specifically interact with the ligand binding domain of the rat TR beta . Several such proteins, called Trips (TR-interacting proteins), were isolated from independent selections carried out either in the presence or absence of T3 . Surprisingly, all of the Trips were dependent on hormone for interaction with the TR, with some interacting only when T3 is present and others only when it is absent . Nearly all of the Trips also show similar ligand-dependent interaction with the retinoid X receptor (RXR), but none interact with the glucocorticoid receptor under any conditions . The sequences of three of the Trips predict specific functional roles: one is an apparent human homolog of a yeast transcriptional coactivator, one is a new member of a class of nonhistone chromosomal proteins, and one contains a conserved domain associated with ubiquitination of specific target proteins . Consistent with the pleiotropic effects of TR and RXR, several other Trips show significant amino acid sequence similarity with proteins involved in various regulatory pathways . The inherent transcriptional activity of the Trips was tested in yeast, and a chimeric protein consisting of a fusion of Trip4 to the bacterial LexA repressor protein is a relatively strong transcriptional activator . Similar LexA fusions to Trip9 and Trip10 had no transcriptional activity on their own but, when coexpressed with both TR and RXR, conferred T3-dependent activation to a reporter gene controlled by LexA binding sites . We suggest that this indirect T3 response provides a novel mechanism for hormonal activation of gene expression, and that studies of the Trips will provide important insights into the specific mechanisms of action of TRs and other receptors.

Mamm Genome, 1995 Feb, 6(2), 84 - 9
Physical structure of human chromosome 21: an analysis of YACs spanning 21q; Ochman H et al.; We have resolved the sizes of the yeast artificial chromosomes (YACs) from an ordered library spanning the entire long arm of Chromosome (Chr) 21 to examine the proximity of sequence-tagged sites (STS) originally used to position these clones . The average insert length was 540 kilobases, and some 18% of the 765 clones have either lost or generated multiple YACs during cultivation . Comparing the sizes of YACs that share common sites allowed the identification of an additional 8% of the clones with large scale additions or deletions . Maximum physical distances between chromosome markers, as established by the co-occurrence of STS on a single YAC, generally agreed with those estimated by other procedures, except for a large region in 21q21 . In addition to providing insights into the structure, mapping and organization of this chromosome, knowledge of the sizes and contents of these clones will greatly facilitate the acquisition of any sequence present in this library.

Biosci Biotechnol Biochem, 1995 Feb, 59(2), 302 - 6
Selenium deficiency as a cause of overload of iron and unbalanced distribution of other minerals; Chareonpong-Kawamoto N et al.; Previous studies have shown that there are hematological abnormalities in selenium (Se)-deficient animals . This study examined the effects of Se deficiency on various minerals in serum and other tissues of male Wistar rats . The animals were given free access to either Torula yeast-based Se-deficient (SeD) diet or Se-adequate (SeA) (containing 0.1 mg Se/kg diet as sodium selenite) diet . Blood was sampled after 12 and 24 weeks, and the rats were killed after 24 weeks, for the analysis of minerals in serum, liver, kidney, heart, and spleen . Analyses showed that Se deficiency affected the concentrations of magnesium, calcium, iron, copper, and zinc in selected tissues and serum . During the entire feeding period, serum iron concentration was 40-58% greater in SeD rats compared with SeA rats . The transferrin saturation with iron was significantly greater in SeD rats than in SeA rats (57-60% versus 30-31%) . Iron concentrations in the tissues ranged from 1.1 to 2.5 times higher in SeD rats than in SeA rats (p < 0.05) . Similarly but to a lesser extent, the concentrations of zinc and magnesium were significantly greater in the serum of SeD rats compared with SeA rats, and the concentrations of calcium was significantly higher in kidney and spleen and of copper in liver, while the concentration of magnesium was significantly lower in liver and kidney . These results suggest that Se deficiency may cause a secondary overload of iron and unbalanced distribution of other minerals.

Hum Mol Genet, 1995 Feb, 4(2), 251 - 5
The DXS423E gene in Xp11.21 escapes X chromosome inactivation; Brown CJ et al.; The DXS423E gene has been localized to Xp11.21 and is expressed in somatic cell hybrids retaining either the human active or inactive X chromosome, demonstrating that DXS423E escapes X chromosome inactivation . The XE169 (DXS1272E or SMCX) gene that escapes X chromosome inactivation is also located in Xp11.21-11.22 and maps within the same YAC as DXS423E . Thus the DXS423E and XE169 genes define a new region in the proximal short arm of the X chromosome that is not subject to X chromosome inactivation, supporting a regional basis for escape from inactivation.

Hum Mol Genet, 1995 Feb, 4(2), 237 - 42
Genomic structure of human mismatch repair gene, hMLH1, and its mutation analysis in patients with hereditary non-polyposis colorectal cancer (HNPCC)
Han HJ, Maruyama M, Baba S, Park JG, Nakamura Y.
Mutation of hMLH1, a gene involved in DNA mismatch repair, is responsible for some families carrying the hereditary non-polypotic colorectal cancer (HNPCC) syndrome . To establish a basis for presymptomatic diagnosis of HNPCC patients who carry germline mutations in this gene, we determined the exon-intron organization of hMLH1 . The results indicated that hMLH1 consists of 19 coding exons spanning approximately 100 kb, and that exons 1-7 contain a region that is highly conserved in the MLH1 and PMS1 genes of yeast . We used PCR-SSCP analysis and DNA sequencing to examine the entire coding region of the MLH1 gene in DNAs of 34 unrelated cancer patients who belong to HNPCC pedigrees . Germline mutations were detectable in eight (24%) of these patients; four of them were missense mutations, one had occurred in an intron where it would affect splicing, and the remaining three were frameshift mutations resulting in truncation of the gene product downstream of the mutation site.

Hum Mol Genet, 1995 Feb, 4(2), 223 - 30
Structure and function of ASP, the human homolog of the mouse agouti gene; Wilson BD et al.; The mouse agouti coat color gene encodes a novel paracrine signaling molecule whose pulsatile expression produces a characteristic pattern of banded pigment in individual hairs . Several spontaneous agouti alleles produce adult-onset obesity and diabetes, and have provided important single-gene animal models for alterations in energy metabolism . Utilizing linkage groups conserved between mice and humans, we have cloned the human homolog of the mouse agouti gene from a human chromosome 20 yeast artificial chromosome known to contain S-adenosyl homocysteine hydrolase (AHCY) . The human agouti gene, named Agouti Signaling Protein (ASP), encodes a 132 amino acid protein, the mRNA for which is expressed in testis, ovary, and heart, and at lower levels in liver, kidney, and foreskin . As predicted by the interactions of mouse agouti with the extension gene (which encodes the melanocyte receptor for alpha-melanocyte stimulating hormone {alpha-MSH}), expression of ASP in transgenic mice produces a yellow coat, and expression of ASP in cell culture blocks the alpha-MSH-stimulated accumulation of cAMP in mouse melanoma cells . The localization of ASP relative to other loci on chromosome 20 excludes it as a candidate for the MODY1 locus, a gene responsible for one form of early-onset non-insulin-dependent diabetes mellitus or maturity-onset diabetes of the young . The expression of ASP in human tissues suggests a function for agouti homologs in species that do not exhibit the characteristic phenotype of banded hairs.

Curr Opin Cell Biol, 1995 Feb, 7(1), 39 - 45
Centrin, centrosomes, and mitotic spindle poles; Salisbury JL; Centrin is a ubiquitous protein component of centrosomes and mitotic spindle poles . cDNA clones encoding centrin have been identified from vertebrate sources, and their sequences demonstrate that centrin is a highly conserved member of the EF-hand superfamily of calcium-binding proteins . Analysis of yeast and Chlamydomonas centrin mutants and experimental studies suggest that centrin has an essential role in the duplication of the centrosomes during the cell cycle, and in microtubule severing.

Curr Opin Genet Dev, 1995 Feb, 5(1), 5 - 11
The genetics of cell cycle checkpoints; Murray AW; Checkpoints help in the prevention of genetic damage by giving cells time to repair damaged structures before proceeding in the cell cycle . Genetic analyses in budding and fission yeast have identified a large number of cell cycle checkpoint genes . Several of these encode proteins related to components of other signal transduction pathways, including protein kinases, lipid kinases, and 14-3-3 proteins . In fission yeast, checkpoints play an important role in keeping cells from entering mitosis before they pass Start.

Bioorg Khim, 1995 Feb, 21(2), 112 - 6
{A tryptic heme peptide of cytochrome c from Candida valida}; Zhuravleva DV et al.; Method for direct isolation of heme peptide from enriched yeast biomass have been developed . This method makes it possible to eliminate the process of cytochrome c purification . Amino acid sequence for the Candida valida heme peptide is proposed . Exact molecular masses for both cytochrome c and its heme peptide are determined by mass-spectrometry.

Vet Immunol Immunopathol, 1995 Feb, 44(3-4), 377 - 87
Phagocytic activity of two lines of chickens divergently selected for antibody production; Kreukniet MB et al.; Differences in phagocytic capacity of two chicken lines selected for high (H) or low (L) antibody response against sheep red blood cells (SRBC) were studied in 8 month old cocks of the seventh selection generation . The H line cocks had significantly higher agglutinin titers after immunization with SRBC than the L line . The total clearance capacity of the phagocytes, measured by the clearance of carbon particles from the blood, did not differ between the lines . The L line cocks had more circulating granulocytes . However, the granulocytes of the H line phagocytized more yeast cells than those of the L line . Neither in immunized nor in non-immunized cocks, were line differences found in the intracellular destruction of antigen by phagocytes, estimated as the superoxide production during phagocytosis and the plasma levels of lysozyme activity and acid phosphatase, before and after immunization . It was concluded that the line difference in antibody response was not due to measurable differences in phagocytic activity.

J Pharm Sci, 1995 Feb, 84(2), 263 - 6
Synthesis and antiinflammatory, analgesic, and antipyretic testing of 4-{1-oxo-(3-substituted aryl)-2-propenyl}-3-phenylsydnones and of 3-{4-{3-(substituted aryl)-1-oxo-2-propenyl}phenyl}sydnones; Satyanarayana K et al.; Two series of styrylcarbonyl 3-phenylsydnone derivatives, 4-{1-oxo-(3-substituted aryl)-2-propenyl}-3-phenylsydnones (series 1, 1-21) and 3-{4-{3-(substituted aryl)-1-oxo-2-propenyl}phenyl}sydnones (series II, 22-40), were synthesized and evaluated pharmacologically at a dose of 100 mg/kg po . Eleven compounds in series I plus one in series II and six in series I plus seven in series II were active in the carrageenan-induced edema and acetic acid-induced writhing assays, respectively . Compound 35 in the latter assay showed activity somewhat similar to that of the positive control drug, aspirin, administered at the same dosage . Compounds 11, 17, and 23 showed activity in both assays, and 23 also was active in the adjuvant-induced arthritis assay.

Biotechnol Appl Biochem, 1995 Feb, 21 ( Pt 1), 101 - 10
Effects of inorganic phosphorus compounds on the hydrolysis of phosphatidylcholine liposomes by phospholipid-deacylating enzymes; Sultana GN et al.; Structural requirements of inorganic phosphorus compounds as specific activators or inhibitors for phospholipase A2 and phospholipase B were investigated using orthophosphate, pyrophosphate and polyphosphate . It was observed that orthophosphate and pyrophosphate stimulated the activities of phospholipase A2 from bee venom, snake (Naja naja) venom and pig pancreas, and also phospholipase B from the yeast Torulaspora delbrueckii . However, polyphosphate was found to act as an inhibitor for phospholipase A2 in the above species and also for phospholipase B from T . delbrueckii . Orthophosphate and pyrophosphate induced gradual aggregation of liposome, but polyphosphate prolonged the lifetime of the liposome, suggesting that orthophosphate and pyrophosphate destabilize the bilayer structure of phosphatidylcholine and polyphosphate stabilizes it.

J Endocrinol, 1995 Feb, 144(2), 243 - 50
Induction of an acute phase response in lambs causes an increase in plasma levels of GH and IGF-I; Moore LG et al.; GH and IGF-I plasma concentrations were measured in lambs during an acute phase response induced by an intrathoracic injection of yeast . The acute phase response was indicated by reduced feed intake, weight loss and an increase in plasma concentrations of the acute phase protein haptoglobin . Intensive blood sampling on day 1 revealed elevated basal concentrations of GH in the yeast-injected group compared with concentrations in pair weight and ad libitum fed control lambs . This suggests that at the beginning of an acute phase response there is an increase in either GH secretion or the half life of GH . No evidence of a specific GH-binding protein in sheep plasma could be detected . IGF-I concentrations in the yeast-injected group remained constant for 3 days then increased to a peak level at day 6 . In contrast, plasma IGF-I concentrations were depressed from days 3 to 6 in the pair weight control group and they were unchanged in the ad libitum fed controls . When the IGF-I concentrations were elevated in the yeast-injected group, this group had a higher daily weight gain despite their lower feed intake compared with the ad libitum fed controls . These results suggest that IGF-I may be associated with the increase in weight in the late stage of an acute phase response during recovery from an infection or injury.(ABSTRACT TRUNCATED AT 250 WORDS)

Vaccine, 1995 Feb, 13(2), 139 - 41
Persistence of anti-HBs antibodies in health care personnel vaccinated with plasma-derived hepatitis B vaccine and response to recombinant DNA HB booster vaccine; Trivello R et al.; Long-term persistence of specific antibodies after primary immunization against HBV infection has been reported . In this study, we evaluated the persistence of anti-HBs in vaccinees 6 years after primary immunization and the response to a booster dose using a recombinant DNA yeast-derived HB vaccine . An 85.4% seroprotection rate was observed after 6 years with a significantly higher seroprotective rate in those subjects who received four doses of vaccine primary immunization as compared with those who received three doses (93.9% versus 67.2%, p < 0.001) . One month after receiving the booster dose, 98.6% of the subjects had an anamnestic type of response . The GMTs were found to decrease progressively with increasing age . The antibody levels after booster dose were higher than those attained at the end of primary immunization and reflected the trend seen before the administration of the booster . These results are consistent with the existence of an effective immunological memory in HB vaccine responders . Subjects who received four doses during primary immunization were better seroprotected and had a higher seroprotection rate after the booster dose.

J Cell Sci, 1995 Feb, 108 ( Pt 2), 675 - 83
CDEBP, a site-specific DNA-binding protein of the 'APP-like' family, is required during the early development of the mouse; Blangy A et al.; A murine protein, termed CDEBP, was previously shown to bind the double-stranded DNA motif GTCACATG, identical to the yeast centromeric element CDEI . The cDNA sequence showed three domains with extensive similarities to the amyloid beta precursor protein (APP) . The protein is homologous over its entire length to the human protein designated APPH . In situ immunofluorescence assays using antibodies raised against distinct parts of CDEBP detected discrete sites of accumulation inside the interphase nucleus, and the bulk of the protein was not associated with mitotic chromosomes . One of the complexes with double-stranded CDEI oligonucleotides detected by gel shift assay was not present when the protein had been selectively removed from nuclear extracts by immunoprecipitation . We reported previously that microinjection into one-cell mouse embryos of DNA fragments including the CDEI sequence results in an early arrest of development with abnormal nuclei containing variable amounts of DNA . The same characteristic figures were observed when embryos were treated with antisense oligonucleotides complementary to parts of the CDEBP coding region . Complexes between the CDEBP protein and CDEI sites in the mouse genome thus appear to play a critical role in the replication/segregation of the embryonic genome.

Biochemistry, 1995 Jan 31, 34(4), 1218 - 23
Second derivative spectroscopy of enolase at high hydrostatic pressure: an approach to the study of macromolecular interactions; Kornblatt JA et al.; Second derivative spectroscopy in the ultraviolet region of proteins has been used to study the polarity of the regions surrounding tyrosine residues . We show here that it can also be a tool to study the degree to which proteins associate and that it can be effectively combined with hydrostatic pressure in order to evaluate equilibrium dissociation constants and reaction volumes . Hydrostatic pressure causes yeast enolase to dissociate . Clear changes in the second derivative spectra of enolase were observed as pressure was increased . At enolase concentrations of about 20 microM, the midpoint of the transition is about 1800 bar . All aspects of the transition are reversible up to 2700 bar . It is likely that the transition observed is the result of enolase dimers dissociating into monomers . The second derivative spectra indicate that one or more tyrosine residues is in an unusually polar environment in the dimer, an environment that is less polar in the monomer . Three tyrosines (6, 11, 130) are near the dimer interface . Tyrosines 6 and 11 are pointing into the water-filled crevice between the subunits and are close to several immobilized waters . All three are close to a network of intersubunit salt bridges and hydrogen bonds . We believe that the average tyrosine polarity in the dimer reflects the exposure of these tyrosines to immobilized water and the fixed dipole of the salt bridge . The water in the crevice between the subunits should be more mobile in the monomer; the salt bridge does not exist in the monomer.(ABSTRACT TRUNCATED AT 250 WORDS)

Nucleic Acids Res, 1995 Jan 25, 23(2), 244 - 7
Structural relationship between DNA polymerases epsilon and epsilon* and their occurrence in eukaryotic cells; Uitto L et al.; Monoclonal antibodies raised against the N-terminal half of human DNA polymerase epsilon bind both to a large > 200 kDa form of DNA polymerase epsilon from HeLa cells and to a small 140 kDa form (DNA polymerase epsilon*) from calf thymus, while antibody against the C-terminal half binds to DNA polymerase epsilon but does not bind to DNA polymerase epsilon* . These results indicate that the two enzymes have common structural motifs in their N-terminal halves, and that DNA polymerase epsilon* is very likely derived from DNA polymerase epsilon by removal of its C-terminal half . DNA polymerase epsilon as well as DNA polymerase epsilon* was detected in extracts from cells of numerous eukaryotic species from yeast to human . The results indicate that DNA polymerase epsilon and its tendency to occur in a smaller form, DNA polymerase epsilon*, are evolutionarily highly conserved and that DNA polymerase epsilon may occur universally in proliferating eukaryotic cells.

Biochim Biophys Acta, 1995 Jan 25, 1260(2), 227 - 9
Characterization of edg-2, a human homologue of the Xenopus maternal transcript G10 from endothelial cells; Hla T et al.; We have isolated by polymerase chain reaction-amplified subtractive hybridization technique, several cDNA clones that are induced by phorbol myristic acetate in human umbilical vein endothelial cells (HUVEC) . One such clone, termed edg-2, was sequenced and was found to encode a human homologue of a Xenopus maternal transcript G10 . The deduced amino acid sequence of edg-2 contains a putative nuclear translocation sequence, an N-terminal acidic domain and a cysteine-rich C-terminal domain containing a putative Zinc-finger structure . The structure of edg-2 polypeptide suggests that it may be a nuclear regulator of transcription . The edg-2 mRNA was expressed ubiquitously in cell lines of epithelial and mesenchymal lineages . In addition, the edg-2 polypeptide sequence is highly conserved in evolution and is expressed by lower organisms such as yeast and C . elegans, suggesting that it may be an important regulator of general nuclear function.

Biochemistry, 1995 Jan 24, 34(3), 973 - 83
Photooxidation of Trp-191 in cytochrome c peroxidase by ruthenium-cytochrome c derivatives; Liu RQ et al.; A novel photoinduced electron-transfer reaction is reported in complexes between resting ferric state cytochrome c peroxidase (CcP) and several horse cytochrome c derivatives labeled at single lysine amino groups with {bis(bipyridine)}(dicarboxybipyridine)ruthenium(II) (Ru-CC) . Photoexcitation of Ru(II) in the 1:1 Ru-27-CC:CcP complex results in formation of a metal-to-ligand charge-transfer state, Ru(II*), which is a strong reducing agent and rapidly transfers an electron to the CC heme Fe(III) with rate constant k1 = 2.3 x 10(7) s-1 . The resulting Ru(III) is a strong oxidizing agent with a redox potential of 1.3 V, and it oxidizes the indole ring of Trp-191 with rate constant k3 = 7 x 10(6) s-1 . The cycle is completed by electron transfer from Fe(II) in CC to the Trp-191 radical in CcP with rate constant k4 = 6.1 x 10(4) s-1 . The Ru group is located close to the interaction domain in the Ru-27-CC:CcP complex, allowing rapid electron transfer with both the heme in CC and Trp-191 in CcP . The electron-transfer reaction was not observed in CcP compound I, where Trp-191 is already oxidized to the radical, or in the W191F mutant, where the indole group is replaced with a phenyl group . The electron-transfer reaction was observed in CcP mutants modified at residues in the heme crevice, R48K, R48L, H52L, M230I, and M231I, but not in D235N which destabilizes the radical on Trp-191 . Increasing the ionic strength results in an increase in the equilibrium dissociation constant K of the Ru-27-CC:CcP complex and an increase in the rate constant k5 for dissociation of the transient intermediate containing Fe(II) CC and the radical form of CcP . Both K and k5 were also increased significantly by the mutations D34N, E290N, and A193F involving residues located in the interaction domain of the crystalline complex between yeast CC and CcP {Pelletier & Kraut (1992) Science 258, 1748-1755} . This new method allows the study of the electron-transfer reaction between CC and the radical on Trp-191 in the complete absence of hydrogen peroxide, and it opens the possibility of measurements at low temperatures in frozen glasses or in crystals.

FEBS Lett, 1995 Jan 23, 358(2), 113 - 8
A novel member of the TRAF family of putative signal transducing proteins binds to the cytosolic domain of CD40; Sato T et al.; CD40 is a member of the tumor necrosis factor receptor (TNF-R) family that regulates B-lymphocyte proliferation, immunoglobulin class-switching, and apoptosis through poorly defined signal transduction mechanisms . Using a yeast two-hybrid method, cDNAs were obtained that encode a novel protein, CD40-associated protein-1 (CAP-1), which binds specifically to the cytosolic domain of CD40 but not TNF-R1, TNF-R2, or Fas . The CAP-1 protein contains a C-terminal domain that shares strong amino acid sequence homology with a unique domain found recently in two putative signal transducing proteins that bind to the TNF-R2 cytosolic tail, TRAF1 and TRAF2 . This C-terminal region of CAP-1 was sufficient to mediate binding to CD40 and homodimerization of CAP-1 proteins . The N-terminal portion of CAP-1 contains a RING finger motif and three zinc finger-like domains similar to those found in several regulatory proteins that interact with DNA or RNA . CAP-1 thus represents a new member of a family of potential signal transducing proteins that contain a conserved domain (the TRAF domain), bind to the cytosolic regions of particular members of TNF-R family proteins, and that can form homo- and heterotypic dimers.

Genomics, 1995 Jan 20, 25(2), 591 - 4
Tandem arrangement of the closely linked desmoglein genes on human chromosome 18; Simrak D et al.; The desmogleins, together with the desmocollins, both members of the cadherin superfamily, are the adhesive proteins of the desmosome type of cell junction, characteristically found in epithelial cells . Three different human desmoglein isoforms are encoded by separate genes (DSG1, DSG2, and DSG3) located on chromosome 18q12.1 . DSG2 has been shown to be the most widely expressed in all desmosome-containing tissues, whereas DSG1 and DSG3 are expressed only in certain tissues, mostly stratified squamous epithelia . The desmoglein isoforms are expressed in a stratification-related manner in human epidermis, DSG1 being suprabasally expressed and DSG3 at a lower level, while DSG2 expression is weak and basal . Yeast artificial chromosome clones carrying all three known human desmoglein genes have now been isolated . The smallest clone containing all three DSG genes was 275 kb, and the three desmoglein genes were clustered within a region of less than 150 kb . From the types of clone obtained and from restriction enzyme analysis the order of the DSG genes and their orientation was deduced to be 5'-DSG1-DSG3-DSG2-3' . There thus appears to be some correspondence between the order of DSG genes and their expression within tissues, raising the intriguing possibility that the organization of the desmoglein gene cluster is required for properly regulated gene expression.

Genomics, 1995 Jan 20, 25(2), 577 - 80
Mapping of ribosomal protein S3 and internally nested snoRNA U15A gene to human chromosome 11q13.3-q13.5; Polakiewicz RD et al.; The mammalian ribosome is a massive structure composed of 4 RNA species and about 80 different proteins . One of these ribosomal proteins, S3, appears to function not only in translation but also as an endonuclease in repair of UV-induced DNA damage . Moreover, the first intron of human RPS3 transcripts is processed to generate U15A, a small nucleolar RNA . We localized the nested RPS3/U15A genes to the immediate vicinity of D11S356 and D11S533 on human chromosome 11q13.3-q13.5 using a combination of somatic cell hybrid analysis, fluorescence in situ hybridization, and YAC/STS content mapping . These findings add to the evidence that genes encoding ribosomal proteins are scattered about the human genome.

Genomics, 1995 Jan 20, 25(2), 568 - 9
Localization of the human phosphatidylinositol-specific phospholipase c beta 3 gene (PLCB3) within chromosome band 11q13; Sinke RJ et al.; In course of the molecular characterization of a human extragonadal germ cell tumor (EGCT)-associated chromosomal translocation, we identified YACs and cosmids from the 11q13 region . The endclone of one of these YACs appeared to contain a stretch of DNA homologous to part of the human phosphatidylinositol-specific phospholipase C beta 3 gene (PLCB3) . Since we considered PLCB3 a candidate gene for these EGCTs, we set out to clone the PLCB3 cDNA, from which the 5' end was still missing, and performed Northern and Southern blot analyses . The localization of PLCB3 to 11q13 was confirmed . In addition, we were able to exclude the gene from involvement in EGCT development.

Genomics, 1995 Jan 20, 25(2), 555 - 8
CLONEPLACER: a software tool for simulating contig formation for ordered shotgun sequencing; Singh GB et al.; This communication describes a software tool that enables one to simulate large-scale regional mapping using an ordered shotgun sequencing approach . The analysis routines that are provided yield an estimate of the depth of coverage of the physical map, the largest contig formed, and the number of gaps remaining at any given juncture in the project . A detailed listing describing the span of each contig within the physical map is also presented . This provides an a priori means of estimating the resources that will be required to undertake any megabase mapping or sequencing project . CLONEPLACER provides the much needed guide to deriving the optimal strategy.

Genomics, 1995 Jan 20, 25(2), 526 - 37
Development and physical analysis of YAC contigs covering 7 Mb of Xp22.3-p22.2; Herrell S et al.; A total of 54 YAC clones have been isolated from the region of Xp22.2-p22.3 extending from the amelogenin gene locus to DXS31 . Restriction analysis of these clones in association with STS contenting and end clone analysis has facilitated the construction of 6 contigs covering a total of 7 Mb in which 20 potential CpG islands have been located . Thirty new STSs have been developed from probe and YAC end clone sequences, and these have been used in the analysis of patients suffering from different combinations of chondrodysplasia punctata, mental retardation, X-linked ichthyosis, and Kallmann syndrome . The results suggest that (1) the gene for chondrodysplasia punctata must lie between the X chromosome pseudoautosomal boundary (PABX) and DXS1145; (2) a gene for mental retardation lies between DXS1145 and the sequence tagged site GS1; and (3) the gene for ocular albinism type 1 lies proximal to the STS G13 . The CpG islands within the YAC contigs constitute valuable markers for the potential positions of genes . Genes found associated with any of these potential CpG islands would be possible candidates for the disease genes mentioned above.

Genomics, 1995 Jan 20, 25(2), 469 - 76
Molecular cloning and chromosomal localization of a pseudogene related to the human acyl-CoA binding protein/diazepam binding inhibitor; Gersuk VH et al.; The acyl-CoA binding protein (ACBP) and the diazepam binding inhibitor (DBI) or endozepine are independent isolates of a single 86-amino-acid, 10-kDa protein . ACBP/DBI is highly conserved between species and has been identified in several diverse organisms, including human, cow, rat, frog, duck, insects, plants, and yeast . Although the genomic locus has not yet been cloned in humans, complementary DNA clones with different 5' ends have been isolated and characterized . These cDNA clones appear to be encoded by a single gene . However, Southern blot analyses, in situ hybridizations, and somatic cell hybrid chromosomal mapping all suggest that there are multiple ACBP/DBI-related sequences in the genome . To identify potential members of this gene family, degenerate oligonucleotides corresponding to highly conserved regions of ACBP/DBI were used to screen a human genomic DNA library using the polymerase chain reaction . A novel gene, DBIP1, that is closely related to ACBP/DBI but is clearly distinct was identified . DBIP1 bears extensive sequence homology to ACBP/DBI but lacks the introns predicted by rat and duck genomic sequence studies . A 1-base deletion in the coding region results in a frameshift and, along with the absence of introns and the lack of a detectable transcript, suggests that DBIP1 is a pseudogene . ACBP/DBI has previously been mapped to chromosome 2, although this was recently disputed, and a chromosome 6 location was suggested . We show that ACBP/DBI is correctly placed on chromosome 2 and that the gene identified on chromosome 6 is DBIP1.

Genomics, 1995 Jan 20, 25(2), 462 - 8
The gene encoding the VP16-accessory protein HCF (HCFC1) resides in human Xq28 and is highly expressed in fetal tissues and the adult kidney; Wilson AC et al.; After herpes simplex virus (HSV) infection, the viral regulatory protein VP16 activates transcription of the HSV immediate-early promoters by directing complex formation with two cellular proteins, the POU-homeodomain transcription factor Oct-1 and the host cell factor HCF . The function of HCF in uninfected cells is unknown . Here we show by fluorescence in situ hybridization and somatic cell hybrid analysis that the gene encoding human HCF, HCFC1, maps to the q28 region of the X chromosome . Yeast artificial chromosome and cosmid mapping localizes the HCFC1 gene within 100 kb distal of the renal vasopressin type-2 receptor (V2R) gene and adjacent to the renin-binding protein gene (RENBP) . The HCFC1 gene is apparently unique . HCF transcripts and protein are most abundant in fetal and placental tissues and cell lines, suggesting a role in cell proliferation . In adults, HCF protein is abundant in the kidney, but not in the brain, a site of latent HSV infection and where HCF levels may influence progression of HSV infection.

Genomics, 1995 Jan 20, 25(2), 447 - 61
A high-resolution integrated physical, cytogenetic, and genetic map of human chromosome 11: distal p13 to proximal p15.1; Fantes JA et al.; We describe a detailed physical map of human chromosome 11, extending from the distal part of p13 through the entirety of p14 to proximal p15.1 . The primary level of mapping is based on chromosome breakpoints that divide the region into 20 intervals . At higher resolution YACs cover approximately 12 Mb of the region, and in many places overlapping cosmids are ordered in contiguous arrays . The map incorporates 18 known genes, including precise localization of the GTF2H1 gene encoding the 62-kDa subunit of TFIIH . We have also localized four expressed sequences of unknown function . The physical map incorporates genetic markers that allow relationships between physical and genetic distance to be examined, and similarly includes markers from a radiation hybrid map of 11 . The cytogenetic location of cosmids has been examined on high-resolution banded chromosomes by fluorescence in situ hybridization, and FLpter values have been determined . The map therefore fully integrates physical, genic, genetic, and cytogenetic information and should provide a robust framework for the rapid and accurate assignment of new markers at a high level of resolution in this region of 11p.

Genomics, 1995 Jan 20, 25(2), 404 - 12
Identification of YAC clones for human chromosome 1p32 and physical mapping of the infantile neuronal ceroid lipofuscinosis (INCL) locus; Hellsten E et al.; Infantile neuronal ceroid lipofuscinosis (INCL, CLN1) is a neurodegenerative disorder in which the biochemical defect is unknown . We earlier assigned the disease locus to chromosome 1p32 in the immediate vicinity of the highly informative HY-TM1 marker by linkage and linkage disequilibrium analysis . Here we report the construction of PFGE maps on the CLN1 region covering a total of 4 Mb of this relatively poorly mapped chromosomal region . We established the order of loci at 1p32 as tel-D1S57-L-myc-HY-TM1-rlf-COL9A2-D1S193-D1S6 2-D1S211-cen by combining data obtained from analysis of a chromosome 1 somatic cell hybrid panel, PFGE, and interphase FISH . We isolated YACs and constructed two separate YAC contigs, the loci L-myc, HY-TM1, rlf, and COL9A2 being present on a 1000-kb contig and the markers D1S193, D1S62, and D1S211 on a YAC contig spanning a maximum of 860 kb . Within the 1000-kb contig we were able to identify five CpG islands in addition to those associated with the earlier cloned genes . The YAC contigs as well as the physical map provide us with tools for the identification of the INCL gene.

Genomics, 1995 Jan 20, 25(2), 421 - 32
A 1.8-Mb YAC contig spanning three members of the receptor tyrosine kinase gene family (Pdgfra, Kit, and Flk1) on mouse chromosome 5; Brunkow ME et al.; We constructed a yeast artificial chromosome (YAC) contig spanning the genes encoding Kit (Kit), the platelet-derived growth factor alpha receptor (Pdgfra), and fetal liver kinase 1 (Flk1), three members of a receptor tyrosine kinase gene family located in the central portion of mouse chromosome 5 . The orientation of YAC clones and the extent of their overlap was determined by "probe content mapping," that is, hybridization analysis of YAC clones using the available gene probes and YAC end sequences . For four YAC clones, which constitute a minimal set spanning 1.8 Mb, a detailed restriction map was constructed . This map, in conjunction with the previously published long-range restriction map, indicates the order, the physical distances, and the relative transcriptional orientations of the Pdgfra, Kit, and Flk1 genes . The YAC clones and corresponding YAC end probes presented here provide an important resource for the molecular analysis of a cluster of developmental mutations, namely dominant white spotting (W), patch (Ph), recessive spotting (rs), and rump-white (Rw), associated with this chromosomal region.

EMBO J, 1995 Jan 16, 14(2), 313 - 20
Heme binds to a short sequence that serves a regulatory function in diverse proteins; Zhang L et al.; Heme is a prosthetic group for numerous enzymes, cytochromes and globins, and it binds tightly, sometimes covalently, to these proteins . Interestingly, heme also potentiates binding of the yeast transcriptional activator HAP1 to DNA and inhibits mitochondrial import of the mammalian delta-aminolevulinate synthase (ALAS) and the catalytic activity of the reticulocyte kinase, HRI . All three of these proteins contain a short sequence, the heme regulatory motif (HRM), that occurs six times adjacent to the HAP1 DNA binding domain, twice in the leader targeting sequence of ALAS and twice near the catalytic domain of the HRI kinase . Here we show that a 10 amino acid peptide containing the HRM consensus binds to heme in the micromolar range, and shifts the heme absorption spectrum to a longer wavelength, a direction opposite to the change caused by cytochromes or globins . Further, we show that a single HRM regulates the acidic activation domains of HAP1 and GAL4 independently of regulation of DNA binding of the transcription factors . These findings thus establish a novel heme binding sequence which is structurally distinct from sequences in globins or cytochromes and which has a regulatory function.

Genes Dev, 1995 Jan 15, 9(2), 182 - 92
A homeo domain protein lacking specific side chains of helix 3 can still bind DNA and direct transcriptional repression; Vershon AK et al.; A series of mutations in the homeo domain of the yeast alpha 2 protein were constructed to test, both in vivo and in vitro, predictions based on the alpha 2-DNA cocrystal structure described by Wolberger et al . (1991) . The effects of the mutations were observed in three different contexts using authentic target DNA sequences: alpha 2 binding alone to specific DNA, alpha 2 binding cooperatively with MCM1 to specific DNA, and alpha 2 binding cooperatively with a1 to specific DNA . As expected, changes in the amino acid residues that contact DNA in the X-ray structure severely compromised the ability of alpha 2 to bind DNA alone and to bind DNA cooperatively with MCM1 . In contrast, many of these same mutations, including a triple change that altered all the "recognition" residues of helix 3, had little or no effect on the cooperative binding of alpha 2 and a1 to specific DNA, as determined both in vivo and in vitro . These results show that the ability of a homeo domain protein to correctly select and repress target genes does not necessarily depend on the residues commonly implicated in sequence-specific DNA binding.

Eur J Biochem, 1995 Jan 15, 227(1-2), 284 - 91
Conversion of phenol derivatives to hydroxylated products by phenol hydroxylase from Trichosporon cutaneum . A comparison of regioselectivity and rate of conversion with calculated molecular orbital substrate characteristics; Peelen S et al.; This study describes the regioselective hydroxylation and the rates of conversion of a series of fluorinated phenol derivatives by phenol hydroxylase from the yeast Trichosporon cutaneum . The natural logarithm of the kcat value for the conversion of the phenolic substrates correlates with the calculated energy of the reactive electrons in the highest occupied molecular orbital of the substrate (r = 0.85) . This observation supports the hypothesis that at physiological pH (7.6) and 25 degrees C, in the absence of monovalent anions, the nucleophilic attack of the electrons in the highest occupied molecular orbital of the substrate on the C(4a)-hydroperoxyflavin enzyme intermediate is of major importance in determining the overall rate of catalysis . Results from 19F-NMR analysis of the incubation mixtures demonstrate for phenols with two identical ortho substituents, that the ortho position which becomes preferentially hydroxylated is the one with the highest density of the reactive electrons in the highest occupied molecular orbital . A halogen substituent at a meta position decreases the chances for hydroxylation at the adjacent ortho position further than expected on the basis of the calculated reactivity . This result indicates a contribution of a protein/substrate dipolar interaction, influencing the time-averaged orientation of the substrate with respect to the reactive C(4a)-hydroperoxyflavin intermediate.

Cell, 1995 Jan 13, 80(1), 155 - 65
Identification and characterization of a spinal muscular atrophy-determining gene; Lefebvre S et al.; Spinal muscular atrophy (SMA) is a common fatal autosomal recessive disorder characterized by degeneration of lower motor neurons, leading to progressive paralysis with muscular atrophy . The gene for SMA has been mapped to chromosome 5q13, where large-scale deletions have been reported . We describe here the inverted duplication of a 500 kb element in normal chromosomes and narrow the critical region to 140 kb within the telomeric region . This interval contains a 20 kb gene encoding a novel protein of 294 amino acids . An highly homologous gene is present in the centromeric element of 95% of controls . The telomeric gene is either lacking or interrupted in 226 of 229 patients, and patients retaining this gene (3 of 229) carry either a point mutation (Y272C) or short deletions in the consensus splice sites of introns 6 and 7 . These data suggest that this gene, termed the survival motor neuron (SMN) gene, is an SMA-determining gene.

Biochem Pharmacol, 1995 Jan 6, 49(1), 39 - 47
Short synthetic peptides exploited for reliable and specific targeting of antibodies to the C-termini of cytochrome P450 enzymes; Edwards RJ et al.; An antibody was raised against a synthetic peptide (Ser-Glu-Asn-Tyr-Lys-Asp-Asn) corresponding to residues 290-296 of the cytochrome P450 enzyme, CYP1A2, of both rat and mouse . A cysteine residue attached to the N-terminus of the peptide during synthesis allowed coupling in a specific orientation via the thiol group to the carrier protein, keyhole limpet haemocyanin . Antiserum raised in rabbits bound specifically to CYP1A2 in the rat and mouse . To determine those amino acid residues involved in binding of the antibody, related peptides of various lengths were synthesised and the binding of the antibody was determined by an enzyme-linked immunosorbent assay . These studies show that the minimum epitope is the C-terminal tripeptide sequence, Lys-Asp-Asn . Other than in rat and mouse CYP1A2, this tripeptide is found as an internal sequence in a large number of proteins including bovine fibronectin, chicken gizzard myosin heavy chain, and the P450 enzymes, rabbit CYP3A6 and human CYP3A4, but the antibody did not bind to any of these proteins . However, the antibody did bind to yeast glucose-6-phosphate dehydrogenase in which the tripeptide sequence is the C-terminus . Antibodies raised against a truncated peptide (Tyr-Lys-Asp-Asn), representing the C-terminal half of the peptide, also bound to glucose-6-phosphate dehydrogenase, but failed to bind to CYP1A2; thus although the C-terminal region of the peptide 290-296 is strongly immunogenic, it appears that it is not this population of antibodies that binds to CYP1A2 . As antibodies were found to bind strongly to the C-terminus of glucose-6-phosphate dehydrogenase, the C-termini of proteins as targets for anti-peptide antibodies were investigated further by immunising rabbits with four 5-residue peptides which represent the C-termini of the P450 enzymes, CYP1A1, CYP1A2, CYP2E1 and CYP2A6 . The peptides were coupled to keyhole limpet haemocyanin through their N-termini via cysteine residues added to the sequences . All four antisera bound specifically to their respective target proteins, as demonstrated by immunoblotting using hepatic microsomal fractions from rat, rabbit and human . It is suggested that this method of antibody production could be of general use for the reliable production of antisera against proteins where their sequence at the C-terminus is known, and such antibodies can be highly specific as they do not bind to internal sequences.

Biochem Biophys Res Commun, 1995 Jan 5, 206(1), 294 - 301
Identification of multiple transcribed sequences from the spinal muscular atrophy region of human chromosome 5; Pizzuti A et al.; We report the isolation and characterization of novel expressed sequences from the spinal muscular atrophy (SMA) region on human chromosome 5q13 . Based on the sequence homology studies these cDNAs were grouped in four classes, one of which shows extensive homologies with the beta-glucuronidase (BG) gene, differing in exon arrangement . The other cDNAs do not show any strong homology with known DNA sequences.

Biochim Biophys Acta, 1995 Jan 5, 1246(1), 53 - 60
Purification and immunochemical characteristics of NADPH-cytochrome P-450 oxidoreductase from tobacco cultured cells; Imaishi H et al.; NADPH-cytochrome P-450 oxidoreductase (EC 1.6.2.4) was purified from the microsomal fraction of tobacco (Nicotiana tabacum) BY2 cells by chromatography on two anion-exchange columns and 2',5' ADP-Sepharose 4B column . The purified enzyme showed a single protein band with a molecular weight of 79 kDa on SDS-PAGE and exhibited a typical flavoprotein redox spectrum, indicating the presence of an equimolar quantity of FAD and FMN . This enzyme followed Michaelis-Menten Kinetics with Km values of 24 microM for NADPH and 16 microM for cytochrome c . An in vitro reconstituted system of the purified reductase with a partially purified tobacco cytochrome P-450 preparation showed the cinnamic acid 4-hydroxylase activity at the rate of 14 pmol min-1 nmol-1 P-450 protein and with a purified rabbit P-4502C14 catalyzed N-demethylation of aminopyrine at the rate of 6 pmol min-1 nmol-1 P-450 protein . Polyclonal antibodies raised against the purified reductase reacted with tobacco reductase but not with yeast reductase on Western blot analysis . Anti-yeast reductase antibodies did not react with the tobacco reductase . This result indicate that the tobacco reductase was immunochemically different from the yeast reductase . The anti-tobacco reductase antibodies totally inhibited the tobacco reductase activity, but not the yeast reductase . Also, Western blot analyses using the anti-tobacco reductase antibodies revealed that leaves, roots and shoots of Nicotiana tabacum plants contained an equal amount of the reductase protein . From these results, it was suggested that there are different antibody binding sites, which certainly participate in enzyme activity, between tobacco and yeast reductase.

Princess Takamatsu Symp, 1995, 25, 185 - 98
Viral co-factors in liver cancer: lessons from hepatitis B virus; Butel JS et al.; Hepatitis B virus (HBV) is a co-factor in some hepatocellular carcinomas (HCC) . Chronic infection with HBV is a risk factor for tumor development, suggesting the accumulation of cellular genetic changes . HBV DNA is frequently found integrated at random sites in HCC, with chromosomal deletions and rearrangements being common at the sites of viral integration . Tumor suppressor gene p53 is frequently altered in HCC . Environmental carcinogens are factors in HCC development in certain geographic locations . HBV encodes a protein (X) known to transactivate viral and cellular genes; the X gene is often retained in HCC . To learn more about X gene function . We employed the yeast two-hybrid genetic system to seek X-interactive proteins . A cellular protein, designated XAP-1, was recovered that interacts specifically with the X protein . XAP-1 is the human homologue of the monkey UV-damaged DNA-binding protein (UV-DDB); the UV-DDB protein functions in DNA repair and is defective in some xeroderma pigmentosum group E patients . The interaction between XAP-1 and HBV X protein was confirmed by several independent methods . This suggests that cellular DNA repair processes may be affected by HBV and that the resulting genetic instability may contribute to hepatocellular carcinogenesis . A unifying model of the molecular basis of HBV involvement in HCC development is presented . Fundamental components of the model are chronic infection by HBV and viral effects on cellular DNA repair . This model has implications for the possible role of HCV infection in the induction of HCV-associated HCC.

Acta Physiol Hung, 1995, 83(3), 243 - 8
A novel spectrophotometric method for the enzymatic determination of NAD+ and NADH; Leskovac V et al.; The theory and practice of a novel spectrophotometric method for the enzymatic determination of NAD+ and NADH is described . The method can not discriminate between NAD+ and NADH, but determines the concentration of the sum of both nucleotides . The method is based on the bleaching of p-nitroso-N,N-dimethylaniline (NMDA) (epsilon 440 nm = 35400 M-1cm-1) with NADH, in the presence of ethanol and yeast alcohol dehydrogenase, under the conditions of enzymatic cycling (ethanol > NDNA > NAD/H) . The initial rates of -NDMA bleaching are proportional to the concentration of NAD+ or NADH, in a broad range from 10 nM to 100 microM.

Eur J Hum Genet, 1995, 3(6), 351 - 6
Seeding of YACs over regions 1q41-q42.3 and 11q14.3-q23 with microdissection clones; Petit J et al.; We describe the use of pooled, region-specific hybridisation probes to screen high-density replica filters of a human genome YAC library . The probes were derived by microdissection of an approximately 30-Mbp region subtending the translocation breakpoint on a der(1)(1;11)(q42.1;q14.3) chromosome . Of 70 microdissection clones used in pools of 4-10, 47 identified a total of 77 YAC recombinants, representing over 50% of the microdissected region . This strategy can easily be adapted to other poorly mapped subchromosomal regions of the human or other mammalian genomes and will provide a solid framework for detailed contig map constructions.

Eur J Hum Genet, 1995, 3(6), 344 - 50
A limited genomic region contains the human REG and REG-related genes; Bartoli C et al.; A glycoprotein expressed in exocrine pancreas (where it has been called lithostathine) and endocrine pancreas (where it has been called the regeneration protein) is encoded by a gene (REG) which maps to 2p12 . A REG-related sequence (REGL) is also located in 2p12 and expressed in the pancreas . Here we describe the physical mapping of these genes within a 100-kb genomic region . A YAC clone was converted into an ordered cosmid contig . We constructed a restriction map of the cosmid contig and localized the loci corresponding to the genes . A third REG-related sequence also maps to this region . Although this sequence was previously described as a pseudogene, we show here that it is also expressed in the pancreas.

Annu Rev Genet, 1995, 29, 553 - 75
Genetic variation and aging; Curtsinger JW et al.; Life span is subject to genetic modification in yeasts, nematodes, fruit flies, mice, humans, and other vertebrates and invertebrates . There are a few single-gene mutants known that extend life span in yeast and nematodes; in other experimental systems the character is treated quantitatively, and generally has a low to moderate heritability . Life span responds to artificial selection in Drosophila and Caenorhabditis . There are many candidate genes presently under investigation, including the anti-oxidizing enzymes and heat-shock proteins . The main evolutionary models of senescence are antagonistic pleiotropy and mutation accumulation, neither of which has substantial experimental support . The incorporation of analytical techniques from demography is playing an increasing role in research on aging.

Annu Rev Genet, 1995, 29, 289 - 303
The Polycomb and trithorax group proteins of Drosophila: trans-regulators of homeotic gene function; Kennison JA; The Polycomb and trithorax group genes encode trans-regulators of homeotic gene function in Drosophila . The Polycomb group genes encode transcriptional repressors, while the trithorax group proteins are positive factors required for homeotic gene function . Among the Polycomb group proteins, the POLYCOMB protein has been most extensively characterized . The POLYCOMB protein contains a chromodomain, a conserved domain found in a Drosophila protein with effects on position-effect variegation . Among the trithorax group proteins characterized, the BRAHMA protein appears to be a subunit of a protein complex conserved from yeast to man (the SNF/SWI complex) that modifies chromatin to facilitate the transcriptional activation by gene-specific DNA-binding proteins . The ZESTE protein may help to activate transcription by bringing distant cis-regulatory elements closer to promoter-bound proteins.

Biochimie, 1995, 77(10), 817 - 25
Chromosomal instability and alteration of telomere repeat sequences; Pommier JP et al.; The very end of the chromosome is called the telomere and is composed of DNA repeat sequences and associated proteins . Genetic and biochemical analyses of this complex, the telosome, lead to the hypothesis that transcription and DNA replication are submitted to position effects mediated by the telomere proximity . Telomere length reduction and alterations of the telomeric chromatin assembly might explain the chromosome instability which occurs during the senescence and the immortalization process in vitro . A particular polymerase, the telomerase, is able to lengthen the telomeres . A telomerase activity was characterized in yeast, Tetrahymena, but also in transformed and in germline cells . We reviewed the involvement of telomeres in the aging process . We proposed that the short size of the telomere repeat at each chromosome could direct the loss of heterozygosity, thus telomere length could play a role in individual and tissular susceptibility to develop cancer . Antitelomerase strategy for cancer therapy is attractive but limited by the short decrease of the telomere length at each cell division.

Essays Biochem, 1995, 30, 77 - 95
Exocytosis; Morgan A; Exocytosis is the fusion of secretory vesicles with the plasma membrane and results in the discharge of vesicle content into the extracellular space and the incorporation of new proteins and lipids into the plasma membrane . Exocytosis can be constitutive (all cells) or regulated (specialized cells such as neurons, endocrine and exocrine cells) . Regulated exocytosis is usually, but not always, triggered by an increase in the cytosolic free Ca2+ concentration . In neurons and endocrine cells, a small proportion of regulated secretory vesicles are ready to fuse with the plasma membrane in response to cell stimulation, but the majority are kept in reserve for subsequent stimulation by linkage to a filamentous network of synapsins (in neurons) or actin (in endocrine cells) . Regulated exocytosis varies greatly in kinetics and Ca2+ dependency between cell types . It is likely that several different Ca(2+)-binding proteins are involved in regulated exocytosis, with synaptotagmin apparently essential for fast exocytosis at synapses . GTP-binding proteins of both the monomeric and heterotrimeric forms are involved in exocytosis, although their precise role is unclear . Intense current interest focuses on the idea that the molecular mechanism of vesicle docking and fusion is conserved from yeast to mammalian brain . The SNARE hypothesis postulates that vesicle SNAREs (synaptobrevin and homologues) mediate docking by binding to target SNAREs (syntaxin/SNAP-25 and homologues), whereupon SNAPs and NSF bind to elicit membrane fusion.

Receptors Channels, 1995, 3(3), 213 - 20
Characterization of a voltage-activated K-channel gene cluster on human chromosome 12p13; Albrecht B et al.; The Shaker related alpha-subunit subfamily of human voltage-activated KCNA-channels is encoded by several genes in the human genome . We have isolated yeast artificial chromosomes (YACs) spanning approximately 1 Mb of human chromosome 12p13 DNA . Molecular biological characterization of the YAC-insert DNAs revealed that three KCNA genes--KCNA1, KCNA5, KCNA6--are clustered within approximately 300 kb of insert-DNA derived from chromosome 12p13 . The KCNA gene cluster has been conserved between mouse and human . The three KCNA genes in the cluster are transcribed from the same DNA strand being arranged in a head-to-tail fashion in the order KCNA6-KCNA1-KCNA5 . Northern blot analyses of the KCNA1, KCNA5 and KCNA6 mRNAs in different human brain areas show that the genes are distinctly expressed . Therefore, they may be transcribed independently of each other.

Crit Rev Oncog, 1995, 6(2), 99 - 115
The growing family of MAP kinases: regulation and specificity; Kortenjann M et al.; The family of MAP kinases consists of several subgroups of serine/threonine protein kinases . Together with their activating kinases, they function to regulate cellular responses to diverse extracellular signals, including osmotic stress, heat shock, proinflammatory cytokines, and mitogens . It is now clear that as in yeast, separate MAP kinase cascades exist in mammalian cells, responding selectively to different stimuli by phosphorylating cytoplasmic components and nuclear transcription factors . Down-regulation of MAP kinase pathways may occur through dephosphorylation by serine/threonine phosphatases, tyrosine phosphatases, or dual-specificity phosphatases and through feedback inhibitory mechanisms that involve the phosphorylation of upstream kinases . The functional integrity of each MAP kinase cascade is thought to be established and maintained by specific molecular interactions both between the kinases and with cytoplasmic anchors that nucleate complex formation . The recent demonstration that a series of pyridinyl-imidazole compounds can bind and inhibit certain MAP kinases suggests that other MAP kinase subgroups may also be susceptible to synthetic compounds . Drugs that selectively down-regulate MAP kinase cascades could prove to be valuable as therapeutic agents in the control of malignant disease.

Reprod Fertil Dev, 1995, 7(4), 669 - 83
Genetic control of mitosis, meiosis and cellular differentiation during mammalian spermatogenesis; Wolgemuth DJ et al.; Gametogenesis in both the male and female mammal represents a specialized and highly regulated series of cell cycle events, involving both mitosis and meiosis as well as subsequent differentiation . Recent advances in our understanding of the genetic control of the eukaryotic cell cycle have underscored the evolutionarily-conserved nature of these regulatory processes . However, most of the data have been obtained from yeast model systems and mammalian cell lines . Furthermore, most of the observations focus on regulation of mitotic cell cycles . In the present paper: (i) aspects of gametogenesis in mammals that represent unique cell-cycle control points are highlighted; (ii) current knowledge on the regulation of the germ cell cycle, in the context of what is known in yeast and other model eukaryotic systems, is summarized; and (iii) strategies that can be used to identify additional cell cycle regulating genes are outlined.

Cell Motil Cytoskeleton, 1995, 32(2), 136 - 44
Dynein family of motor proteins: present status and future questions; Gibbons IR; Analysis of sequence relationships in dynein heavy chains shows that dynein motor proteins comprise a single homologous family with three main branches, cytoplasmic dynein, axonemal dynein, and a third branch represented by DYH1B that lies between the other two . In all branches of the family the dynein heavy chain has four copies of the P-loop motif for a nucleotide-binding site spaced approximately 300 residues apart in its midregion, with the amino acid sequence GPAGTGKT in the P-loop of the hydrolytic ATP-binding site . Cytoplasmic dyneins appear more primitive in that the heavy chain usually occurs as a homodimer, with traces of the early evolution of its four P-loop motifs by gene duplication being recognizable . In the axonemal subfamily the heavy chain occurs as heterodimers or heterotrimers encoded by multiple genes, and their non-hydrolytic P-loop motifs are much more divergent with little trace of their origin by gene duplication . The DYH1B subfamily is more closely related to the cytoplasmic dyneins in sequence, but appears related to axonemal dyneins in function since it becomes upregulated during reciliation and has not been found in organisms, such as yeast and Dictyostelium, that are totally without cilia or flagella.

Hum Mutat, 1995, 6(4), 303 - 10
Analysis of mutational changes at the HLA locus in single human sperm; Huang MM et al.; Using a simple and efficient single sperm PCR and direct sequencing method, we screened for HLA-DPB1 gene mutations that may give rise to new alleles at this highly polymorphic locus . More than 800 single sperm were studied from a heterozygous individual whose two alleles carried 16 nucleotide sequence differences clustered in six polymorphic regions . A potential microgene conversion event was detected . Unrepaired heteroduplex DNA similar to that which gives rise to postmeiotic segregation events in yeast was observed in three cases . Control experiments also revealed unusual sperm from DPB1 homozygous individuals . The data may help explain allelic diversity in the MHC and suggest that a possible source of human mosaicism may be incomplete DNA mismatch repair during gametogenesis.

Cell Motil Cytoskeleton, 1995, 32(1), 26 - 36
Genomic structure of a cytoplasmic dynein heavy chain gene from the nematode Caenorhabditis elegans; Lye RJ et al.; We report the cloning and sequencing of genomic DNA encoding a cytoplasmic dynein heavy chain from the nematode Caenorhabditis elegans . In a contiguous stretch of 35,103 bp of DNA from the left arm of linkage group I, we have found a gene that is predicted to encode a protein of 4,568 amino acids . This gene is composed of 15 exons and 14 relatively short introns, and it has significant homology to the other dynein heavy chains in the databases . The deduced molecular mass of the derived polypeptide is 512,624 Da . As with other dynein heavy chains that have been sequenced to date, it contains four GXXGXGK(S/T) motifs that form part of a consensus sequence for the nucleotide triphosphate-binding domains . Comparison of the axonemal and cytoplasmic dynein heavy chains shows that regions of homology among all dyneins are clustered in the carboxyl terminal two-thirds of the polypeptide, whereas the amino terminal one-third of the heavy chains may contain domains that specify functions that differ between the axonemal and cytoplasmic forms of the dynein heavy chain.

Blood Cells Mol Dis, 1995, 21(3), 207 - 16
A strategy for cloning the hereditary hemochromatosis gene; Beutler E et al.; Selective hybridization of small intestine and liver cDNA libraries was carried out using yeast artificial chromosomes (YACs) surrounding D6S105, the microsatellite that appears to be close to the gene for hereditary hemochromatosis (HFE) . Of 14 candidate probes hybridizing with these YACs, only one, designated . LD5-1, detected abnormalities in southern blots of patients with hemochromatosis . Two different abnormalities . were detected in 3 of 55 patients with hemochromatosis with the LD5-1 probe, and one of these was detected in one of 44 normal subjects . The gene that hybridizes with this probe is located about 300-400 kb centromeric of D6S105 . It is transcribed into mRNA that is about 8.5 kb in length in many tissues, including peripheral blood leukocytes . The available sequence indicates tha it codes for a zinc finger protein . We propose that there is a reasonable probability that LD5-1 hybridizes with the gene for hereditary hemochromatosis.

Acta Vet Scand, 1995, 36(4), 553 - 62
Flow-cytometric studies of the phagocytic capacities of equine neutrophils; Johannisson A et al.; Methodological aspects of flow-cytometric evaluation of the phagocytic properties of equine neutrophils were elucidated . The kinetics of attachment and ingestion were studied, and the phagocytic process was more rapidly completed when serum-opsonized yeast cells were used than with use of IgG-opsonized yeast cells . Trypan blue was successfully used to quench fluorescence of non-ingested yeast cells . There were only minor differences in the kinetics of phagocytosis between quenched and unquenched samples, indicating that attachment is rapidly followed by ingestion . Trypan blue quenching caused loss of cells with light scattering properties of granulocytes, although this did not affect the determined frequencies of truly phagocytic neutrophils . Aggregation of yeast cells proved to be a disturbance but not an obstacle to the determination of frequencies of actively phagocytic cells . Flow cytometry is well suited for studies of phagocytosis of yeast cells by equine neutrophils, and the trypan blue quenching provides a means of eliminating false-positive events due to aggregation of yeast cells . The main advantage of the flow-cytometric method is the possibility of rapid processing of a large number of samples, making the method useful for studies of herds.

Tsitologiia, 1995, 37(8), 813 - 9
{The physical mapping of human chromosomes . The use of the polymerase chain reaction on unique DNA sequences with a known location on chromosome 3}; Svetlova MP et al.; 16 pairs of oligonucleotide primers, complementary to unique DNA sequences of human chromosome 3, were synthesized . For 10 of these, fragments of expected length were generated in polymerase chain reaction (PCR) . These fragments may be used as markers for detailed physical mapping of this chromosome . The above primers were used in PCR in order to analyse a hybrid mice-human cell line which contained presumably a fragment of human chromosome 3 . The presence of human DNA in the hybrid line has been shown, but no ultimate evidence was received to confirm its location in human chromosome 3 . By means of primers, complementary to the butyrylcholinesterase gene (BCHE), pools of clones from the yeast total library of human DNA were analysed, and then the pool and later the individual {correction of undividual} clone, containing a fragment of BCHE gene, were identified.

Intervirology, 1995, 38(1-2), 63 - 74
Hepatitis B virus core particles as epitope carriers; Pumpens P et al.; HBV core (HBc) particle is one of the most intensively studied particulate carriers for the insertion of foreign peptide sequences . Recombinant HBc protein expressed from the cloned gene undergoes the correct folding in a large variety of bacterial, yeast, insect and mammalian cells . Unique assembly properties and shape of 30/34-nm HBc particles allow substantial insertions into their primary structure without loss of their capsid-forming ability . N- and C-terminal regions, as well as the immunodominant loop in the middle of the molecule are widely accepted as targets for the introduction of foreign epitopes, ensuring retention and even enhancement of the original immunological activity of inserted sequences . Special sets of display vectors have been constructed on the basis of the cloned HBc gene . Epitope sequences of viral (BLV, FeLV, FMDV, HBV, HCV, HIV-1, HRV2, MCMV, PV-1, SIV) and nonviral (human chorionic gonadotropin) origin have been studied as model display moieties.

Intervirology, 1995, 38(1-2), 16 - 23
Mutational analysis of HBsAg assembly; Prange R et al.; The 20-nm noninfectious empty envelope particles carrying the hepatitis B surface antigen are secreted in large excess from hepatocytes during a hepatitis B infection . Similar particles produced in cell lines or yeast are an efficient vaccine against hepatitis B virus . We have analyzed the assembly of 20-nm particles using a mutagenesis approach . Specific mutations were introduced into the S gene and the preS region encoding the viral envelope proteins using recombinant DNA techniques . The mutant genes were expressed in cell lines to identify the amino acid residues that are critical for the assembly and the secretion of the 20-nm particles.

Nucleic Acids Symp Ser, 1995, (33), 115 - 9
Structural specificity of nuclease from wheat chloroplasts stroma; Gabryszuk J et al.; A single-strand-specific nuclease from wheat chloroplasts (ChS nuclease) was tested as a tool for RNA secondary and tertiary structure investigations, using yeast tRNA(Phe) and yeast tRNA(Asp) as models . In tRNA(Phe) the nuclease introduced main primary cleavages at positions U33, A35 and A36 in the anticodon-loop and G18 and G19 in the D-loop . In tRNA(Asp) the main primary cleavages occurred at positions U33, G34 and U35 in the anticodon-loop and the lower one at position C20:1 in the D-loop . No primary cleavages were observed within the double-stranded stems . Because ChS nuclease has (i) a low molecular weight, (ii) a wide pH range of action (5.0 to 7.5) (iii) no divalent cation requirement in the reaction mixture and (iv) can be obtained as a pure protein in rather large quantities it appeared to be a very good tool for secondary and tertiary structural studies of RNAs.

J Comput Biol, 1995 Winter, 2(4), 557 - 72
Challenges in integrating biological data sources; Davidson SB et al.; Scientific data of importance to biologists reside in a number of different data sources, such as GenBank, GSDB, SWISS-PROT, EMBL, and OMIM, among many others . Some of these data sources are conventional databases implemented using database management systems (DBMSs) and others are structured files maintained in a number of different formats (e.g., ASN.1 and ACE) . In addition, software packages such as sequence analysis packages (e.g., BLAST and FASTA) produce data and can therefore be viewed as data sources . To counter the increasing dispersion and heterogeneity of data, different approaches to integrating these data sources are appearing throughout the bioinformatics community . This paper surveys the technical challenges to integration, classifies the approaches, and critiques the available tools and methodologies.

Southeast Asian J Trop Med Public Health, 1995, 26 Suppl 1, 34 - 43
Clinical and molecular cytogenetics and gene mapping: principles and techniques; Francke U; This article reviews the history of human cytogenetics with respect to technical advances from chromosome banding to molecular cytogenetics . Technologies such as in situ hybridization, chromosome painting, comparative genomic hybridization and interphase cytogenetics and their applications are discussed . The assignments of genes to chromosome regions by somatic cell genetics is illustrated by molecular analyses of somatic cell hybrid panels . The generation of complete physical maps of human chromosomes, by radiation hybrid mapping of sequence-tagged sites and establishment of chromosome-specific yeast artificial chromosome (YAC) banks and clone overlaps (contigs), is exemplified by studies of chromosome 18 . The last section outlines the recent and future advances in clinical cytogenetics made possible by progress in molecular genetics.

Biochimie, 1995, 77(7-8), 549 - 61
Design of ruthenium-cytochrome c derivatives to measure electron transfer to cytochrome c peroxidase; Liu RQ et al.; A new technique has been introduced to measure interprotein electron transfer which involves photoexcitation of a tris(bipyridine)ruthenium (Ru) complex covalently attached to one of the proteins . Four different strategies have been developed to specifically attach Ru to protein lysine amino groups, histidine imidazole groups, and cysteine sulhydryl groups . These strategies have been used to prepare more than 20 different singly-labeled Ru-cytochrome c derivatives . The new ruthenium photoexcitation technique has been used to study the mechanism for electron transfer between cytochrome c and cytochrome c peroxidase . Laser excitation of a complex between Ru-cytochrome c and cytochrome c peroxidase compound I results in formation of Ru(II*) which is a strong reducing agent, and rapidly transfers an electron to heme c Fe(III) to form Fe(II) . The heme c Fe(II) then rapidly transfers an electron to the Trp-191 radical cation in CMPI . The rate constant for this reaction is 6 x 10(4) s-1 for a horse Ru-cytochrome c derivative labeled at lysine 27, and greater than 10(6) s-1 for yeast Ru-cytochrome c derivatives . A second laser flash results in electron transfer from heme c to the oxyferryl heme in cytochrome c peroxidase compound II with a rate constant of 350 s-1 . The ruthenium photoreduction technique has been used to study the interaction domain between the two proteins, the pathway for electron transfer to the radical cation and the oxyferryl heme, and the specific residues in the heme crevice which control the electron transfer properties of the Trp-191 radical cation and the oxyferryl heme.

Ciba Found Symp, 1995, 191, 7 - 17; discussion 17-23
Transcriptional control by steroid hormones: the role of chromatin; Truss M et al.; The mouse mammary tumour virus (MMTV) promoter contains a complex hormone-responsive unit composed of four hormone-responsive elements, a nuclear factor I (NFI) binding site and two octamer motifs . All these sites are required for optimal hormonal induction . Although synergism has been found between hormone receptors and octamer transcription factor 1 (Oct-1/OTF-1), we were unable to detect a positive interaction between receptors and NFI in vitro . In chromatin, the MMTV hormone-responsive unit is contained in a phased nucleosome . The precise positioning of the DNA double helix on the surface of the histone octamer precludes binding of NFI and Oct-1/OTF-1 to their cognate sequences, while still allowing recognition of two hormone-responsive elements by the hormone receptors . Hormone treatment leads to a characteristic change in chromatin structure that makes the centre of the nucleosome more accessible to digestion by DNase I and facilitates binding of receptors, NFI and Oct-1/OTF-1 to the nucleosomally organized promoter . The MMTV promoter functions in yeast in a hormone receptor-dependent and NFI-dependent fashion . Depletion of nucleosomes activates hormone-independent transcription from the MMTV promoter . These results imply that nucleosome positioning not only represses hormone-independent transcription, but also enables binding of a full complement of transcription factors to the hormone-responsive unit after hormone induction.

Pharmacol Ther, 1995, 67(3), 323 - 50
Protein kinases and phosphatases that act on histidine, lysine, or arginine residues in eukaryotic proteins: a possible regulator of the mitogen-activated protein kinase cascade; Matthews HR; Phosphohistidine goes undetected in conventional studies of protein phosphorylation, although it may account for 6% of total protein phosphorylation in eukaryotes . Procedures for studying protein N- kinases are described . Genes whose products are putative protein histidine kinases occur in a yeast and a plant . In rat liver plasma membranes, activation of the small G-protein, Ras, causes protein histidine phosphorylation . Cellular phosphatases dephosphorylate phosphohistidine . One eukaryotic protein histidine kinase has been purified, and specific proteins phosphorylated on histidine have been observed . There is a protein arginine kinase in mouse and protein lysine kinases in rat . Protein phosphohistidine may regulate the mitogen-activated protein kinase cascade.

Microbios, 1995, 83(337), 261 - 70
Reduced uptake of nickel by a nickel resistant strain of Candida utilis; Ross IS; A nickel resistant strain of the yeast Candida utilis was obtained by selection of spontaneous mutants on solid medium containing 3 mM Ni2+ . In time-course experiments, non-growing suspensions of the mutant strain took up approximately 50% less nickel than the parent strain over 30 min incubation in MES buffer plus glucose at pH 5.5 . Efflux of nickel from either strain was negligible . Uptake was reduced by omission of glucose from the uptake buffer in both strains but this was much more marked in the parent strain . Uptake of nickel in the presence of equimolar concentration of Mg2+ was reduced by 80% in both parent and mutant strains . It was concluded that resistance to nickel was due to an alteration in a transport system primarily responsible for uptake of magnesium.

Microbios, 1995, 83(336), 167 - 74
The pattern of respiratory burst of leucocytes in patients with Echinococcus granulosus; al-Tuwaijri AS et al.; The function of polymorphonuclear leucocytes (PMN) of patients with hydatidosis was investigated . The patients were divided into three categories according to the characteristics of the cyst (calcified, alive and dead cyst) . Healthy blood donors were used as a control group . The oxidative activity of PMN was determined by chemiluminescence (CL) assay . Reduction of ferricytochrome C was used to measure the superoxide (O2-) production . Phagocytosis was monitored by opsonized yeast uptake . The results showed that the CL response, O2- production and phagocytic index of PMN, significantly increased in patients with dead cysts compared with healthy subjects while in patients with live cysts there was a marked reduction . No significant changes were noticed in patients with calcified cysts . These data indicate that the PMN of infected patients were in an activated state both functionally and metabolically.

Int Rev Cytol, 1995, 162B, 75 - 139
Nuclear matrix isolated from plant cells; Moreno Diaz de la Espina SM; Residual nuclear matrices can be successfully obtained from isolated nuclei of different monocot and dicot plant species using either high ionic or low ionic extraction protocols . The protein composition of isolated nuclear matrices depends on the details of isolation protocols . They are stable and present in all cases, a tripartite organization with a lamina, nucleolar matrix, and internal matrix network, and also maintain some of the basic architectural features of intact nuclei . In situ preparations demonstrate the continuity between the nuclear matrix and the plant cytoskeleton . Two-dimensional separation of isolated plant nuclear matrix proteins reveals a heterogeneous polypeptide composition corresponding rather to a complex multicomponent matrix than to a simple nucleoskeletal structure . Immunological identification of some plant nuclear matrix components such as A and B type lamins, topoisomerase II, and some components of the transcription and splicing machineries, internal intermediate filament proteins, and also specific nucleolar proteins like fibrillarin and nucleolin, which associate to specific matrix domains, establish a model of organization for the plant nuclear matrix similar to that of other eukaryotes . Components of the transcription, processing, and DNA-anchoring complexes are associated with a very stable nucleoskeleton . The plant matrix-attached regions share structural and functional characteristics with those of insects, vertebrates, and yeast, and some of them are active in animal cells . In conclusion, the available data support the view that the plant nuclear matrix is basically similar in animal and plant systems, and has been evolutionarily conserved in eukaryotes.

Int Rev Cytol, 1995, 162B, 257 - 302
Nuclear pore complex proteins; Bastos R et al.; The nuclear envelope forms the boundary between the nucleus and the cytoplasm and as such regulates the exchange of macromolecules between the two compartments . The channels through the nuclear envelop that actually mediate this macromolecular traffic are the nuclear pore complexes . These are extremely elaborate structures which in vertebrate cells exhibit a mass of approximately 120 MDa . They are thought to be composed of as many as 100 distinct polypeptide subunits . A major challenge in the field of nucleocytoplasmic transport is to identify these subunits and to determine their functions and interactions in the context of the three-dimensional structure of the nuclear pore complex . It is the aim of this review to summarize what is currently known of the 20 or so nuclear pore complex proteins that have been described in either vertebrate or yeast cells.

Artif Cells Blood Substit Immobil Biotechnol, 1995, 23(6), 681 - 92
Enzyme replacement therapy in ENU2 phenylketonuric mice using oral microencapsulated phenylalanine ammonia-lyase: a preliminary report; Safos S et al.; The presence of an extensive enterorecirculation of amino acids between the intestine and the body allows the removal of systemic phenylalanine in PKU rats by oral microencapsulated phenylalanine ammonia lyase . The work presented in this article has the main goal of assessing the feasibility of yeast phenylalanine ammonia-lyase (PAL) loaded collodion microcapsules in reducing elevated plasma phenylalanine concentrations to standard levels in genetically mutated PKU mice, within a 30 day time frame . The distinguishing aspect from previous studies lies in the available animal model . Rather than artificial induction of elevated phenylalanine plasma levels, the mice representing the human phenylketonuric condition, are mutated strains which are deficient in the enzyme phenylalanine hydroxylase . The first in vivo study established a method for orally feeding microcapsules, both control and enzyme loaded, over 30 consecutive days, by mixing with soft, unripened cheese . Under this unique regime a decrease of 51.3% +/- 9.02% in phenylalanine plasma levels was observed after 23 days . Reduction in the phenylalanine plasma levels to within the desired maintenance range of 250-1000 umol/L was observed in 2 out of 4 PAL treated mice, with only 50% of the PAL dose used in previous rat studies by Chang's group . The second animal study confirmed the finding in the first in vivo study that there is no significant decrease in the plasma phenylalanine levels within the first seven days . This may be due to the severely deteriorated physical condition of the ENU mice used, the PAL enzyme preparations available or the fact that normal mice contain 10 times the amount of phenylalanine hydroxylase as compared with humans, thus requiring larger doses of PAL in order to be effective in a shorter time span.

Vestn Khir Im I I Grek, 1995, 154(2), 57 - 60
{A first trial of the use of human recombinant interleukin (rIL-2) in patients with tumorous diseases}; Grinev MV et al.; Clinical approbation of human recombinant yeast human interleukin-2 (rIL-2) was carried out in 10 patients with III-IV stages of tumor that have undergone 65 intravenous drop by drop infusions of the drug as a course of 5-11 injections in the dosage of 1-8 mln/un . The drug toxicity was shown in 4 mln and especially, in 8 mln/un dose administration . That's why the dose of 1-2 mln/un is recommended . This dose was not followed by any serious complications, and the number of slightly complicated cases was significantly decreased as compared to similar rIL-2 drug made by the "Cetus Corporation" firm . Immunostimulating effect of yeast rIL-2 was found which appeared to be able to reach it's maximum by 3-4 administrations, with it's following disappearance or inversion, which may cause immunosuppression.

Rom J Intern Med, 1995 Jan-Jun, 33(1-2), 113 - 20
The influence of "selenium organicum" upon the hepatic function of carbon tetrachloride poisoned rats; Ianas O et al.; The hepatoprotective action of the Romanian preparation Orgasel containing selenium (Se) 5.01 mg/100 g autolysated yeast powder, was tested on adult Wistar rats poisoned with carbon tetrachloride (CCl4) . The hepatotoxic agent (a 20% CCl4 solution in oil) was administered i.p . in a single dose of 0.3 ml CCl4 solution/100 g body weight, and the preparation tested (autolysate of seleniated yeast) was administered by gavage in 4 doses (of 100 mg Se powder/100 g animal each) along 2 days . After 48 hrs the animals were sacrificed, then their blood and liver were collected . The treatment with Orgasel significantly reduced the organs, morphological changes (fat liver degeneration, splenomegaly, testicle degeneration) induced by CCl4 poisoning in the rat, an effect found also at the biological parameters levels studied in plasma and liver . In the plasma, the high lipid peroxide concentrations, the increased activity of alkaline phosphatases, and the reduced antioxidative activity generated by CCl4 have been statistically significant brought to the normal range after Orgasel administration . At the liver this treatment significantly decreased the lipid peroxides production, the total lipids and cholesterol concentrations, and statistically significant increased the enzymes activity (alkaline phosphatases, GPT) . The results obtained after Orgasel administration proved that this preparation has a global beneficial action upon the organism in the poisoned rat, as well as a strong antioxidative effect, confirming once again the essential role of Se in maintaining cells' integrity.

Mol Med, 1995 Jan, 1(2), 194 - 205
Genomic organization and sequence of the human NRAMP gene: identification and mapping of a promoter region polymorphism; Blackwell JM et al.; BACKGROUND: Murine Nramp is a candidate for the macrophage resistance gene Ity/Lsh/Bcg . Sequence analysis of human NRAMP was undertaken to determine its role in man . MATERIALS AND METHODS: A yeast artificial chromosome carrying NRAMP was subcloned and positive clones sequenced . The transcriptional start site was mapped using 5' RACE PCR . Polymorphic variants were amplified by PCR . Linkage analysis was used to map NRAMP . RESULTS: NRAMP spans 12kb and has 15 exons encoding a 550 amino acid protein showing 85% identity (92% similarity) with Nramp . Two conserved PKC sites occur in exon 2 encoding the Pro/Ser rich SH3 binding domain, and in exon 3 . Striking sequence similarities (57 and 53%) were observed with yeast mitochondrial proteins, SMF1 and SMF2, especially within putative functional domains: exon 6 encoding the second transmembrane spanning domain, site of the murine susceptibility mutation; and exon 11 encoding a conserved transport motif . No mutations comparable to the murine susceptibility mutation were found . The transcriptional initiation site mapped 148 bp 5' of the translational initiation codon . 440bp of 5' flanking sequence contained putative promoter region elements: 6 interferon-gamma response elements, 3 W-elements, 3 NF kappa B binding sites and 1 AP-1 site . Nine purine-rich GGAA core motifs for the myeloid-specific PU.1 transcription factor were identified, two combining with imperfect AP1-like sites to create PEA3 motifs . TATA, GC and CCAAT boxes were absent . A possible enhancer element containing the Z-DNA forming dinucleotide repeat t(gt),ac(gt),ac(gt),g was polymorphic (4 alleles; n = 4,9,10,11), and was used to map NRAMP to 2q35 . CONCLUSIONS: This analysis provides important resources to study the role of NRAMP in human disease.

Arch Tierernahr, 1995, 48(1-2), 83 - 8
{Effect of ethoxyquin on the utilization of selenium in growing pigs}; Ji C et al.; In a 2 x 3 factorial arrangement with 30 male castrates (German Landrace; 13.7 +/- 1.7 kg b.w.), after a 2 week feeding period with a maize/torula yeast basal diet poor in selenium and without vitamin E supplementation, the effect of two ethoxiquin concentrations (0 and 150 mg/kg) und 3 selenium concentrations (0.075, 0.10 and 0.125 mg/kg) on growth, the activity of the Se dependent GSH-Px in the erythrocytes and the Se concentrations in liver, kidney, heart and diaphragm was measured after 4 weeks on the experimental diets . Under the experimental conditions chosen ethoxiquin had no effect of any of the parameters studied . Selenium supplementation significantly increased the enzyme activity and the Se concentrations in the organs and tissues analyzed.

Arch Tierernahr, 1995, 48(1-2), 71 - 81
{Chromium supplements in the feed for growing pigs and meat quality}; Wenk C et al.; An experiment with 40 female growing pigs from 27.4 to 106.5 kg body weight (BW) in individual pens was conducted to evaluate the effect of different chromium supplements (Cr-chloride, Cr-yeast and Cr-picolinate) according to 0.5 ppm Cr in the diet compared with a control diet without any additional chromium . The influence on growth performance and carcass as well as meat composition was studied . In comparison with the control diet body weight gain and feed conversion ratio in the finishing period (60 to 106.5 kg BW) were significant increased and lowered respectively in the treatment with Cr-chloride and with the other Cr supplements tendentially . The results of the carcass composition as well as the fatty acid profile of neutral and complex lipids in the muscle (M . longissimus dorsi) at the 10th rib did not indicate a statistically significant effect of the Cr supplements . Furthermore energy utilization on the base of digestibility was not affected . Concerning the Longissimus muscle area and the intramuscular fat content there were positive tendencies of the investigated Cr supplements.

Plant Mol Biol, 1995 Jan, 27(2), 339 - 50
A homologue of the MAP/ERK family of protein kinase genes is expressed in vegetative and in female reproductive organs of Petunia hybrida; Decroocq-Ferrant V et al.; The mitogen activated protein (MAP) kinase pathway of eukaryotes is stimulated by many growth factors and is required for the integration of multiple cellular signals . In order to study the function of MAP kinases during plant ovule development we have synthesized a Petunia hybrida ovule-specific cDNA library and screened for MAP protein kinase-related sequences using a DNA probe obtained by PCR . A full-length cDNA clone was identified (PMEK for Petunia hybrida MAP/ERK-related protein kinase) and shown to encode a protein related to the family of MAP/ERK protein kinases . Southern blot analysis showed that PMEK is a member of a small multigene family in P . hybrida . The cDNA codes for a protein (PMEK1) of 44.4 kDa with an overall sequence identity of 44% to the products of the mammalian ERK/MAP kinase gene, and the budding yeast KSS1 and FUS3 genes . PMEK1 displays 96 and 80% identity respectively with the tobacco NTF3 and Arabidopsis ATMPK1 kinases, and only 50% to the more distantly related plant MAP kinase MsERK1 from alfalfa . The two phosphorylation sites found in the loop between subdomain VII and VIII in all the other MAP kinases are also present in PMEK1 . RNA gel blot and RT-PCR analyses demonstrated that PMEK1 is expressed in vegetative organs and preferentially accumulated in female reproductive organs of P . hybrida . In situ hybridization experiments showed that in the reproductive organs PMEK1 is expressed only in the ovary and not in the stamen.

Plant Mol Biol, 1995 Jan, 27(2), 263 - 75
Classification and expression of a family of cyclin gene homologues in Brassica napus; Szarka S et al.; In order to investigate the role of cell division in plant development, we isolated several plant genes which encode homologues of animal and yeast cell cycle regulators known as cyclins . Through the use of degenerate primers and the polymerase chain reaction (PCR) we isolated a Brassica sequence which showed homology to the 'cyclin box' functional domain found within cyclin proteins . Southern blot analysis indicated that Brassica napus has a large number of genes containing cyclin box-related sequences . This was further supported by the isolation of cyclin box sequences from six different genomic clones . In addition, we have isolated two different cyclin cDNA clones, BnCYC1 and BnCYC2, from a Brassica napus shoot apical cDNA library . Both of the cDNA clones contain a 'destruction box' regulatory domain similar to animal mitotic cyclins . Northern blot analysis using BnCYC2 shows mRNA levels which correlate well with the level of cell division in various tissues . Messenger RNA abundance was highest in 1-3 mm leaves, root tips and shoot apices . The mRNA detected using BnCYC1 was restricted to young leaves and the shoot apex, suggesting divergent, organ-specific roles for cyclin family members . The results demonstrate that the plant cyclin gene family is more extensive than previously demonstrated and consists of genes expressed in all dividing tissues as well as a subset of developmentally specific members.

Heredity, 1995 Jan, 74 ( Pt 1), 80 - 90
Evolution in heterogeneous environments: genetic variability within and across different grains in Tribolium castaneum; Via S et al.; The course of adaptation to heterogeneous environments is influenced by the magnitude of genetic variation for ecologically important characters within each environment and the extent of genotype x environment interaction . Using the genetic correlation between the expression of characters in different environments as a measure of genotype x environment interaction is particularly useful for evolutionary interpretation . In this study, we estimated patterns of genetic variability and cross-environment genetic correlations for pupal weight and development time in two strains of the flour beetle Tribolium castaneum in five flours (wheat with brewer's yeast, wheat, rice, corn and oat) . Wheat plus yeast is the standard medium in which the strains have been reared for hundreds of generations; other flours are novel environments . The results indicated moderate levels of genetic variation within the various flours for pupal weight but not for development time . Performance varied considerably across flours, with the highest performance for both strains found in the standard medium and the poorest in oat flour . The genetic correlations of pupal weight across flours in both strains were generally not significantly different from + 1 . This suggests that evolution of body size in different flours cannot proceed independently, and that improved performance in the novel flours may produce declines in fitness in the standard environment.

Cancer Genet Cytogenet, 1995 Jan, 79(1), 1 - 7
Identification of the chromosome 12 translocation breakpoint region of a pleomorphic salivary gland adenoma with t(1;12)(p22;q15) as the sole cytogenetic abnormality; Kools PF et al.; Cell line Ad-312/SV40, which was derived from a primary pleomorphic salivary gland adenoma with t(1;12)(p22;q15), was used in fluorescence in situ hybridization (FISH) analysis to characterize its translocation breakpoint region on chromosome 12 . Results of previous studies have indicated that the chromosome 12 breakpoint in Ad-312/SV40 is located proximally to locus D12S8 and distally to the CHOP gene . We here describe two partially overlapping yeast artificial chromosome (YAC) clones, Y4854 (500 kbp) and Y9091 (460 kbp), which we isolated in the context of a chromosome walking project with D12S8 and CHOP as starting points . We present a composite long-range restriction map encompassing the inserts of these two YAC clones and show by FISH analysis that both YACs span the chromosome 12 breakpoint as present in Ad-312/SV40 cells . Subsequently, we have isolated cosmid clones corresponding to various sequence-tagged sites (STSs) mapping within the inserts of these YAC clones . These included cRM51, cRM69, cRM85, cRM90, cRM91, cRM110, and cRM111 . In FISH studies, cosmid clones cRM85, cRM90, and cRM111 appeared to map distally to the chromosome 12 breakpoint, whereas cosmid clones cRM51, cRM69, cRM91, and cRM110 were found to map proximally to it . These results assign the chromosome 12 breakpoint in Ad-312/SV40 to a DNA region of less than 165 kbp . FISH evaluation of the chromosome 12 breakpoints in five other pleomorphic salivary gland adenoma cell lines indicated that these are located proximally to the one in Ad-312/SV40, at a distance of more than 0.9 Mbp from STS RM91 . These results, while pinpointing a potentially critical region on chromosome 12, also provide evidence for the possible involvement of 12q13-q15 sequences located elsewhere.

Hypertension, 1995 Jan, 25(1), 6 - 13
Evaluation of the SA locus in human hypertension; Nabika T et al.; The SA gene is expressed at 10-fold greater levels in the kidney of the spontaneously hypertensive rat compared with the normotensive Wistar-Kyoto rat . The gene is linked to blood pressure levels in a number of crosses involving the spontaneously hypertensive rat and other strains of genetically hypertensive rats . To assess its role in human hypertension, a human SA cDNA was cloned from a liver library . The cDNA was 1513 bp in length and exhibited a high identity with the published rat SA cDNA sequence in the coding region . A microsatellite marker was developed from a yeast artificial chromosome clone containing SA and mapped by linkage to human chromosome 16p13.11-12.3 . Polymerase chain reaction amplification of human genomic DNA revealed two introns located in the SA gene, one of which contains a frequent polymorphism due to a single nucleotide substitution (cytosine to thymidine at residue 79 of the intron) . Association and linkage studies in a large sample of hypertensive patients, normotensive control subjects, and multiplex sibships with these markers and other microsatellites in close proximity to SA revealed no evidence favoring involvement of the gene in the disease in humans . The methodology used in this study can be applied to the evaluation of other novel candidate genes obtained from investigations of experimental models of hereditary hypertension.

Am J Hum Genet, 1995 Jan, 56(1), 202 - 9
Refinement of the spinal muscular atrophy locus by genetic and physical mapping; Wang CH et al.; We report the mapping and characterization of 12 microsatellite markers including 11 novel markers . All markers were generated from overlapping YAC clones that span the spinal muscular atrophy (SMA) locus . PCR amplification of 32 overlapping YAC clones shows that 9 of the new markers (those set in italics) map to the interval between the two previous closest flanking markers (D5S629 and D5S557): cen-D5S6-D5S125-D5S435-D5S1407- D5S629-D5S1410-D5S1411/D5S1412-D5S1413- D5S1414-D5Z8-D5Z9-CATT1-D5Z10/D5Z6- D5S557-D5S1408-D5S1409-D5S637-D5S351-MA P1B-tel . Four of these new markers detect multiple loci in and out of the SMA gene region . Genetic analysis of recombinant SMA families indicates that D5S1413 is a new proximal flanking locus for the SMA gene . Interestingly, among the 40 physically mapped loci, the 14 multilocus markers map contiguously to a genomic region that overlaps, and perhaps helps define, the minimum genetic region encompassing the SMA gene(s).

Hum Genet, 1995 Jan, 95(1), 82 - 8
Isolation and characterization of a cosmid contig for the GCPS gene region; Vortkamp A et al.; The zinc finger gene GLI3 has been shown to be involved in the embryonal development of the limbs and skull . Mutations in GLI3 lead to the development of the human Greig cephalopolysyndactyly syndrome (GCPS) and the mouse mutations extra toes (Xt) and anterior digit deformity (add) . The GCPS locus on human chromosome 7p13 has recently been isolated in a yeast artificial chromosome (YAC) contig . Here, we describe the establishment of a cosmid contig that was derived from two of the YAC clones, that spans 550 kb of human DNA, and that includes the GLI3 gene . In this contig, three GCPS translocation breakpoints have been mapped to distinct EcoRI fragments in the 3' half of the gene . In addition, exon-carrying fragments have been identified and the size of the GLI3 gene could be determined as at least 280 kb . The gene is flanked by a CpG island that lies on the 5' side and that is in close proximity to the first exon detected by the cloned GLI3 cDNA . Further upstream, five segments were found that have been conserved between man and mouse . In the mouse, this region has been characterized as the transgene integration site resulting in the add phenotype . Both the CpG island and the conserved regions are probable candidates for a search for GLI3 promoter and control elements.

Hum Genet, 1995 Jan, 95(1), 18 - 21
Positional cloning of cDNAs from the human chromosome 3p21-22 region identifies a clustered organization of zinc-finger genes; Calabro V et al.; The human 3p21-22 region is frequently involved in karyotype rearrangements associated with malignancies . The high frequency of allelic loss in this region has been associated with virtually all small cell lung carcinomas and many renal carcinomas . These findings suggest that at least one tumor-suppressor gene might be located in 3p21-22 . We have recently reported the isolation of a 750-kb yeast artificial chromosome (YAC) contig from 3p21-22 . Here, we describe three new genes isolated from the 3p YAC contig by using a cDNA hybridization selection . Remarkably, the three new genes encode zinc-finger proteins, indicating the presence of a cluster of zinc-finger genes in human chromosome 3p21.

Hum Genet, 1995 Jan, 95(1), 119 - 22
Five new microsatellite polymorphisms at the q21 region of human chromosome 21; Bosch A et al.; Five clones, containing polymorphic CA-repeat sequences, have been isolated from a specific human chromosome 21 phage library and have been localised to band q21 of chromosome 21 using a somatic cell hybrid panel . These highly repetitive sequences (D21S1263, D21S1264, D21S1415, D21S1417 and D21S1420) have been characterised in the CEPH reference parents and have heterozygosities ranging from 0.30 to 0.81 and an average polymorphism information content (PIC) of 0.62 . The relative order of these markers, based on the somatic cell hybrid panel, is cen-D21S1417, D21S1420-D21S1263, D21S1415-D21S1264-tel . The most polymorphic marker (D21S1264) has been included in the chromosome 21 genetic map . They have also been localised in the CEPH/Genethon YAC panel, providing a refined localisation of these polymorphic sequences . These five CA-repeat markers should provide a better characterisation of the q21 region of chromosome 21.

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