Yeasts

Immunobiology, 1996 Jul, 195(2), 220 - 30
Anti-candidial activity of natural killer (NK) and lymphokine activated killer (LAK) lymphocytes in vitro; Gulay Z et al.; The natural cytotoxic effects of peripheral blood lymphocytes (PBL) on Candida stellatoidea and several other Candida species were examined by a colony forming inhibition (CFI) assay . Peripheral blood mononuclear cells (PBMC), were incubated with C . stellatoidea yeast cells . After the incubation period the colony-forming ability of the yeast was significantly reduced . In similar experiments, six different Candida species (C . albicans, C . krusei, C . stellatoidea, C . tropicalis, C . pseudotropicalis, C . guillermondii) were used as target cells . There was no statistically significant difference in the anticandidial activities of PBL against the Candida species used . It was demonstrated that a fraction of lymphocytes, natural killer cells (NK), had the major natural anti-candidial activity by using anti-Leu M1 (CD 15) and anti-Leu 11b (CD 16) monoclonal antibodies (mAbs) plus complement (C') . It was observed that inhibition of colony-forming ability of C . stellatoidea was significantly (78-96%) reduced when anti-Leu 11b plus C' were used . In addition, the colony formation inhibition capacity of NK cells was increased by recombinant human interleukin-2 (rhIL-2) while anti-interferon-gamma (IFN-gamma) had no effect . Besides the fact that NK cells are among those responsible for natural immunity against Candida species, this colony-forming inhibition assay performed with C . stellatoidea yeast cells as target and monocyte-depleted PBMC as effector cells, is a simple method to assess NK cell activity.

J Med Vet Mycol, 1996 Jul-Aug, 34(4), 293 - 7
Random amplified polymorphic DNA analysis of culture collection strains of Candida species; Zeng S et al.; Random amplified polymorphic DNA (RAPD) profiles, obtained when screening culture collection strains of Candida spp., showed several species to be highly heterogeneous . The RAPD-defined groups were as follows: C . catenulata, two groups among five strains; Debaryomyces hansenii (anamorph C . famata), three groups among five strains; C . sake, three groups among three strains; C . intermedia, two groups among two strains; C . rugosa, two groups among three strains and C . parapsilosis, three groups among four strains . The five strains of Issatchenkia orientalis (anamorph C . krusei) belonged to a single RAPD-defined group . The two strains of the teleomorphic yeast Arxiozyma telluris had distinct RAPD profiles but neither resembled profiles of its anamorph, C . pintolopesii . The two varieties of C . pintolopesii had different RAPD profiles . Researchers are cautioned that organisms with the same name may not be closely related.

Bull Cancer, 1996 Jul, 83(7), 527 - 34
{Apropos of the studies of Lewis, Nusslein-Volhard and Wieschaus, 1995 Nobel prize winners, on the genetic mechanisms of embryonic development of drosophila . A model for human cancer progression}; Cillo C; EB Lewis, C Nusslein-Volhard and E Wieschaus were the winners of the Nobel prize in 1995 for the discovery of genes controling the embryonic development in drosophila . Drosophila development is dependent on sequential activities of three types of genes: the maternal genes, the segmentation genes, and the homeotic genes which are responsible for the segment identity and finally for the building of the body . Mutations of these genes are spectacular because they affect the body structure formed from individual segments . Therefore, the molecular processes regulating the development of inferior organisms such as yeast or more complex as the vertebrates were elucidated by these three researchers . These early biological mechanisms regulate the cell life through interactions with neighbouring cells . We speculate that any alteration of these processes might be implicated in cancer . Understanding of these molecular mechanisms which control cell interactions in cancer constitutes a basis for definition of new prognostic markers and putatively novel therapeutic approaches.

Mol Biochem Parasitol, 1996 Jul, 79(1), 1 - 12
Current status of the Plasmodium falciparum genome project; Dame JB et al.; The Plasmodium falciparum Genome Project is a collaborative effort by many laboratories that will provide detailed molecular information about the parasite, which may be used for developing practical control measures . Initial goals are to prepare an electronically indexed clone bank containing partially sequenced clones representing up to 80% of the parasite's genes and to prepare an ordered set of overlapping clones spanning each of the parasite's 14 chromosomes . Currently, clones of genomic DNA, prepared as yeast artificial chromosomes, are arranged into contigs covering approximately 70% of the genome of parasite clone 3D7, gene sequence tags are available from more than contigs covering approximately 70% of the genome of parasite clone 3D7, gene sequence tags are available from more than 20% of the parasite's genes, and approximately 5% of the parasite's genes are tentatively identified from similarity searches of entries in the international sequence databases . A total of > 0.5 Mb of P . falciparum sequence tag data is available . The gene sequence tags are presently being used to complete YAC contig assembly and localize the cloned genes to positions on the physical map in preparation for sequencing the genome . Routes of access to project information and services are described.

Int J Pept Protein Res, 1996 Jul, 48(1), 31 - 47
Chemical synthesis and purification of proteins: a methodology; Ball HL et al.; Classical stepwise solid-phase peptide synthesis (SPPS) has been used successfully for the synthesis of proteins up to 150 residues in length, although usually with poor yields and homogeneity . The major limitation has been the inability to separate chromatographically similar deletion and truncated impurities from the target sequence . We have developed a highly effective protocol for stepwise SPPS and 'one-step' purification of small proteins . to demonstrate the effectiveness of the methodology we synthesised the 101 residue chaperonin 10 protein from Rattus norvegicus (Rat Cpn 10) using three different chemical protocols . Highly homogeneous Rat Cpn10 was obtained using an optimised synthetic strategy and one-step purification procedure (method C), involving (i) HBTU/HOBt activation, (ii) N-(2-chlorobenzyloxycarbonyloxy)succinimide as capping agent and (iii) the incorporation of a reversible Fmoc-based chromatographic probe, derivatised with a lipophilic group for fast one-step RP purification, to give an overall yield of 9.6% . Analysis by ESI-MS indicated that the product was virtually free of deletion impurities, while RP-HPLC under four different conditions and CZE indicated that the protein was 100 and 84% pure, respectively . The spontaneous folding of Rat Cpn10 into its biologically active form was found to correlate well with the degree of purity as assessed by chromatography, ESI-MS and sequencing, since 29 (A), 55 (B) and 81% (C) of correctly folded heptameric structure was obtained . The degree of homogeneity was also reflected in the ability of purified Rat Cpn10 to facilitate the refolding of yeast enolase.

J Rheumatol, 1996 Jul, 23(7), 1233 - 6
Generation of inorganic pyrophosphate from extracellular adenosine triphosphate by human serum and plasma; Park W et al.; OBJECTIVE: To quantify inorganic pyrophosphate (PPi) production from extracellular adenosine triphosphate (ATP) by human serum or plasma . METHODS: Serial measurements of ATP hydrolysis (t1/2) were performed by the luciferase method from a starting concentration of 1 microM in serum or platelet-poor plasma incubated under physiologic conditions . ATP was then pumped into another sample of each specimen using the rate constant derived from the ATP t1/2 of that specimen . Trace (32P) gamma ATP was added at the start of the infusion; conversion to (32P) inorganic orthophosphate (Pi) and to (32P) PPi was determined by precipitation of Pi as reduced phosphomolybdate before and after treatment with yeast pyrophosphatase . RESULTS: ATP was hydrolyzed by all serum and plasma specimens; the rate of hydrolysis in serum and plasma from the same blood sample was nearly identical . PPi was the major product, averaging 71% . CONCLUSION: PPi is the major product of ATP catabolism in serum and platelet-poor plasma.

Fam Med, 1996 Jul-Aug, 28(7), 493 - 5
The use of over-the-counter antifungal vaginitis preparations by college students; Lipsky MS et al.; BACKGROUND AND OBJECTIVES: This study examined college students' perceptions of the over-the-counter (OTC) availability of vaginal antifungal preparations . METHODS: A cross-sectional survey of 258 college students participated in the study over a 3-month period . Students were surveyed about their use of OTC antifungal preparations and their experiences with product effectiveness, benefits, and risks . RESULTS: Among 157 women who experienced a yeast infection, 110 (71%) had used OTC antifungal agents . Approximately 92% reported that the products cured their infection . Most students believed the OTC agents were a good idea because of the convenience and cost savings . CONCLUSIONS: The consumer group, which consisted of young female college students, felt they had assimilated the introduction of OTC antifungal preparations into their personal health care safely and effectively.

Br J Pharmacol, 1996 Jul, 118(5), 1183 - 91
The inhibition of antigen-induced eosinophilia and bronchoconstriction by CDP840, a novel stereo-selective inhibitor of phosphodiesterase type 4; Hughes B et al.; 1 . The novel tri-aryl ethane CDP840, is a potent and selective inhibitor of cyclic AMP phosphodiesterase type 4 (PDE 4) extracted from tissues or recombinant PDE 4 isoforms expressed in yeast (IC50S: 4-45 nM) . CDP840 is stereo-selective since its S enantiomer (CT 1731) is 10-50 times less active against all forms of PDE 4 tested while both enantiomers are inactive (IC50S: > 100 microM) against PDE types 1, 2, 3 and 5 . 2 . Oral administration of CDP840 caused a dose-dependent reduction of interleukin-5 (IL-5)-induced pleural eosinophilia in rats (ED50 = 0.03 mg kg-1) . The eosinophils in pleural exudates from CDP840-treated animals contained higher levels of eosinophil peroxidase (EPO) than cells from control animals, suggesting a stabilizing effect on eosinophil degranulation . CDP840 was approximately equi-active with the steroid dexamethasone in this model and was 10-100 times more potent than the known PDE 4-selective inhibitors rolipram and RP73401 . The activity of CDP840 was not influenced by adrenalectomy, beta-sympathomimetics or beta-sympatholytics . 3 . Antigen-induced pulmonary eosinophilia in sensitized guinea-pigs was reduced dose-dependently by CDP840 (0.01-1 mg kg-1, i.p.) and intracellular EPO levels were significantly higher . CDP840 was more potent in these activities than CT1731 or rolipram and comparable in potency to RP73401 . 4 . Rolipram or CDP840 were less active than dexamethasone in preventing neutrophil accumulation, or exudate formation in carrageenan-induced pleurisy in rats and thus do not exhibit general anti-inflammatory activity . 5 . In sensitized guinea-pigs, aerosols of the antigen ovalbumin caused a dose-dependent bronchoconstriction demonstrated by an increase in pulmonary inflation pressure . Administration of CDP840 (0.001-1.0 mg kg-1, i.p.), 1 h before antigen challenge, resulted in dose-dependent reduction in response to antigen . This activity was not due to bronchodilatation since higher doses of CDP840 (3 mg kg-1) did not significantly change the bronchoconstrictor response to histamine . Rolipram was approximately 10 times less active than CDP840 in preventing antigen-induced bronchoconstriction . 6 . These results confirm the observations that selective PDE 4 inhibitors reduce antigen-induced bronchoconstriction and pulmonary eosinophilic inflammation . CDP840 is more potent than rolipram in inhibiting native or recombinant PDE 4 . Unlike the recently described potent PDE 4 inhibitor RP73401, CDP840 is more active than rolipram in the rat IL-5 model following oral administration . The novel series of tri-aryl ethanes, of which CDP840 is the lead compound, could be the basis of an orally active prophylactic treatment for human asthma.

J Neurobiol, 1996 Jul, 30(3), 349 - 58
Expression of a developmentally regulated gene, Mng10, in identified neurosecretory cells in the CNS of Manduca sexta; Meszaros M et al.; We are interested in the molecular events underlying the development of the nervous system of Manduca sexta during the final 24 h of the pupal molt . In this article we describe a gene, Mng10, that is expressed in the abdominal nervous system of M . sexta and is developmentally regulated over this 24-h period . In situ hybridization analysis shows that the transcript is localized predominantly to a single pair of uniquely identifiable neurosecretory neurons, the NS-L1 cells in the abdominal ganglia . Mng10 is a single copy gene encoding a 229 amino acid protein with a predicted molecular mass of 26 kDa . At the amino acid level the protein shows 34% identity to the yeast transcription unit, Yer082 . Northern blot analysis shows that the transcript of Mng10 is very rare, comprising about 0.001% of the poly (A)+ RNA from the CNS and is detectable at 4 h but not 24 h prior to pupal ecdysis . One of the physiological events that develops over the final 24 h of the pupal molt is the ability of the nervous system to respond to the neuropeptide eclosion hormone . In this context, it is interesting to note that the NS-L1 cells are members of the group of 50 neurons that show increased cGMP immunoreactivity when the nervous system is exposed to the neuropeptide eclosion hormone.

Curr Biol, 1996 Jul 1, 6(7), 848 - 54
HIV Rev uses a conserved cellular protein export pathway for the nucleocytoplasmic transport of viral RNAs; Fritz CC et al.; BACKGROUND: The structural proteins of human immunodeficiency virus type 1 (HIV-1) are encoded by intron-containing mRNAs that normally are retained in the nucleus . A viral regulatory protein, Rev, specifically induces the accumulation of these transcripts in the cytoplasm . Rev is an RNA-binding protein that also contains an 'effector' domain . The Rev effector domain has recently been shown to function as an autonomous nuclear export signal (NES) that, when fused to a foreign protein, will cause its rapid nuclear export . We and others have recently reported the cloning of a human protein (hRIP/Rab), that specifically interacts with the effector domain of Rev . RESULTS: Here we show that the NESs contained within two cellular proteins, PKI and I kappa B, which are not involved in RNA metabolism, also interact with hRIP . Fusion of these cellular sequences to the Rev RNA-binding domain reconstitutes a functional Rev protein . In addition to hRIP, these NESs also bind to several nuclear pore complex (NPC) . We show that this protein export pathway is highly conserved by demonstrating that mammalian NESs also function in yeast . CONCLUSIONS: Our results indicate that the HIV-1 Rev protein evolved to take advantage of a cellular protein export pathway in order to allow the nucleocytoplasmic transport of unspliced viral RNA . Our data suggest a model in which the export substrate is translocated through the NPC by sequential interactions with different nucleoporins . Finally, our experiment suggests a mechanism by which I kappa B can downregulate nuclear NF kappa B activity by causing its rapid export from the nucleus.

Biosci Biotechnol Biochem, 1996 Jul, 60(7), 1193 - 4
Conversion of glutathione into cadystins and their analogs catalyzed by carboxypeptidase Y; Imai K et al.; Cadystins induced in a fission yeast treated with Cd2+ are the higher homologs of glutathione . In the present work, glutathione was incubated with Carboxypeptidase Y at a high substrate concentration . The reaction afforded not only the degraded product, but also cadystins and their analogs . A possible transformation pathway for glutathione by this enzyme is proposed.

J Struct Biol, 1996 Jul-Aug, 117(1), 1 - 15
Intermediate filament protein polymerization: molecular analysis of Drosophila nuclear lamin head-to-tail binding; Stuurman N et al.; Polymerization of intermediate filament proteins results from interactions among several distinct binding sites on the constituent proteins . Nuclear lamin head-to-tail polymers arise from one such interaction . We studied this binding using Drosophila lamin Dm0-derived fragments containing either the NH2-terminal or COOH-terminal binding site with a combination of co-immunoprecipitation, yeast two-hybrid, analytical ultracentrifugation, and electron microscopic assays . Fragment binding and full-length lamin head-to-tail polymerization were similar to each other in morphology, buffer requirements, and inhibition after phosphorylation with cdc2 kinase . Deletion analysis localized the binding sites to the ends of the rod domain that are highly conserved among all intermediate filament proteins . Point mutants, defective in binding, were isolated . Two were identical to point mutations in specific human keratin genes known to affect keratin assembly and to cause genetic skin diseases . Results further indicate that the binding sites only function in specific sequence contexts and that binding can be modulated by elements outside the binding sites (like the cdc2 kinase phosphorylation site) . Our data indicate that one type of interaction in intermediate filament protein polymerization is the longitudinal binding of dimers via the conserved end segments of the coiled-coil rod domain.

FEBS Lett, 1996 Jul 1, 389(2), 153 - 6
Kinetic and spectral properties of pea cytosolic ascorbate peroxidase; Marquez LA et al.; Sufficient highly purified native pea cytosolic ascorbate peroxidase was obtained to characterize some of its kinetic and spectral properties . Its rate constant for compound I formation from reaction with H2O2 is 4.O x 10(7) M-1 s-1, somewhat faster than is typical for peroxidases . Compound I has the typical optical spectrum of an iron(IV)-porphyrin-pi-cation radical, despite considerable homology with yeast cytochrome c peroxidase . The rate constant for compound I reduction by ascorbate is extremely fast (8.0 x 10(7) M-1 S-1 at pH 7.8), again in marked contrast to the behavior of the yeast enzyme . The pH-rate profile for compound I formation indicates a pKa value of 5.0 for a group affecting the active site reaction.

Eur J Immunol, 1996 Jul, 26(7), 1613 - 20
The mouse immunoglobulin kappa locus contains about 140 variable gene segments; Kirschbaum T et al.; In continuation of our efforts to elucidate the immunoglobulin kappa locus of the mouse we analyzed 46 yeast artificial chromosomes (YACs) containing V kappa, J kappa and C kappa genes . The YACs, which were derived from DNA of C57BL/6 and C3H mice, ranged from 0.3-1.9 Mb in size . On the basis of hybridization with probes specific for the V kappa gene families a group of 13 YACs was selected for detailed analysis . The V kappa genes of the YACs were then characterized by hybridization to the family-specific probes and by the sizes of the EcoRI fragments on which they were found . This way evidence was obtained for 140 different V kappa gene signals on the YACs . Of these 63 had been characterized before on clones from a cosmid library of total mouse DNA (I . Zocher et al., Eur . J . Immunol . 1995 . 25: 3326-3331) and 22 others were found now on cosmid clones derived from the YACs . Six V kappa genes of the previous study which were not found on the YACs are probably located outside of the kappa locus . The YACs were arrayed in a unique order establishing a YACs panel which most likely contains the whole kappa locus . The cosmid contigs and solitary cosmid clones which contain the 63 plus 22 V kappa gene signals mentioned above comprise about 2.0 Mb . Assuming that the remaining 55 V kappa genes are spaced at the same average distance of 24 kb, one may extrapolate to a locus size of 3.3 Mb.

Physiol Rev, 1996 Jul, 76(3), 651 - 85
Regulation of protein transport to the nucleus: central role of phosphorylation; Jans DA et al.; Nuclear protein transport is integral to eukaryotic cell processes such as differentiation, transformation, and the control of gene expression . Although the targeting role of nuclear localization signals (NLSs) has been known for some time, more recent results indicate that NLS-dependent nuclear protein import is precisely regulated . Phosphorylation appears to be the main mechanism controlling the nuclear transport of a number of proteins, including transcription factors such as NFkappaB, c-rel, dorsal, and SWI5 from yeast . Cytoplasmic retention factors, intra- and intermolecular NLS masking, and NLS masking by phosphorylation are some of the mechanisms by which phosphorylation specifically regulates nuclear transport . Even nuclear localization of the archetypal NLS-containing simian virus 40 large tumor antigen (T-ag) is regulated, namely by the "CcN motif," which comprises the T-ag NLS ("N") determining ultimate subcellular destination, a casein kinase II site ("C") 13 amino acids NH2-terminal to the NLS modulating the rate of nuclear import, and a cyclin-dependent kinase site ("c") adjacent to the NLS regulating the maximal level of nuclear accumulation . The CcN motif appears to be a special form of phosphorylation-regulated NLS (prNLS), where phosphorylation at site(s) close to the NLS specifically regulates NLS function . The regulation of nuclear transport through phosphorylation and prNLSs appears to be common in eukaryotic cells from yeast and plants to higher mammals.

Microbiology, 1996 Jul, 142 ( Pt 7), 1597 - 604
Cell wall protein and glycoprotein constituents of Aspergillus fumigatus that bind to polystyrene may be responsible for the cell surface hydrophobicity of the mycelium; Penalver MC et al.; Cell surface hydrophobicity (CSH) of Aspergillus fumigatus grown both in complex medium (yeast extract/peptone/dextrose; YPD) and minimal (Vogel's N) medium was monitored by assessing attachment of polystyrene microspheres to the cell surface . It was found that mature mycelium was hydrophobic . Treatment of intact mycelium with beta-mercaptoethanol (beta ME) abolished binding of the microspheres to hyphal elements, and coating of the microspheres with beta ME extracts from mycelium inhibited their attachment to intact mycelial cells . A . fumigatus mycelium was tagged in vivo with biotin and treated with beta ME . The beta ME extracts were analysed by SDS-PAGE and Western blotting with both peroxidase-conjugated-ExtrAvidin and concanavalin A (ConA) . This procedure allowed identification of cell wall surface proteins and glycoproteins . Rabbit polyclonal antisera were raised against beta ME extracts obtained from cells grown in YPD and Vogel's N media . These antisera defined some major cell-wall-bound antigens . SDS-PAGE and Western blotting analysis of the cell wall material released by beta ME and adsorbed on polystyrene microspheres revealed about 19 protein species with apparent molecular masses ranging from 20 to 70 kDa, and two high-molecular-mass glycoproteins of 115 and 210 kDa . Treatment of cells grown in YPD, but not those grown in Vogel's N medium, with beta ME released a 55 kDa polypeptide able to adsorb to polystyrene microspheres that was detectable with the antisera . The ability to bind to polystyrene particles exhibited by several protein and glycoprotein species released by beta ME treatment suggested that these cell wall moieties possess exposed hydrophobic domains that could be responsible for the CSH of mycelium.

Br J Haematol, 1996 Jul, 94(1), 105 - 11
The der(21)t(12;21) chromosome is always formed in a 12;21 translocation associated with childhood acute lymphoblastic leukaemia; Kobayashi H et al.; We studied 116 patients (93 children and 23 adults) with acute lymphoblastic leukaemia (ALL) using fluorescence in situ hybridization (FISH) with the yeast artificial chromosome (YAC) clone, 964c10, which includes the recently described ETS-like gene, TEL, on 12p13 . FISH revealed that nine of the patients had a t(12;21), which had not been previously detected . The nine patients were all children, seven boys and two girls, aged 1-10 years (median 3 years), had an early B immunophenotype, and achieved complete remission, although two of them experienced haematological relapse . In addition to the t(12;21), FISH also revealed that three of the nine had a del(12p) in the other homolog of chromosome 12 or in the der(12) chromosome itself, and that two others had 12p translocations in the other chromosome 12 homolog . Although chromosomal rearrangements associated with the t(12;21) were heterogenous and complex, fusion of the sequences from chromosomes 12 and 21 on the der(21)t(12;21) chromosomes was consistent, suggesting that the TEL-AML1 gene fusion on the der(21) chromosome may be critical in leukaemogenesis and that FISH or reverse transcriptase-polymerase chain reaction (RT-PCR) targeted to the chimaeric sequences on the der(21) will be most useful in detecting the t(12;21) or following a patient with the t(12;21), which is one of the most frequent chromosomal rearrangements in both Caucasian and Asian childhood ALL.

Appl Biochem Biotechnol, 1996 Jul, 60(1), 41 - 8
Biotransformation of uridine monophosphate (UMP) and glucose to uridine diphosphate-glucose (UDPG) by Candida saitoana KCTC7249 cells; Ko JH et al.; The present study investigates the biotransformation of glucose with uridine monophosphate (UMP) to obtain sugar nucleotide, UDP-glucose (UDPG), by the dried cells of Candida saitoana KCTC7249 . The biotransformation was optimized by varying the concentrations of substrates and phosphate ion . UDPG (24 mM) was biotransformed from 200 mM glucose and 37.5 mM UMP by dried cells of C . saitoana . The glucose yields about 64% UDP-glucose, based on UMP concentration . The addition of glucose-1-phosphate to the reaction mixture accelerated the formation of UDPG from a concentration of UMP . The structure of UDP-glucose obtained was determined with 13C NMR and FAB mass spectra . These results indicate that the yeast-dried cells could be used for the production of nucleotide sugars for donor molecules of complex carbohydrate synthesis.

Infect Immun, 1996 Jul, 64(7), 2716 - 23
Natural immune response to the C-terminal 19-kilodalton domain of Plasmodium falciparum merozoite surface protein 1; Shi YP et al.; We have characterized the natural immune responses to the 19-kDa domain of merozoite surface protein 1 in individuals from an area of western Kenya in which malaria is holoendemic . We used the three known natural variant forms of the yeast-expressed recombinant 19-kDa fragment that are referred to as the E-KNG, Q-KNG, and E-TSR antigens . T-cell proliferative responses in individuals older than 15 years and the profile of immunoglobulin G (IgG) antibody isotypes in individuals from 2 to 74 years old were determined . Positive proliferative responses to the Q-KNG antigen were observed for 54% of the individuals, and 37 and 35% of the individuals responded to the E-KNG and E-TSR constructs, respectively . Considerable heterogeneity in the T-cell proliferative responses to these three variant antigens was observed in different individuals, suggesting that the 19-kDa antigen may contain variant-specific T epitopes . Among responses of the different isotypes of the IgG antibody, IgG1 and IgG3 isotype responses were predominant, and the prevalence and levels of the responses increased with age . We also found that a higher level of IgG1 antibody response correlated with lower parasite density among young age groups, suggesting that IgG1 antibody response may play a role in protection against malaria . However, there was no correlation between the IgG3 antibody level and protection . Furthermore, we observed that although the natural antibodies cross-reacted with all three variant 19-kDa antigens, IgG3 antibodies in 12 plasma samples recognized only the E-KNG and Q-KNG constructs and not the E-TSR antigen . This result suggests that the fine specificity of IgG3 antibodies differentiates among variant-specific natural B-cell determinants in the second epidermal growth factor domain (KNG and TSR) of the antigen.

Nucleic Acids Res, 1996 Jul 1, 24(13), 2551 - 9
XPC and human homologs of RAD23: intracellular localization and relationship to other nucleotide excision repair complexes; van der Spek PJ et al.; The xeroderma pigmentosum syndrome complementation group C (XP-C) is due to a defect in the global genome repair subpathway of nucleotide excision repair (NER) . The XPC protein is complexed with HHR23B, one of the two human homologs of the yeast NER protein, RAD23 (Masutani at al . (1994) EMBO J . 8, 1831-1843) . Using heparin chromatography, gel filtration and native gel electrophoresis we demonstrate that the majority of HHR23B is in a free, non-complexed form, and that a minor fraction is tightly associated with XPC . In contrast, we cannot detect any bound HHR23A . Thus the HHR23 proteins may have an additional function independent of XPC . The fractionation behaviour suggests that the non-bound forms of the HHR23 proteins are not necessary for the core of the NER reaction . Although both HHR23 proteins share a high level of overall homology, they migrate very differently on native gels, pointing to a difference in conformation . Gel filtration suggests the XPC-HHR23B heterodimer resides in a high MW complex . However, immunodepletion studies starting from repair-competent Manley extracts fall to reveal a stable association of a significant fraction of the HHR23 proteins or the XPC-HHR23B complex with the basal transcription/repair factor TFIIH, or with the ERCC1 repair complex . Consistent with a function in repair or DNA/chromatin metabolism, immunofluorescence studies show all XPC, HHR23B and (the free) HHR23A to reside in the nucleus.

Arch Environ Contam Toxicol, 1996 Jul, 31(1), 120 - 7
Toxicity and oxidative stress of different forms of organic selenium and dietary protein in mallard ducklings; Hoffman DJ et al.; Concentrations of over 100 ppm (mg/kg) selenium (Se) have been found in aquatic plants and insects associated with irrigation drainwater and toxicity to fish and wildlife . Composition of diet for wild ducklings can vary in selenium-contaminated environments . Earlier studies have compared toxicities and oxidative stress of Se as selenite to those of seleno-DL-methionine (DL) in mallards (Anas platyrhynchos) . This study compares DL, seleno-L-methionine (L), selenized yeast (Y) and selenized wheat (W) . Day-old mallard ducklings received an untreated diet (controls) containing 75% wheat (22% protein) or the same diet containing 15 or 30 ppm Se in the above forms except for 30 ppm Se as W . After 2 weeks, blood and liver samples were collected for biochemical assays and Se analysis . All forms of selenium caused significant increases in plasma and hepatic glutathione peroxidase activities . Se as L at 30 ppm in the diet was the most toxic form, resulting in high mortality (64%) and impaired growth (>50%) in survivors and the greatest increase in ratio of oxidized to reduced hepatic glutathione (GSH) . Se as both L and DL decreased the concentrations of hepatic GSH and total thiols . Se as Y accumulated the least in liver (approximately 50% of other forms) and had less effect on GSH and total thiols . In a second experiment, in which the basal diet was a commercial duck feed (22% protein), survival was not affected by 30 ppm Se as DL, L, or Y and oxidative effects on GSH metabolism were less pronounced than with the wheat diet.

Hum Genet, 1996 Jul, 98(1), 7 - 11
Identification and characterization of NF1-related loci on human chromosomes 22, 14 and 2; Hulsebos TJ et al.; Neurofibromatosis type 1 (NF1) is a frequent hereditary disorder . The disease is characterized by a very high mutation rate (up to 1/10000 gametes per generation) . NF1-related loci in the human genome have been implicated in the high mutation rate by hypothesizing that these carry disease-causing mutations, which can be transferred to the functional NF1 gene on chromosome arm 17q by interchromosomal gene conversion . To test this hypothesis, we want to identify and characterize the NF1-related loci in the human genome . In this study, we have localized an NF1-related locus in the most centromeric region of the long arm of chromosome 22 . We demonstrate that this locus contains sequences homologous to cDNAs that include the GAP-related domain of the functional NF1 gene . However, the GAP-related domain itself is not represented in this locus . In addition, cosmids specific to this locus reveal, by in situ hybridization, NF1-related loci in the pericentromeric region of chromosome arm 14q and in chromosomal band 2q21 . These cosmids will enable us to determine whether identified disease-causing mutations are present at the chromosome 22-associated NF1-related locus.

Hum Genet, 1996 Jul, 98(1), 16 - 21
Fine mapping of the 1q21 breakpoint of the papillary renal cell carcinoma-associated (X;1) translocation; Weterman MA et al.; A combination of Southern blot analysis on a panel of tumor-derived somatic cell hybrids and fluorescence in situ hybridization (FISH) techniques was used to map a series of DNA markers relative to the 1q21 breakpoint of the renal cell carcinoma (RCC)-associated (X;1)-(p11;q21) translocation . This breakpoint maps between several members of the S100 family which are clustered in the 1q21 region and a conserved region between man and mouse containing the markers SPTA1-CRP-APCS-FcER1A-ATP1A2-APOA2 . The location of the breakpoint coincides with the transition of a region of synteny of human chromosome 1 with mouse chromosomes 3 and 1.

Genes Dev, 1996 Jul 1, 10(13), 1683 - 98
A nuclear cap-binding complex facilitates association of U1 snRNP with the cap-proximal 5' splice site; Lewis JD et al.; The mechanism by which intron-containing RNAs are recognized by the splicing machinery is only partly understood . A nuclear cap-binding complex (CBC), which specifically recognizes the monomethyl guanosine cap structure carried by RNA polymerase II transcripts, has previously been shown to play a role in pre-mRNA splicing . Using a combination of splicing complex and psoralen cross-linking analysis we demonstrate that CBC is required for efficient recognition of the 5' splice site by U1 snRNP during formation of E (early) complex on a pre-mRNA containing a single intron . However, in a pre-mRNA containing two introns, CBC is not required for splicing of the cap distal intron . In this case, the presence of an intact polypyrimidine tract in the cap-proximal intron renders splicing of the cap-distal intron independent of CBC . These results support models in which the splice sites in a pre-mRNA are originally recognized by interactions spanning exons . The defects in splicing and U1 snRNP binding caused by CBC depletion can be specifically reversed by recombinant CBC . In summary, efficient recognition of the cap-proximal 5' splice site by U1 snRNP is facilitated by CBC in what may be one of the earliest steps in pre-mRNA recognition . Data in Colot et al . (this issue) indicate that this function of CBC is conserved in humans and yeast.

J Gerontol A Biol Sci Med Sci, 1996 Jul, 51(4), B280 - 3
Failure to confirm increased longevity in Drosophila melanogaster submitted to a food restriction procedure; Le Bourg E et al.; Several studies have shown that, contrary to what occurs in rodents and in some invertebrate species, food restriction has no positive effect on longevity in Drosophila melanogaster . However, Chippindale et al . (1993) reported that flies subjected to food restriction, by modulating the yeast level, could live longer . In the present study we used the same yeast levels as Chippindale et al . in an attempt to confirm these results . No positive effect of food restriction on longevity could be observed in either sex in mated and virgin flies.

J Virol, 1996 Jul, 70(7), 4299 - 310
Binding of the human immunodeficiency virus type 1 Gag polyprotein to cyclophilin A is mediated by the central region of capsid and requires Gag dimerization; Colgan J et al.; The cellular peptidyl-prolyl isomerase cyclophilin A (CyPA) is incorporated into human immunodeficiency virus type 1 (HIV-1) virions via direct contacts with the HIV-1 Gag polyprotein . Disruption of the Gag-CyPA interaction leads to the production of HIV-1 particles lacking CyPA; these virions are noninfectious, indicating that contacts between CyPA and Gag are necessary for HIV-1 replication . Here, we have used the yeast two-hybrid system in conjunction with an in vitro binding assay to identify the minimal domain of Gag required for binding to CyPA . Analysis of a panel of gag deletion mutants in the two-hybrid system indicated that a region spanning the central portion of the capsid (CA) domain was sufficient for interactions with CyPA, but discrepancies between results obtained in different fusion protein contexts suggested that multimerization of Gag might also be necessary for binding to CyPA . Consistent with a requirement for multimerization, the binding of Gag to CyPA in vitro required a region within the nucleocapsid (NC) domain shown previously to be important for Gag self-association . Substitution of a heterologous dimerization motif for the region from NC also promoted specific binding to CyPA, confirming that interactions with CyPA are dependent on Gag multimerization . Fusion of the heterologous dimerization motif to a 100-amino-acid domain from CA was sufficient for binding to CyPA in vitro . These results define the minimal CyPA-binding domain within Gag and provide insight into the mechanism by which CyPA is incorporated into HIV-1 virions.

J Virol, 1996 Jul, 70(7), 4228 - 36
A conserved domain of the Epstein-Barr virus nuclear antigens 3A and 3C binds to a discrete domain of Jkappa; Zhao B et al.; EBNA-3C can affect the LMP-1 promoter in both a positive and a negative manner through distinct DNA sequence elements . The viral transactivator EBNA-2 normally binds DNA indirectly via Jkappa to activate transcription, but this activation is prevented in the presence of EBNA-3C . The DNA element recognized by Jkappa is both required and sufficient for this inhibition . Jkappa clones isolated in a yeast two-hybrid screen using EBNA-3C as bait allowed us to delineate the sequences of both proteins mediating the interaction . Two isoforms of Jkappa that differ in exon 1, Jkappa-1 and RBP-2N, interact with EBNA-3C, suggesting that exon 1 is not required for this interaction; indeed, clones with deletion of the N-terminal third of Jkappa interacted as efficiently with EBNA-3C as full-length Jkappa clones . A Jkappa domain as small as 56 amino acids was sufficient to bind to EBNA-3C . A 74-amino-acid domain of EBNA-3C, conserved in all three EBNA-3 family members, was sufficient to interact with Jkappa . A specific mutation in this conserved domain suppressed the ability of EBNA-3C to downregulate transcription . Accordingly, EBNA-3A was also able to interact with Jkappa and downregulate Jkappa-mediated transcription as efficiently as EBNA-3C . The ability of the EBNA-3 proteins to prevent Jkappa from binding to DNA in vitro and suppress transactivation via Jkappa DNA elements suggests that the EBNA-3 proteins act analogously to the Drosophila protein Hairless.

EMBO J, 1996 Jul 1, 15(13), 3394 - 402
The hbrm and BRG-1 proteins, components of the human SNF/SWI complex, are phosphorylated and excluded from the condensed chromosomes during mitosis; Muchardt C et al.; In yeast, the SNF/SWI complex is believed to regulate transcription by locally altering the chromatin structure . At the present time, three human homologues of yeast SNF/SWI proteins have been characterized: hbrm and BRG-1, homologues of SNF2/SWI2, and hSNF5, a homologue of SNF5 . We show here that, during mitosis, hbrm and BRG-1 are phosphorylated and excluded from the condensed chromosomes . In this phase of the cell cycle, the level of hbrm protein is also strongly reduced, whereas the level of BRG-1 remains constant . The mitotic phosphorylation of hbrm and BRG-1 is found not to disrupt the association of these proteins with hSNF5 but correlates with a decreased affinity for the nuclear structure in early M phase . We suggest that chromosomal exclusion of the human SNF/SWI complex at the G2-M transition could be part of the mechanism leading to transcriptional arrest during mitosis.

EMBO J, 1996 Jul 1, 15(13), 3229 - 37
An essential ubiquitin-conjugating enzyme with tissue and developmental specificity in th nematode Caenorhabditis elegans; Zhen M et al.; The ubc-2 gene in Caenorhabditis elegans encodes a ubiquitin-conjugating enzyme (E2) homologous to yeast UBC4 and UBC5 . UBC4 and UBC5 are individually dispensable class I E2 enzymes involved in the degradation of short-lived and abnormal proteins . Transgenic analysis using ubc-2-lacZ fusions and in situ immunofluorescence indicate that ubc-2 is abundantly expressed in most tissues of embryos and early larvae, but becomes specific to the nervous system in L4 larvae and adults . This suggests that the functions of this type of E2 are developmentally regulated in C.elegans . This hypothesis is supported by antisense analysis, which shows that blocking the expression of ubc-2 has a more severe effect in early developmental stages than in later stages . Through complementation of previously identified essential genes in the vicinity of ubc-2, we demonstrate that ubc-2 corresponds to let-70, a gene essential for C.elegans larval development . One let-70(ubc-2) allele contains a His75-->Tyr substitution, while another has an altered splice donor site.

Mol Cell Biol, 1996 Jul, 16(7), 3317 - 26
Identification of proteins that interact with exon sequences, splice sites, and the branchpoint sequence during each stage of spliceosome assembly; Chiara MD et al.; We have carried out a systematic analysis of the proteins that interact with specific intron and exon sequences during each stage of mammalian spliceosome assembly . This was achieved by site-specifically labeling individual nucleotides within the 5' and 3' splice sites, the branchpoint sequence (BPS), or the exons with 32P and identifying UV-cross-linked proteins in the E, A, B, or C spliceosomal complex . Significantly, two members of the SR family of splicing factors, which are known to promote E-complex assembly, cross-link within exon sequences to a region approximately 25 nucleotides upstream from the 5' splice site . At the 5' splice site, cross-linking of the U5 small nuclear ribonucleoprotein particle protein, U5(200), was detected in both the B and C complexes . As observed in yeast cells, U5(200), also cross-links to intron/exon sequences at the 3' splice site in the C complex and may play a role in aligning the 5' and 3' exons for ligation . With label at the branch site, we detected three distinct proteins, designated BPS72,BpS70, and BPS56, which replace one another in the E, A, and C complexes . Another dynamic exchange was detected with pre-mRNA labeled at the AG dinucleotide of the 3' splice site . In this case, a protein, AG100,cross-links in the A complex and is replaced by another protein, AG75, in the C complex . The observation that these proteins are specifically associated with critical pre-mRNA sequence elements in functional complexes at different stages of spliceosome assembly implicates roles for these factors in key recognition events during the splicing pathway.

Virology, 1996 Jul 1, 221(1), 44 - 53
The transactivation and DNA binding domains of the BPV-1 E2 protein have different roles in cooperative origin binding with the E1 protein; Winokur PL et al.; The bovine papillomavirus E2 transactivator protein enhances the ability of the E1 protein to bind to the viral origin of replication which contains an E1 binding site flanked by two E2 binding sites . To determine which regions and functions of the E2 protein are important for this cooperative interaction, a series of mutated E2 proteins were assayed for their ability to enhance E1 origin-specific binding . Cooperative origin binding required at least one E2 DNA binding site, an intact functional E2 DNA binding domain, and an intact transactivation domain . The hinge region of the E2 proteins was dispensable for this activity . To further examine the role of the E2 C-terminal domain, a series of chimeric proteins were generated that substituted the yeast GAL4 DNA binding domain for the E2 DNA binding domain . These chimeric proteins were able to cooperatively bind to a hybrid origin that contained GAL4 binding sites in place of the E2 binding sites . These studies indicate that the E2 transactivation domain is sufficient for interaction with the E1 protein and that the E2 DNA binding domain is required for interaction with origin DNA sequences.

Genomics, 1996 Jul 1, 35(1), 241 - 3
Gene structure and chromosome localization to 7q21.3 of the human rod photoreceptor transducin gamma-subunit gene (GNGT1); Scherer SW et al.; The transducin gamma-subunit gene (GNGT1) encodes a member (gamma1) of the family of heterotrimeric G-protein gamma-subunits that is specific to rod photoreceptors . In this report we have determined the complete structure of the GNGT1 gene and have localized it to human chromosome 7q21.3 using somatic cell hybrid and yeast artificial chromosome analysis.

Genomics, 1996 Jul 1, 35(1), 172 - 81
Characterization and genomic mapping of genes and pseudogenes of a new human protein tyrosine phosphatase; Zhao Z et al.; Previously described protein tyrosine phosphatases (PTPs) are classified into three types according to their sequence homology and structural features . Here we describe the characterization of genes and pseudogenes of a member of a fourth type of PTP, designated protein tyrosine phosphatase 4A (PTP4A) . The 167-amino-acid human PTP4A bears the signature active site of all PTPs, but does not show any other sequence homology to any of the previously described PTPs . Two cDNAs encoding PTP4A that differed in their noncoding regions were isolated . Another cDNA that has a high level of sequence identity with these two cDNAs and a deletion in the coding region was also isolated . Northern analysis using a probe from a common 3'-untranslated region of the cDNAs recognized mRNAs of about 2 and 4 kb . Both species of mRNA were seen in all human adult and fetal tissues tested . Fluorescence in situ hybridization mapping of the corresponding yeast artificial chromosome clones and sequence-tagged site analysis suggested that one of the PTP4A coding genes is located at 1p35 and the other is on chromosome 11 . A processed pseudogene for PTP4A was found in the BRCA1 region of 17q21 and shares 96% sequence identity to one of the PTP4A coding cDNAs . Our studies also suggest the existence of another processed pseudogene on chromosome 11.

Genomics, 1996 Jul 1, 35(1), 118 - 28
Physical mapping of the bloom syndrome region by the identification of YAC and P1 clones from human chromosome 15 band q26.1; Straughen J et al.; The gene for Bloom syndrome (BLM) has been mapped to human chromosome 15 band q26.1 by homozygosity mapping . Further refinement of the location of BLM has relied upon linkage-disequilibrium mapping and somatic intragenic recombination . In combination with these mapping approaches and to identify novel DNA markers and probes for the BLM candidate region, a contiguous representation of the 2-Mb region that contains the BLM gene was generated and is presented here . YAC and P1 clones from the region have been identified and ordered by using previously available genetic markers in the region along with newly developed sequence-tagged sites from radiation-reduced hybrids, polymorphic dinucleotide repeat loci, and end sequences of YACs and P1s . A long-range restriction map of the 2-Mb region that allowed estimation of the distance between polymorphic microsatellite loci is also reported . This map and the DNA markers derived from it were instrumental in the recent identification of the BLM gene.

Genomics, 1996 Jul 1, 35(1), 109 - 17
Direct cloning of DNA sequences from the common fragile site region at chromosome band 3p14.2; Rassool FV et al.; Despite several lines of evidence suggesting that common chromosomal fragile sites are biologically important as hot spots for recombination, their structure remains unknown . We showed previously that the plasmid pSV2neo preferentially integrates into bands containing fragile sites in cells transfected under conditions of fragile site induction . Here we report the isolation and characterization of the DNA sequences from two such independent integrations into 3p14.2, a common fragile site (FRA3B) . These FRA3B region sequences were shown to lie within a 1330-kb YAC, 850A6, approximately 350 kb telomeric of the breakpoint of t(3;8), a constitutional rearrangement . The two integration sites are 10 kb apart, but each integration is associated with a deletion . We have constructed a partial genomic contig of the integration sites and deleted regions spanning approximately 85 kb . Analysis of the DNA sequences immediately surrounding the plasmid integrations revealed no known coding sequences or repeat structures resembling the (CGG)n motif characteristic of the rare fragile sites . In addition, by Southern blotting analysis, none of the phage clones isolated from the FRA3B region were found to contain CGG repeats . Fluorescence in situ hybridization analysis of genomic clones from this contig to metaphase cells induced to express breaks demonstrated hybridization adjoining the chromosome breaks, and occasionally the hybridization signal spanned the break . The results imply that breakage occurs at variable positions within a large region (at least on the order of 85 kb) . Together, these data suggest that the structure of FRA3B differs from that of rare fragile sites.

Genomics, 1996 Jul 1, 35(1), 94 - 100
Physical mapping and genomic structure of the human TNFR2 gene; Beltinger CP et al.; The tumor necrosis factor receptor 2 (TNFR2) gene localizes to 1p36 . 2, a genomic region characteristically deleted in neuroblastomas and other malignancies . In addition, TNFR2 is the principal mediator of the effects of TNF on cellular immunity, and it may cooperate with TNFR1 in the killing of nonlymphoid cells . Therefore, we undertook an analysis of the genomic structure and precise physical mapping of this gene . The TNFR2 gene is contained on 10 exons that span 26 kb . Most of the functional domains of TNFR2 are encoded by separate exons, and each of the repeats of the extracellular cysteine-rich domain is interrupted by an intron . The genomic structure reveals a close relationship to TNFR1, another member of the TNFR superfamily . Based on electrophoretic analysis of yeast artificial chromosomes, TNFR2 maps within 400 kb of the genetic marker D1S434 . In addition, we have identified a new polymorphic dinucleotide repeat within intron 4 of TNFR2 . The genetic sequence information and exon-intron boundaries we have determined will facilitate mutational analysis of this gene to determine its potential role in neuroblastoma, as well as in other cancers with characteristic deletions or rearrangements of 1p36.

Genomics, 1996 Jul 1, 35(1), 87 - 93
A 350-kb cosmid contig in 3p14.2 that crosses the t(3;8) hereditary renal cell carcinoma translocation breakpoint and 17 aphidicolin-induced FRA3B breakpoints; Paradee W et al.; The constitutive fragile site at human chromosomal band 3p14.2, FRA3B, has been described as the most active common fragile site in the human genome . FRA3B is cytologically indistinguishable from the chromosome 3 breakpoint observed in the hereditary renal cell carcinoma (hRCC) translocation t(3;8) (p14.2;q24.13) . Previous work demonstrated that a 1330-kb YAC clone, YC850A6, spans both the t(3;8) translocation and FRA3B and also encompasses FRA3B-associated breakpoints induced in hamster-human hybrids . This YAC was used to construct a multi-hit cosmid library . Screening of this library resulted in a 350-kb cosmid contig that extends distally from the t(3;8) translocation breakpoint . Seventeen aphidicolin-induced 3p14 . 2 breakpoints derived from hamster-human hybrids were mapped within this cosmid contig . These breakpoints were found to localize as two distinct clusters, separated by 200 kb, which lie on either side of a region of frequent breakage within FRA3B as defined by FISH analysis using cosmids from the contigs . The most proximal of the breakpoint clusters lies approximately 100 kb distal to the hRCC t(3;8) breakpoint . The distribution of these breakpoints, together with the region of frequent chromosomal breakage mapped by FISH analysis, further confirms the position of FRA3B and helps to define the extent over which its fragility is exerted . These data indicate that FRA3B comprises several hundred kilobases of DNA sequence within 3p14.2 . The 350-kb contig and the cosmid library constructed from YAC YC850A6 will be essential for further characterization of the region surrounding FRA3B and in experiments to determine the molecular basis of the fragility of FRA3B.

Genomics, 1996 Jul 1, 35(1), 71 - 8
Efficient construction of a physical map by fiber-FISH of the CLN5 region: refined assignment and long-range contig covering the critical region on 13q22; Klockars T et al.; The variant form of late infantile neuronal ceroid lipofuscinosis (vLINCL, locus definition CLN5) represents a progressive brain disease with autosomal recessive inheritance . We have previously assigned the CLN5 locus to chromosome 13q21.1-q32 between markers D13S160 and D13S162 by linkage analysis in Finnish families . The information on ancient recombination events obtained from linkage disequilibrium provided an efficient tool for further refining the assignment of the CLN5 locus . Isolation of two novel (CA)n markers, COLAC1 and AC224, resulted in a dramatic restriction of the critical DNA region . We utilized the Fiber-FISH technique to orient and order the large DNA clones isolated by STSs and were able to eliminate almost totally the restriction digestion and PFGE step in the construction of the long-range DNA contig . Both linkage disequilibrium data and Fiber-FISH analyses assigned the CLN5 locus to a well-defined 200-kb region . Here we report a complete physical map of about 350 kb covering the critical chromosomal region of CLN5, which will facilitate the final isolation of the CLN5 gene.

Genomics, 1996 Jul 1, 35(1), 46 - 54
Physical mapping of chromosome 8p22 markers and their homozygous deletion in a metastatic prostate cancer; Bova GS et al.; Numerous studies have implicated the short arm of chromosome 8 as the site of one or more tumor suppressor genes inactivated in carcinogenesis of the prostate, colon, lung, and liver . Previously, we identified a homozygous deletion on chromosome 8p22 in a metastatic prostate cancer . To map this homozygous deletion physically, long-range restriction mapping was performed using yeast artificial chromosomes (YACs) spanning approximately 2 Mb of chromosome band 8p22 . Subcloned genomic DNA and cDNA probes isolated by hybrid capture from these YACs were mapped in relation to one another, reinforcing map integrity . Mapped single-copy probes from the region were then applied to DNA isolated from a metastatic prostate cancer containing a chromosome 8p22 homozygous deletion and indicated that its deletion spans 730-970 kb . Candidate genes PRLTS (PDGF-receptor beta-like tumor suppressor) and CTSB (cathepsin B) are located outside the region of homozygous deletion . Genethon marker D8S549 is located approximately at the center of this region of homozygous deletion . Two new microsatellite polymorphisms, D8S1991 and D8S1992, also located within the region of homozygous deletion on chromosome 8p22, are described . Physical mapping places cosmid CI8-2644 telomeric to MSR (macrophage scavenger receptor), the reverse of a previously published map, altering the interpretation of published deletion studies . This work should prove helpful in the identification of candidate tumor suppressor genes in this region.

Cell, 1996 Jun 28, 85(7), 1077 - 88
Site-specific ribose methylation of preribosomal RNA: a novel function for small nucleolar RNAs; Kiss-Laszlo Z et al.; Eukaryotic cells contain many fibrillarin-associated small nucleolar RNAs (snoRNAs) that possess long complementarities to mature rRNAs . Characterization of 21 novel antisense snoRNAs from human cells followed by genetic depletion and reconstitution studies on yeast U24 snoRNA provides evidence that this class of snoRNAs is required for site-specific 2'-O-methylation of preribosomal RNA (pre-rRNA) . Antisense sno-RNAs function through direct base-pairing interactions with pre-rRNA . The antisense element, together with the D or D' box of the snoRNA, provide the information necessary to select the target nucleotide for the methyltransfer reaction . The conclusion that sno-RNAs function in covalent modification of the sugar moieties of ribonucleotides demonstrates that eukaryotic small nuclear RNAs have a more versatile cellular function than earlier anticipated.

J Biol Chem, 1996 Jun 28, 271(26), 15322 - 9
p120 Ras GTPase-activating protein interacts with Ras-GTP through specific conserved residues; Miao W et al.; Previous structural studies of RasGAP have failed to clearly localize sites of Ras interaction to individual amino acids . Hypothesizing that sites of interaction with Ras-GTP would be conserved, 11 of the most highly conserved amino acid residues of RasGAP were changed by mutation . Each mutant protein was purified as a glutathione S-transferase catalytic domain fusion and analyzed for protein stability, Ras GTPase stimulating activity, affinity for Ras-GTP, and when possible, secondary structure . The majority of conserved positions were found to be important structurally but with no direct role in Ras interactions . However, Arg786, Lys831, and Arg925 were observed to be essential for binding to Ras-GTP but not for protein structure . RasGAP residues 890-902 (block 3A) were observed to be homologous to residues 1540-1552 of the yeast adenylyl cyclase with amino acid substitutions in both regions resulting in increased affinity for Ras . This is the first example of a conserved Ras interaction motif in distinct Ras effector proteins . Our data are supportive of a model for GAP/Ras-GTP association in which the conserved, positively charged Arg786, Lys831, and Arg925 residues form salt bridges with the conserved, negatively charged residues in the Ras effector loop.

Biochem Biophys Res Commun, 1996 Jun 25, 223(3), 729 - 34
Difference in the mechanism of interaction of Raf-1 and B-Raf with H-Ras; Shinkai M et al.; Ras is known to possess multiple cellular targets including Raf-1 . Here, we measured both direct binding of various H-Ras mutants to two representative mammalian Ras targets, Raf-1 and B-Raf, and the activity of the mutants to stimulate Raf-1 and B-Raf, and analysed the difference in their Ras-interaction mechanisms . B-Raf was shown to share almost the same H-Ras binding-specificity with Raf-1 by examining binding of the H-Ras mutants to Raf-1 and B-Raf in the yeast two-hybrid and in vitro binding assays . Mutants, Y32F, A59E, and V45E bound to Raf-1 in Sf9 cells coexpressing them, but failed to activate Raf-1 . On the other hand, Y32F activated B-Raf in a cell-free system which consisted of rat brain cytosol and recombinant MEK . These results suggest that there is a subtle structural difference in requirements for the interaction of Ras with Raf-1 and B-Raf.

Biochem Biophys Res Commun, 1996 Jun 25, 223(3), 701 - 5
Molecular interaction between TLE1 and the carboxyl-terminal domain of HES-1 containing the WRPW motif; Grbavec D et al.; Groucho is a protein implicated in Notch signaling and involved in segmentation and neural development in Drosophila . Groucho forms transcription complexes with the basic helix-loop-helix proteins encoded by the hairy/Enhancer of split ("hairy-like") gene family . These interactions are mediated by the carboxyl-terminal WRPW motif of Hairy-like proteins . We are interested in determining whether Groucho and its mammalian homologues, the TLE proteins, perform conserved functions . We show that TLE1 interacts with HES-1, a murine homologue of Drosophila Hairy-like proteins, both in the yeast two-hybrid assay and in an interaction assay based on glutathione S-transferase fusion proteins . These results show that Groucho/TLE proteins and Hairy-like/HES proteins are involved in similar interactions in Drosophila and mammals and further suggest that these proteins perform conserved cellular functions.

Oncogene, 1996 Jun 20, 12(12), 2713 - 7
Tissue-specific expression, evolutionary conservation and localization of the cph proto-oncogene on Syrian hamster chromosome X; Velasco JA et al.; Treatment of Syrian hamster embryo fibroblasts with a single dose of 3-methylcholanthrene caused the activation of the transforming potential of cellular sequences (Notario et al, Oncogene 5: 1425-1430, 1990), which were subsequently isolated by cosmid rescue techniques, and further identified as a novel oncogene, termed cph because of its involvement in the carcinogenic progression of hamster embryo cells (Velasco et al, Oncogene 9: 2065-2069, 1994) . We have analysed the expression of the cph proto-oncogene in adult Syrian hamster tissues by northern hybridization using cph-specific genomic probes . The three cph transcripts expressed in normal and neoplastic Syrian hamster embryo cells in culture (5.0, 3.5 and 2.0 kb) were also present in most adult tissues, although different mRNA species, most likely resulting from alternative splicing events, were expressed in testes . The highest steady-state level of cph mRNA was found in kidney, whereas cph expression was nearly undetectable in skin and skeletal muscle . Southern blot analyses of DNAs from other eucaryotic organisms were performed under moderate stringency conditions with a Syrian hamster-specific cph probe . Discrete cph-hybridizing sequences were present in genomes from yeast to mammalian species, including humans, thus demonstrating that cph is a highly conserved gene in eucaryotic evolution . Using fluorescence in situ hybridization (FISH), we have determined also the chromosomal localization of the cph proto-oncogene in the hamster genome . FISH experiments demonstrated that cph is a single copy gene, localized on the euchromatic short arm of the X chromosome, at region Xpa7 . Because chromosome X is frequently involved in structural alterations in neoplastic Syrian hamster cells transformed by chemical carcinogens and oncogenic viruses, the localization of the cph locus on this chromosome supports the notion that the cph oncogene plays a role in the malignant conversion of chemically transformed hamster fibroblasts . The wide range of tissue-specific expression and species-specific distribution of cph strongly suggest that the normal function of the cph protein product(s) may be essential for metabolic processes involved in the regulation of cell proliferation and survival.

Oncogene, 1996 Jun 20, 12(12), 2563 - 71
RN-tre identifies a family of tre-related proteins displaying a novel potential protein binding domain; Matoskova B et al.; Eps8 is a recently identified SH3-containing substrate for tyrosine kinase receptors . To understand the role of eps8 in receptor-mediated signaling, we cloned cDNAs encoding proteins that bind to its SH3 domain . One of these cDNAs predicts the synthesis of an 828 amino acid protein with homology to the N-terminal region of the tre oncogene . We designated this protein RN-tre for Related to the N-terminus of tre . RN-tre is ubiquitously expressed and maps to 10p13, a region known to be involved in translocations in various leukemias . In addition, a 10p13 monosomy syndrome, characterized by developmental alterations, has been reported . The regional homology between RN-tre and tre, which is limited to their N-terminal portion, prompted us to investigate the origin of the tre oncogene transcriptional unit . We were able to show that tre is the fusion product of a 5' genetic element, homologous to RN-tre and a 3' element, encoding a de-ubiquinating enzyme . Moreover, we identified, within the N-terminus of RN-tre and tre, a domain (named TrH, for Tre Homology), which is conserved within several proteins from yeast to mammals and has protein-binding properties in vitro.

EMBO J, 1996 Jun 17, 15(12), 2997 - 3005
IQGAP1, a calmodulin-binding protein with a rasGAP-related domain, is a potential effector for cdc42Hs; Hart MJ et al.; Proteins that associate with the GTP-bound forms of the Ras superfamily of proteins are potential effector targets for these molecular switches . A 195 kDa protein was purified from cell lysates by affinity chromatography on immobilized cdc42Hs-GTP and a corresponding cDNA was isolated . Sequence analysis revealed localized identities to calponin, the WW domain, unconventional myosins and to the rasGAP-related domain (GRD) contained in IRA, NF-1, SAR1 and rasGAP . p195 was found to be identical to IQGAP1, a protein previously reported to bind ras . Purified recombinant p195/IQGAP1 bound to and inhibited the GTPase activity of cdc42Hs and rac whereas no interaction with ras was detected . The C-terminal half of IQGAP1 containing the GRD bound to cdc42 and rac in a GRD-dependent fashion, but a smaller fragment containing only the GRD did not . Cdc42 was also co-immunoprecipitated from cell lysates with antibody specific to p195/IQGAP1 . Calmodulin also co-immunoprecipitated with p195/IQGAP1 and was found to associate with fragments containing the IQ domain . Expression of a cDNA fragment encoding the GRD inhibited the CDC24/CDC42 pathway in yeast, but no effect on ras was observed . In mammalian cells, both endogenous and ectopically expressed p195/IQGAP1 were localized to lamellipodia and ruffling cell membranes, where co-localization with actin was apparent . These results suggest that IQGAP1 is an effector target for cdc42Hs and may mediate the effects of this GTPase on cell morphology.

Genomics, 1996 Jun 15, 34(3), 422 - 5
Characterization of a translocation-associated deletion defines the candidate region for the gene responsible for branchio-oto-renal syndrome; Kalatzis V et al.; Fluorescence in situ hybridization analysis of an 8q translocation breakpoint, dir ins(8)(q24.11;q13.3;q21.13), carried by an individual presenting with Branchio-Oto-Renal (BOR) syndrome, resulted in the identification of an associated deletion . The generation of a YAC contig and the isolation of overlapping recombinant P1 and lambda phage clones from the region allowed further characterization of this deletion . Its size was estimated to be between 470 and 650 kb, and it was flanked by the two polymorphic markers D8S1060 and D8S1807 . This mapping led us to reevaluate the localization of the gene responsible for BOR syndrome and has now focused the search for the BOR gene to within the limits of this deletion.

Genomics, 1996 Jun 15, 34(3), 381 - 8
Genomic structure and precise mapping of a thymic regulatory region on mouse chromosome 17 revealed by a c-myc transgene insertion; Lavenu A et al.; In one transgenic strain harboring a human c-myc proto-oncogene construct, the transgene was actively and exclusively expressed in the thymus, where it contributed to the development of lymphoma that corresponded to CD4(+)CD8(+) cells . Here, we have pursued the analysis of transgene expression in healthy transgenic mice and show that transgene activation occurs in the thymus 3 days before birth, at a time when CD4(+)CD8(+) lymphocytes emerge . In the adult, its expression is restricted to the CD4(+)CD8(+) cells . The region flanking the transgene insertion site was isolated and made it possible to map the preintegration locus, hereafter called Tsil (for thymus-specific integration locus) on chromosome 17 between D17Rp11e and Ras12-3 . A YAC that contains both Tsil and the Pim2 locus, previously shown to be involved in progression of T-cell lymphoma, was isolated . Analysis of Tsil offers a unique opportunity to identify a regulatory region or a gene that might play an important role in T-cell maturation.

Biochem J, 1996 Jun 15, 316 ( Pt 3), 723 - 7
Isolation and characterization of a 30 kDa protein with antifungal activity from leaves of Engelmannia pinnatifida; Huynh QK et al.; During the course of screening plants for novel antifungal activity, we found that a high-molecular-mass fraction of an extract from leaves of Engelmannia pinnatifida exhibited potent and broad-spectrum antifungal activity . In this study a 30 kDa protein from E . pinnatifida leaves was purified to homogeneity by ammonium sulphate precipitation, gel filtration, Mono-Q and C18 reverse-phase column chromatographies . The purified protein showed potent antifungal activity against various plant pathogens with as little as 50 ng . The N-terminal amino acid sequence of the purified protein was determined as XXTKFDFFTLALQXPAXF, where X indicates an unidentified residue . This sequence showed 35-50% sequence identity with purified style glycoproteins associated with self-incompatibility from wild tomato, tobacco and petunia, a phosphate-starvation-induced ribonuclease from cultured tomato cells and the SIR 63.4 kDa protein from yeast.

Genes Dev, 1996 Jun 15, 10(12), 1503 - 15
Xe-p9, a Xenopus Suc1/Cks homolog, has multiple essential roles in cell cycle control; Patra D et al.; The small Suc1/Cks protein is a ubiquitous subunit of Cdk/cyclin complexes, but its precise function has remained unclear . We have isolated a Xenopus homolog, Xe-p9, of the Suc1/Cks protein by virtue of its ability to rescue a fission yeast mutant that enters mitosis prematurely . To assess its functional role in cell cycle control, we have both overexpressed p9 in Xenopus egg extracts and immunodepleted the protein from these extracts . We found that addition of recombinant His6-p9 to egg extracts results in a pronounced delay of mitosis that can be attributed to an inhibition of the tyrosine dephosphorylation of the inactive Cdc2/cyclin B complex . In immunodepletion studies, we observed that the consequences of removing p9 from egg extracts depend on the stage of the cell cycle . Specifically, in the case of interphase extracts, the removal of p9 abolishes the entry into mitosis as a result of a failure in the activation of the Cdc2/cyclin B complex by tyrosine dephosphorylation . Furthermore, mitotic extracts lacking p9 fail to exit mitosis because of a defect in the destruction of cyclin B . Collectively, these results indicate that p9 has multiple essential roles in the cell cycle by governing the interaction of the Cdc2/cyclin B complex with both positive and negative regulators.

Cell, 1996 Jun 14, 85(6), 829 - 39
cul-1 is required for cell cycle exit in C . elegans and identifies a novel gene family; Kipreos ET et al.; The gene cul-1 (formerly lin-19) is a negative regulator of the cell cycle in C . elegans . Null mutations cause hyperplasia of all tissues . cul-1 is required for developmentally programmed transitions from the G1 phase of the cell cycle to the GO phase or the apoptotic pathway . Moreover, the mutant phenotype suggests that G1-to-S phase progression is accelerated, overriding mechanisms for mitotic arrest and producing abnormally small cells . Significantly, diverse aspects of cell fate and differentiation are unaffected in cul-1 mutants . cul-1 represents a conserved family of genes, designated cullins, with at least five members in nematodes, six in humans, and three in budding yeast.

J Mol Biol, 1996 Jun 14, 259(3), 337 - 48
hnRNP A1 selectively interacts through its Gly-rich domain with different RNA-binding proteins; Cartegni L et al.; Heterogeneous nuclear ribonucleoproteins (hnRNPs) are abundant nuclear polypeptides, most likely involved in different steps of pre-mRNA processing . Protein A1 (34 kDa), a prominent member of the hnRNP family, seems to act by modulating the RNA secondary structure and by antagonizing some splicing factors (SR proteins) in splice-site selection and exon skipping/inclusion . A role of A1 in the nucleo-cytoplasmic transport of RNA has also been proposed . These activities might depend not only on the RNA-binding properties of the protein but also on specific protein-protein interactions . Here we report that A1 can indeed selectively interact, in vitro, both with itself and with other hnRNP basic "core" proteins . Such selective binding is mediated exclusively by the Gly-rich C-terminal domain, where a novel protein-binding motif constituted by hydrophobic repeats can be envisaged . The same domain is necessary and sufficient to promote specific interaction in vivo, as assayed by the yeast two-hybrid assay . Moreover, an in vitro interaction with some SR proteins was also observed . These observations suggest that diverse and specific protein-protein interactions might contribute to the different functions of the hnRNP A1 protein in mRNA maturation.

J Mol Biol, 1996 Jun 14, 259(3), 317 - 24
Topoisomerase II-mediated DNA cleavage: evidence for distinct regions of enzyme-DNA contacts; Alsner J et al.; To determine the specific interaction sites of topoisomerase II within the DNA region defined by the footprint of the enzyme, we have investigated the cleavage reaction on double-stranded DNA substrates containing nicks and deletions . Topoisomerase II-mediated cleavage of the DNA substrates is suicidal as the enzyme is unable to religate the cleaved DNA due to diffusion of the small nucleotides 5' to the cleavage position . Thus, suicidal cleavage is obtained with substrates having one, two or three nucleotides 5' to the cleavage position . The enzyme requires interaction with three distinct regions of double-stranded DNA for cleavage to occur, one region spanning the eight nucleotides located around the cleavage position and two distal regions each spanning approximately six nucleotides . A model is proposed, where these data are taken to imply that two distinct regions of interactions exist between each topoisomerase II subunit and its DNA substrate . The model is discussed in relation to the recently solved three-dimensional structure of yeast topoisomerase II.

Proc Natl Acad Sci U S A, 1996 Jun 11, 93(12), 6203 - 8
Genomic cloning of methylthioadenosine phosphorylase: a purine metabolic enzyme deficient in multiple different cancers; Nobori T et al.; 5'-Deoxy-5'-methylthioadenosine phosphorylase (methylthioadeno-sine: ortho-phosphate methylthioribosyltransferase, EC 24.2.28; MTAP) plays a role in purine and polyamine metabolism and in the regulation of transmethylation reactions . MTAP is abundant in normal cells but is deficient in many cancers . Recently, the genes for the cyclin-dependent kinase inhibitors p16 and p15 have been localized to the short arm of human chromosome 9 at band p21, where MTAP and interferon alpha genes (IFNA) also map . Homozygous deletions of p16 and p15 are frequent malignant cell lines . However, the order of the MTAP, p16, p15, and IFNA genes on chromosome 9p is uncertain, and the molecular basis for MTAP deficiency in cancer is unknown . We have cloned the MTAP gene, and have constructed a topologic map of the 9p21 region using yeast artificial chromosome clones, pulse-field gel electrophoresis, and sequence-tagged-site PCR . The MTAP gene consists of eight exons and seven introns . Of 23 malignant cell lines deficient in MTAP protein, all but one had complete or partial deletions . Partial or total deletions of the MTAP gene were found in primary T-cell acute lymphoblastic leukemias (T-ALL) . A deletion breakpoint of partial deletions found in cell lines and primary T-ALL was in intron 4 . Starting from the centromeric end, the gene order on chromosome 9p2l is p15, p16, MTAP, IFNA, and interferon beta gene (IFNB) . These results indicate that MTAP deficiency in cancer is primarily due to codeletion of the MTAP and p16 genes.

Proc Natl Acad Sci U S A, 1996 Jun 11, 93(12), 6048 - 52
Mutations in the rotated abdomen locus affect muscle development and reveal an intrinsic asymmetry in Drosophila; Martin-Blanco E et al.; In bilateral animals, the left and right sides of the body usually present asymmetric structures, the genetic bases of whose generation are still largely unknown {CIBA Foundation (1991) Biological Asymmetry and Handedness, CIBA Foundation Symposium 162 (Wiley, New York), pp . 1-327} . In Drosophila melanogaster, mutations in the rotated abdomen (rt) locus cause a clockwise helical rotation of the body . Even null alleles are viable but exhibit defects in embryonic muscle development, rotation of the whole larval body, and helical staggering of cuticular patterns in abdominal segments of the adult . rotated abdomen is expressed in the embryonic mesoderm and midgut but not in the ectoderm; it encodes a putative integral membrane glycoprotein (homologous to key yeast mannosyltransferases) . Mesodermal cells defective in O-glycosylation lead to an impaired larval muscular system . We propose that the staggering of the adult abdominal segments would be a consequence of the relaxation of intrinsic rotational torque of muscle architecture, preventing the colateral alignment of the segmental histoblast cells during their proliferation at metamorphosis.

Proc Natl Acad Sci U S A, 1996 Jun 11, 93(12), 6043 - 7
A specific protein carboxyl methylesterase that demethylates phosphoprotein phosphatase 2A in bovine brain; Lee J et al.; Phosphoprotein phosphatase 2A (PP2A) is one of the four major protein serine/threonine phosphatases found in all eukaryotic cells . We have shown that the 36-kDa catalytic subunit of PP2A is carboxyl methylated in eukaryotic cells, and we have previously identified and purified a novel methyltransferase (MTase) that is responsible for this modification . Here, we describe a novel protein carboxyl methyl-esterase (MEase) from bovine brain that demethylates PP2A . The enzyme has been purified to homogeneity as a monomeric 46-kDa soluble protein . The MEase is highly specific for PP2A . It does not catalyze the demethylation of other protein or peptide methylesters . Moreover, MEase activity is dramatically inhibited by nanomolar concentrations of okadaic acid, a specific inhibitor of PP2A . From these results, we conclude that PP2A methylation is controlled by two specific enzymes, a MTase and a MEase . Since PP2A methylation is highly conserved in eukaryotes ranging from human to yeast, it is likely that this system plays an important role in phosphatase regulation.

Hum Gene Ther, 1996 Jun 10, 7(9), 1103 - 9
Gene targeting to the centromeric DNA of a human minichromosome; Raimondi E et al.; A human supernumerary minichromosome (MC), previously identified as a derivative of chromosome 9, has been introduced into Chinese hamster ovary (CHO) cells by means of cell fusion . A hybrid clone containing the MC as the only free human chromosome was isolated . A selectable marker gene (neo) inserted into a yeast artificial chromosome (YAC) has been successfully targeted to the MC centromeric DNA via co-transfection with chromosome-9-specific alpha satellite DNA . In situ hybridization and Southern blotting experiments demonstrated that the intact neo gene was integrated into the MC centromeric DNA . Studies on the clonal distribution and on the stability of the MC either in the presence or in the absence of the selective agent have been carried out . The MC is susceptible to further manipulations and may thus represent a model for the construction of a large-capacity vector for somatic gene therapy.

J Biol Chem, 1996 Jun 7, 271(23), 13300 - 3
Identification of a novel syntaxin- and synaptobrevin/VAMP-binding protein, SNAP-23, expressed in non-neuronal tissues; Ravichandran V et al.; The specificity of vesicular transport is regulated, in part, by the interaction of a vesicle-associated membrane protein termed synaptobrevin/VAMP with a target compartment membrane protein termed syntaxin . These proteins, together with SNAP-25 (synaptosome-associated protein of 25 kDa), form a complex which serves as a binding site for the general membrane fusion machinery . Synaptobrevin/VAMP and syntaxin are ubiquitously expressed proteins and are believed to be involved in vesicular transport in most (if not all) cells . However, SNAP-25 is present almost exclusively in the brain, suggesting that a ubiquitously expressed homolog of SNAP-25 exists to facilitate transport vesicle/target membrane fusion in other tissues . Using the yeast two-hybrid system, we have identified a 23-kDa protein from human B lymphocytes (termed SNAP-23) that binds tightly to multiple syntaxins and synaptobrevins/VAMPs in vitro . SNAP-23 is 59% identical with SNAP-25 . Unlike SNAP-25, SNAP-23 was expressed in all tissues examined . These findings suggest that SNAP-23 is an essential component of the high affinity receptor for the general membrane fusion machinery and an important regulator of transport vesicle docking and fusion in all mammalian cells.

J Biol Chem, 1996 Jun 7, 271(23), 13304 - 7
Mouse p170 is a novel phosphatidylinositol 3-kinase containing a C2 domain; Virbasius JV et al.; Phosphatidylinositol (PI) 3-kinases catalyze the formation of 3'-phosphoinositides, which appear to promote cellular responses to growth factors and such membrane trafficking events as insulin-stimulated translocation of intracellular glucose transporters . We report here the cloning of a novel PI 3-kinase, p170, from cDNA of insulin-sensitive mouse 3T3-L1 adipocytes . Mouse p170 utilizes PI and to a limited extent PI 4-P as substrates, in contrast to the PI-specific yeast VPS34 homolog PtdIns 3-kinase and the p110 PI 3-kinases, which phosphorylate PI, PI 4-P, and PI 4,5-P2 . Mouse p170 is also distinct from PtdIns 3-kinase or the p110 PI 3-kinases in exhibiting a 10-fold lower sensitivity to wortmannin . Unique structural elements of p170 include C-terminal sequences strikingly similar to the phosphoinositide-binding C2 domain of protein kinase C isoforms, synaptotagmins, and other proteins . These features of mouse p170 are shared with a recently cloned Drosophila PI 3-kinase, DmPI3K_68D . Together, these proteins define a new class of PI 3-kinase likely influenced by cellular regulators distinct from those acting upon p110- or VPS34-like PI 3-kinases.

J Biol Chem, 1996 Jun 7, 271(23), 13448 - 53
Differential responsiveness of a splice variant of the human type I interferon receptor to interferons; Cook JR et al.; Chinese hamster ovary cells containing the yeast artificial chromosome F136C5 (alphaYAC) respond to all type I human interferons including IFN-alphaA, IFN-beta, and IFN-omega . The alphaYAC contains at least two genes encoding interferon-alpha receptor (IFN-alphaR) chains that are required for response to type I human interferons: Hu-IFN-alphaR1 and Hu-IFN-alphaR2 . We previously isolated a splice variant of the Hu-IFN-alphaR1 chain designated Hu-IFN-alphaR1s . Chinese hamster ovary cells containing a disrupted alphaYAC, which contains a deletion in the human IFNAR1 gene, were transfected with expression vectors for the Hu-IFN-alphaR1 and Hu-IFN-alphaR1s chains . With these cells, two type I interferons have been identified which can interact with the splice variant (Hu-IFN-alphaR1s) and with the Hu-IFN-alphaR1 chains: Hu-IFN-alphaA and IFN-omega . Two other type I interferons, Hu-IFN-alphaB2 and Hu-IFN-alphaF, are capable of signaling through the Hu-IFN-alphaR1 chain only and cannot utilize the splice variant Hu-IFN-alphaR1s . Hu-IFN-alphaR1 and Hu-IFN-alphaR1s differ in that the latter is missing a single subdomain of the receptor extracellular domain encoded by exons 4 and 5 of the IFNAR1 gene . These results therefore indicate that different type I interferons require different subdomains of the Hu-IFN-alphaR1 receptor chain, and that the splice variant chain (Hu-IFN-alphaR1s) is functional.

Biochim Biophys Acta, 1996 Jun 3, 1307(1), 31 - 4
Cloning of a cDNA encoding a human homologue of CDC47, a member of the MCM family; Kiyono T et al.; A complementary DNA (cDNA) clone encoding a 719-amino acid (aa) protein was isolated, which has a 49% aa identity with budding yeast CDC47, a member of the MCM family . Antiserum raised against a C-terminal polypeptide bacterially produced from the cDNA clone detected a cellular protein about 80 kDa, which coincides with the size of the in vitro transcription/translation product of the cDNA . These results indicate that the cDNA covers the entire coding region of a human homologue of CDC47.

J Ind Microbiol, 1996 Jun, 16(6), 348 - 50
Effect of inoculum level of xylitol production from rice straw hemicellulose hydrolysate by Candida guilliermondii; Roberto IC et al.; The effect of inoculum level on xylitol production by Candida guilliermondii was evaluated in a rice straw hemicellulose hydrolysate . High initial cell density did not show a positive effect in this bioconversion since increasing the initial cell density from 0.67 g L-1 to 2.41 g L-1 decreased both the rate of xylose utilization and xylitol accumulation . The maximum xylitol yield (0.71 g g-1) and volumetric productivity (0.56 g L-1 h-1) were reached with an inoculum level of 0.9 g L-1 . These results show that under appropriate inoculum conditions rice straw hemicellulose hydrolysate can be converted into xylitol by the yeast C . guilliermondii with efficiency values as high as 77% of the theoretical maximum.

Pharmacol Res, 1996 Jun, 33(6), 367 - 73
Oxaceprol, an atypical inhibitor of inflammation and joint damage; Ionac M et al.; Oxaceprol, an established therapeutic agent for osteoarthritis, had no effect on macrophage prostaglandin E2 release in vitro and inhibited carrageenan paw oedema at high doses (18-150 mg/kg p.o.) . In the same dose range, oxaceprol was comparable to indomethacin (3 mg/kg p.o.) as an inhibitor of yeast hyperalgaesia and at 6-50 mg/kg/day p.o . had a mild, variable inhibitory effect on cotton pellet granuloma formation . In adjuvant arthritic rats, oxaceprol (6-54 mg/kg/day p.o.) given therapeutically had no effect on the primary paw oedema response, but inhibited secondary lesions in the ears and tail . Histologically, oxaceprol markedly inhibited inflammatory cell infiltration and bone damage in the adjuvant-injected paw . In contrast to indomethacin, oxaceprol was more effective at inhibiting periarticular soft tissue inflammation but did not affect cartilage breakdown in this model . Oxaceprol has a clearly different spectrum of action to NSAIDs such as indomethacin and may act by inhibiting leucocyte infiltration and late connective tissue changes during inflammatory joint disease.

Comp Biochem Physiol A Physiol, 1996 Jun, 114(2), 175 - 87
Mechanical responses of chromium-deficient developing rat heart; Penefsky ZJ et al.; Mechanical responses were compared between controls, developing Sprague-Dawley rat papillary muscle and age-matched weanlings fed with Torula yeast, a food source deficient in chromium . At 8 weeks postnatal, deficient rats differed in significant ways from their normal counterparts . Deficient rats in contrast to controls weighed less, their interval-force (I-F) relationship was more negative and their inotropic response to high calcium concentrations was greater . At this time, however, deficient and control rats responded equally to alpha (phenylephrine) and beta (isoproterenol) agonists . At 10 weeks of age, the controls exhibited a less negative I-F and a negative inotropic response to high calcium concentrations while the response to alpha and beta agonists was unchanged . In contrast, at 10 weeks of age, the chromium-deficient rats exhibited a highly negative I-F response and significant inotropic response to high calcium concentrations . The response of the deficient hearts to beta-agonists diminished . At 13 weeks postnatal, control hearts showed only a 10-15% negative I-F response, a persistent response to catecholamines and negative inotropic responses to high calcium concentrations . In deficient hearts, the negative I-F response was reduced and the response to beta-agonists was further diminished but a positive inotropic response to phenylephrine and high calcium concentrations persisted . These observations in deficient animals are explained in terms of a retarded development of the calcium handling elements in the heart and a lack of an insulin-like growth factor.

Protein Eng, 1996 Jun, 9(6), 499 - 505
Effect of replacing helical glycine residues with alanines on reversible and irreversible stability and production of Aspergillus awamori glucoamylase; Chen HM et al.; To decrease irreversible thermoinactivation of Aspergillus awamori glucoamylase, five Gly residues causing helix flexibility were replaced with Ala residues . Mutation of Gly57 did not affect thermostability . Mutation of Gly137 doubled it at pHs 3.5 and 4.5 but barely changed it at pH 5.5 . The Gly139-->Ala mutation did not change thermostability at pH 3.5, improved it at pH 4.5 and worsened it at pH 5.5 . The Gly 137/Gly139-->Ala/Ala mutation gave 1.5-2-fold increased thermostabilities at pHs 3.5-5.5 . Mutations of Gly251 and Gly383 decreased it at all pHs . Gly137-->Ala and Gly137/Gly139-->Ala/Ala glucoamylases are the most stable yet produced by mutation . Guanidine treatment at pH 4.5 decreased the reversible stabilities of Gly137-->Ala, Gly139-->Ala and Gly137/Gly139-->Ala/Ala glucoamylases at infinite dilution while not changing those of Gly251-->Ala and Gly383-->Ala glucoamylases, which is, in general, opposite to what occurred with thermoinactivation . Mutation of Gly57 greatly improved the extracellular glucoamylase production by yeast, that of Gly137 barely affected it and those of Gly139 and of both Gly137 and Gly139 strongly impeded it . These observations suggest that alpha-helix rigidity can affect reversible and irreversible glucoamylase stability differently, that the effects of multiple mutations within one alpha-helix to improve stability are not always additive and that even single mutations can strongly affect extracellular enzyme production.

Immunopharmacology, 1996 Jun, 33(1-3), 217 - 21
Removal and restoration of epithelial chloride secretory activity of kinins by gene manipulation; Cuthbert A et al.; Kinins are known to stimulate electrogenic chloride secretion in many mammalian epithelia, including those of the airways and the alimentary tract . In this study the chloride secretory activity of lysylbradykinin (LBK) on murine colonic epithelium has been examined, specifically to discover the primary and final effector mechanisms in this process, i.e., which kinin receptors are involved and which chloride channels are responsible for chloride secretion . The approach used was to modify the mice genetically and assess the effects on kinin mediated chloride secretion using voltage clamping at zero potential . Briefly, LBK increased SCC in mouse colon by approximately 150 microA cm-2 with an EC50 of approximately 5 nM . In null CF mice LBK, 1 microM had no effect on chloride secretion, but reduced SCC due to K+ secretion . This effect is normally masked in wild-type tissues by dominant chloride secretion, but can be shown to occur to the same extent by measuring K+ secretion with radioisotopes . Null CF mice produce no cftr, but CFTR was introduced into CF mice by injecting a YAC containing the human CF gene into the pronucleus of CF zygotes . Colonic epithelia from mice with the incorporated YAC showed the same sensitivity to LBK as wild-type tissues and achieved the same maximal chloride secretory response . Colonic epithelia from mice in which the B2r gene had been disrupted showed no response to LBK at normally supramaximally effective concentrations, although responses to other secretagogues were normal . Similarly des-Arg-BK caused no acute chloride secretory response in colonic epithelia from B2 knockout mice, however small responses appeared if tissues were incubated in vitro for 3-6 h . It is concluded that cftr chloride channels and B2rs are required for electrogenic chloride secretion . Further CFTR can replace cftr with no effect on either the sensitivity or extent of chloride secretion . In vitro, colonic epithelia may generate B1rs which, upon activation, have a minor effect on chloride secretory activity.

Genome Res, 1996 Jun, 6(6), 504 - 14
Construction of a YAC contig encompassing the Usher syndrome type 1C and familial hyperinsulinism loci on chromosome 11p14-15.1; Ayyagari R et al.; The Usher syndrome type 1C (USH1C) and familial hyperinsulinism (HI) loci have been assigned to chromosome 11p14-15.1, within the interval D11S419-D11S1310 . We have constructed a yeast artificial chromosome (YAC) contig, extending from D11S926 to D11S899, which encompasses the critical regions for both USH1C and HI and spans an estimated genetic distance of approximately 4 cM . A minimal set of six YAC clones constitute the contig, with another 22 YACs confirming the order of sequence-tagged sites (STSs) and position of YACs on the contig . A total of 40 STSs, including 10 new STSs generated from YAC insert-end sequences and inter-Alu PCR products, were used to order the clones within the contig . This physical map provides a resource for identification of gene transcripts associated with USH1C, HI, and other genetic disorders that map to the D11S926-D11S899 interval.

Genome Res, 1996 Jun, 6(6), 478 - 91
Transcription mapping in a 700-kb region around the DXS52 locus in Xq28: isolation of six novel transcripts and a novel ATPase isoform (hPMCA5); Heiss NS et al.; The chromosomal band Xq28 has been a focus of interest in human genetics because > 20 hereditary diseases have been mapped to this region . However, about two-thirds of the disease genes remain uncloned . The region around the polymorphic DXS52 locus (ST14) within Xq28 lies in the candidate regions for several as-yet-uncloned disease genes . So far, only four melanoma antigen genes (MAGE) and the human biglycan (BGN) gene, have been mapped within the 700-kb stretch around DXS52, suggesting that more genes may reside in this region . By combining exon trapping and direct cDNA selection methods, we sought to identify novel transcripts around the DXS52 locus . In addition to recovering the MAGE and BGN genes, we isolated and mapped six putative novel genes (XAP103-XAP108), the caltractin gene, and a gene encoding a novel Ca(2+)-transporting ATPase isoform (hPMCA5) . The newly isolated sequences were considered as representing parts of putative genes if they contained at least one unique exon-trap product and/or at least one expressed sequence tag (EST) from sequence data bases and if, in addition, they showed evidence of expressed RT-OCT and/or Northern blot analysis . Our data facilitated the integration of the transcription map with the physical map around the DXS52 locus . Future analysis of the novel genes as candidates for Barth syndrome (BTHS) and chondrodysplasia punctata (CDPX2) is in progress.

Genes Chromosomes Cancer, 1996 Jun, 16(2), 94 - 105
Narrowing the critical region for a rhabdoid tumor locus in 22q11; Biegel JA et al.; Rhabdoid tumor is a rare malignant neoplasm of childhood that may occur in various locations, including the central nervous system and the kidney . Previous cytogenetic studies of primary rhabdoid tumors have demonstrated monosomy or deletion of chromosome 22 and have implicated the presence of a rhabdoid tumor suppressor gene that maps to 22q . We have employed fluorescence in situ hybridization to narrow the region for this locus in four rhabdoid tumor cell lines with translocations or deletions involving chromosome segment 22q11 . The completion of a cosmid and yeast artificial chromosome contig spanning the immunoglobulin lambda gene locus to BCR has allowed us to map a critical region for a rhabdoid tumor gene to a 500 kb span of chromosome segment 22q11.

Chem Biol, 1996 Jun, 3(6), 491 - 7
Cytochrome c folding triggered by electron transfer; Mines GA et al.; BACKGROUND: Experimental and theoretical studies of protein folding suggest that the free-energy change associated with the folding process is a primary factor in determining folding rates . We have recently developed a photochemical electron-transfer-triggering method to study protein-folding kinetics over a wide range of folding free energies . Here, we have used this technique to investigate the relationship between folding rate and free-energy change using cytochromes c from horse (h-cyt c) and yeast (y-cyt c), which have similar backbone folds but different amino-acid sequences and, consequently, distinct folding energies . RESULTS: The folding free energies for oxidized and reduced h-cyt c and y-cyt c are linear functions of the denaturant (guanidine hydrochloride) concentration, but the concentration required to unfold half of the protein is 1.5 M lower for y-cyt c . We measured the folding rates of reduced h-cyt c and y-cyt c over a range of guanidine hydrochloride concentrations at two temperatures . When driving forces are matched at the appropriate denaturant concentrations, the two homologs have comparable folding rates . The activation free energies for folding h-cyt c and y-cyt c are linearly dependent on the folding free energies . The slopes of these lines are similar (approximately 0.4) for the two proteins, suggesting an early transition state along the folding reaction coordinate . CONCLUSIONS: The free-energy relationships found for h-cyt c and y-cyt c folding kinetics imply that the height of the barrier to folding depends upon the relative stabilities of the unfolded and folded states . The striking correspondence in rate/free-energy profiles for h-cyt c and y-cyt c suggests that, despite low sequence homology, they follow similar folding pathways.

Curr Opin Struct Biol, 1996 Jun, 6(3), 317 - 21
Hammering away at RNA global structure; Hagerman PJ et al.; A major goal of the study of RNA tertiary structure is an understanding of the rules relating sequence and global conformation . This goal has been furthered during the past year for two important structural elements: yeast tRNAPhe and the self-cleaving hammerhead RNA . In both cases, a combination of solution and crystallographic studies has yielded strongly concordant views of their global conformations.

J Med Vet Mycol, 1996 Jun-Jul, 34(3), 175 - 80
Differential expression of the 45 kDa protein (actin) during the dimorphic transition of Sporothrix schenckii; Han-Yaku H et al.; We investigated the gene expression of a protein during the dimorphic transition from yeast to mycelial form of Sporothrix schenckii . Yeast cells were converted to mycelial cells in Sabouraud glucose broth at 25 degrees C . After 1, 3 and 5 days of culture, the intermediate form of cells between yeast and mycelium was obtained, and after 7 days these cells were morphologically similar to the mycelial cells . Proteins having the molecular weight of 45 kDa were found to by synthesized preferentially by intermediate form of day 7 and mycelial cells . On the other hand, the 45 kDa proteins were predominantly translated by the RNA isolated from the intermediate of day 7 and mycelial cells using in vitro cell-free translation assay . The 45 kDa proteins synthesized by mycelial cells were found to be identical with the 45 kDa translation products directed by the mRNA isolated from the intermediate and mycelial cells by V8 protease peptide mapping . The 45 kDa protein was considered to be actin by Western blot analysis using an anti-actin monoclonal antibody . These results suggest that the intermediate form of day 7 has the same phenotypes in the morphology and biosynthesis of actin as those of mycelial cells . The expression of the actin gene may be involved in the dimorphic transition of S . schenckii.

Trends Microbiol, 1996 Jun, 4(6), 246 - 51
Role of cell-surface molecules of Blastomyces dermatitidis in host-pathogen interactions; Klein BS et al.; The fungal pathogen Blastomyces dermatitidis produces an adhesin (WI-1) in yeast stages, which contains repetitive regions that bind host-cell receptors . Adhesin and glucan may modulate fungal interactions with macrophages; their level of expression is altered in hypovirulent mutants . Adhesin is also involved in immune responses, and may be important in eliciting the clearance of the fungus.

Curr Opin Immunol, 1996 Jun, 8(3), 412 - 8
Phosphatidylinositol 3-kinase related kinases; Abraham RT; Studies in yeast, files and mammalian cells have uncovered a novel family of signal-transducing kinases which bear an evolutionary relationship to phosphatidylinositol 3-kinase . These phosphatidylinositol 3-kinase related enzymes play critical roles in DNA repair, V(D)J recombination and cell-cycle checkpoints, and their dysfunction leads to clinical manifestations ranging from immunodeficiency to cancer.

Curr Biol, 1996 Jun 1, 6(6), 651 - 4
Cell division: why daughters cannot be like their mothers; Chang F et al.; A cell-fate determinant that segregates asymmetrically at cell division has been identified in budding yeast . Possible mechanisms for this asymmetric segregation are suggested by the identification of mutants in genes encoding cortically localized proteins.

Mol Endocrinol, 1996 Jun, 10(6), 631 - 41
Evidence for the direct interaction of the insulin-like growth factor I receptor with IRS-1, Shc, and Grb10; Dey BR et al.; We have used the yeast two-hybrid system to study the interaction between the IGF-I receptor and two putative substrates, IRS-1 and Shc . In addition, we have identified Grb10 as a protein that binds to the insulin-like growth factor I (IGF-I) receptor . This two-hybrid system (the interaction trap) utilizes a hybrid protein containing the LexA DNA-binding domain fused to the intracellular portion of the IGF-I receptor (LexA-IGFIR beta) and hybrids containing an activation domain fused to either IRS-1 (Ad-IRS-1), Shc (Ad-Shc), or a cDNA library . A positive interaction of LexA-IGFIR beta with the activation domain hybrid results in activation of reporter genes, LacZ and LEU2, in the yeast . Western blotting of extracts from transformed yeast demonstrated that the LexA-IGFIR beta fusion protein was expressed and phosphorylated on tyrosine residues . Coexpression of LexA-IGFIR beta with Ad-IRS-1 resulted in strong activation of both reporter genes; activation did not occur with a kinase-negative receptor mutant . IRS-1 residues 160-516 were sufficient for this strong interaction . Coexpression of LexA-IGFIR beta with Ad-Shc also resulted in strong activation of both LacZ and LEU2 reporter genes . This interaction was also dependent upon a tyrosine kinase-active receptor and required tyrosine 950 in the juxtamembrane region of the receptor . An N-terminal fragment of Shc (amino acids 1-232) interacted almost as strongly as full-length Shc whereas the Shc SH2 domain only activated the more sensitive LEU2 reporter . Full-length Shc was phosphorylated on tyrosine when coexpressed with IGFIR beta but not when coexpressed with the kinase-negative receptor mutant . To identify additional proteins that interact with the IGFIRs, a human fetal brain cDNA library was screened using the interaction trap system . This analysis identified partial cDNAs for Grb10 . Coexpression of LexA-IGFIR beta with Ad-Grb10 resulted in strong activation of both LacZ and LEU2 reporter genes; this interaction was dependent upon a tyrosine kinase-active receptor but did not require tyrosine 950.

Hum Mol Genet, 1996 Jun, 5(6), 827 - 33
Identification of a gene disrupted by a microdeletion in a patient with X-linked retinitis pigmentosa (XLRP); Roepman R et al.; The gene for the most frequent from of X-linked retinitis pigmentosa (XLRP), RP3, has been assigned by genetic and physical mapping to a segment of less than 1000 kbp, which is flanked by the marker DXS1110 and the ornithine transcarbamylase (OTC) gene . In search of microdeletions, we have screened the DNA of 30 unrelated patients with XLRP by employing a representative set of YAC-derived DNA fragments that were generated by restriction enzyme digestion and PCR amplification . In one of these patients, a 6.4 kbp microdeletion was detected which was not present in the DNA of 444 male controls . A cosmid contig spanning the deletion was constructed and used to isolate cDNAs from retina-specific libraries . Exons corresponding to these expressed sequences as well as other putative exons were identified by sequencing more than 30 kbp of the critical region . So far, no point mutations in these putative exon sequences have been identified.

Recenti Prog Med, 1996 Jun, 87(6), 271 - 4
Vaccination against hepatitis B virus in children and adolescents in a pediatric hospital; Catania G et al.; Vaccination against hepatitis B has become compulsory in Italy and is routinely administered in outpatient clinics of Local Health Service . In the setting of a pediatric hospital 205 children (less than 10-year-old) and 144 adolescents (10-19-year-old) have been vaccinated with anti-HBV recombinant yeast-derived vaccine (Engerix B 10 U.I . at 0.1 and 6 months in children and 20 U.I . in adolescents) . Anti-HBs titers were evaluated 1 month after least dose . Geometric mean titer (GMT) was 9500 mU/I in children and 18,000 in adolescents; 99.85% of subjects had a protective anti-HBs titer (10 mU/I or more) at one month after the third dose and 89.1% had titers of 1000 mU/I or more . Only 9.7% of subjects had adverse events, mainly (75%) at the injection site, anyway of trivial importance, without any difference of sex and age . Anti-hepatitis B yeast-derived vaccine is highly immunogenic, without relevant adverse events; the hospital setting is appropriate to participate to the program of vaccination in pediatric age.

Am J Physiol, 1996 Jun, 270(6 Pt 1), C1647 - 55
Is gamma-actin a regulator of amino acid transport?
Lin G, McCormick JI, Johnstone RM.
A mutated yeast cell line incapable of growth in minimal medium with proline as the sole nitrogen source was restored to normal growth by transfection with a cDNA from mouse Ehrlich cells . The cloned cDNA (E51) was found to be 90% homologous to gamma-actin . Immediately after transfection with E51 cDNA, both alpha-aminoisobutyric acid (AIB) and proline uptake in the mutated yeast were increased, particularly at pH 5 . The expression of the same E51 cDNA also enhanced amino acid uptake in Xenopus laevis oocytes after injection into the Xenopus nuclei . A mutated mammalian lymphocyte cell line (GF-17), deficient in system A transport, also showed increased Na(+)-dependent transport after transfection with E51 cDNA . Whereas the mock transfected GF-17 cells failed to grow in the selection medium, the transfectants with E51 cDNA grew better than the untransfected cells . The data are consistent with the conclusion that expression of E51 cDNA can modify inactive, endogenous amino acid transporters, permitting substantial amino acid uptake in cells deficient in amino acid transporter(s) and permitting rapid cell growth . The data suggest that the gamma-actin-like protein coded for by E51 cDNA may play a significant regulatory role in amino acid transport.

J Cell Biochem, 1996 Jun 1, 61(3), 459 - 66
Mammalian CAP interacts with CAP, CAP2, and actin; Hubberstey A et al.; We previously identified human CAP, a homolog of the yeast adenylyl cyclase-associated protein . Previous studies suggest that the N-terminal and C-terminal domains of CAP have distinct functions . We have explored the interactions of human CAP with various proteins . First, by performing yeast two-hybrid screens, we have identified peptides from several proteins that interact with the C-terminal and/or the N-terminal domains of human CAP . These peptides include regions derived from CAP and BAT3, a protein with unknown function . We have further shown that MBP fusions with these peptides can associate in vitro with the N-terminal or C-terminal domains of CAP fused to GST . Our observations indicate that CAP contains regions in both the N-terminal and C-terminal domains that are capable of interacting with each other or with themselves . Furthermore, we found that myc-epitope-tagged CAP coimmunoprecipitates with HA-epitope-tagged CAP from either yeast or mammalian cell extracts . Similar results demonstrate that human CAP can also interact with human CAP2 . We also show that human CAP interacts with actin, both by the yeast two-hybrid test and by coimmunoprecipitation of epitope-tagged CAP from yeast or mammalian cell extracts . This interaction requires the C-terminal domain of CAP, but not the N-terminal domain . Thus CAP appears to be capable of interacting in vivo with other CAP molecules, CAP2, and actin . We also show that actin co-immunoprecipitates with HA-CAP2 from mammalian cell extracts.

Curr Opin Cell Biol, 1996 Jun, 8(3), 331 - 9
Biochemistry and regulation of pre-mRNA splicing; Adams MD et al.; During the past year, significant advances have been made in the field of pre-mRNA splicing . It is now clear that members of the serine-arginine-rich protein family are key players in exon definition and function at multiple steps in the spliceosome cycle . Novel findings have been made concerning the role of exon sequences, which function as both constitutive and regulated enhancers of splicing, in trans-splicing and as targets for tissue-specific control of splicing patterns . By combining biochemical approaches in human and yeast extracts with genetic analysis, much has been learned about the RNA-RNA and RNA-protein interactions that are necessary to assemble the various complexes that are found along the pathway to the catalytically active spliceosome.

Biochem J, 1996 Jun 1, 316 ( Pt 2), 681 - 4
An H(+)-ATPase regulates cytoplasmic pH in Pneumocystis carinii trophozoites; Docampo R et al.; Pneumocystis carinii is an opportunistic fungus which causes interstitial pneumonia in patients with acquired immunodeficiency syndrome (AIDS) . Cytoplasmic pH (pHi) regulation in short-term-cultured P . carinii trophozoites was studied using the fluorescent dye 2',7'-bis-(2-carboxyethyl)-5-(-6)-carboxyfluorescein . With an extracellular pH of 7.4, the mean baseline pHi of P . carinii trophozoites was 7.40 +/- 0.10 (n = 8) . This steady-state pHi was not significantly affected in the absence of extracellular Na+ or K+ . Moreover, steady-state pHi was maintained in the nominal absence of HCO3- and was not affected by the Cl-/HCO(3-)-exchanger inhibitor 4, 4'-di-isothiocyanato-dihydrostilbene-2, 2'-disulphonic acid (100 microM), or the Na+/H(+)-exchanger inhibitor N-ethyl-N-isopropylamiloride (100 microM) . In contrast, the general inhibitors of ATPases, N-ethylmaleimide (1 mM), and dicyclohexylcarbodi-imide (100 microM), and the inhibitor of yeast H(+)-ATPase, diethylstilbestrol (12.5-100 microM), decreased pHi, while the K+/H(+)-ATPase inhibitor omeprazole (50-400 microM), and the vacuolar-type H(+)-ATPase inhibitor bafilomycin A1 (1-5 microM) only produced a dose-dependent acidification of the cells when used at high concentrations . In addition, steady-state pHi depended on the availability of cellular ATP, since it was decreased by the ATP synthase inhibitors oligomycin (1 microgram/ml) and sodium azide (1 mM), and by the uncoupler of oxidative phosphorylation carbonyl cyanide p-trifluorophenylhydrazone (1 microM), agents that were able to deplete significantly the intracellular ATP levels . Taken together, these results are consistent with an important role of an H(+)-ATPase similar to those found in other fungi in the regulation of pHi homoeostasis in P . carinii trophozoites.

J Cell Biol, 1996 Jun, 133(6), 1293 - 305
Actin organization, bristle morphology, and viability are affected by actin capping protein mutations in Drosophila; Hopmann R et al.; Regulation of actin filament length and orientation is important in many actin-based cellular processes . This regulation is postulated to occur through the action of actin-binding proteins . Many actin-binding proteins that modify actin in vitro have been identified, but in many cases, it is not known if this activity is physiologically relevant . Capping protein (CP) is an actin-binding protein that has been demonstrated to control filament length in vitro by binding to the barbed ends and preventing the addition or loss of actin monomers . To examine the in vivo role of CP, we have performed a molecular and genetic characterization of the beta subunit of capping protein from Drosophila melanogaster . We have identified mutations in the Drosophila beta subunit-these are the first CP mutations in a multicellular organism, and unlike CP mutations in yeast, they are lethal, causing death during the early larval stage . Adult files that are heterozygous for a pair of weak alleles have a defect in bristle morphology that is correlated to disorganized actin bundles in developing bristles . Our data demonstrate that CP has an essential function during development, and further suggest that CP is required to regulate actin assembly during the development of specialized structures that depend on actin for their morphology.

FEMS Microbiol Lett, 1996 Jun 1, 139(2-3), 223 - 28
Characterisation of the Aspergillus nidulans frA1 mutant: hexose phosphorylation and apparent lack of involvement of hexokinase in glucose repression; Ruijter GJ et al.; Hexose phosphorylation was studied in Aspergillus nidulans wild-type and in a fructose non-utilising mutant (frA) . The data indicate the presence of at least one hexokinase and one glucokinase in wild-type A . nidulans, while the frA1 mutant lacks hexokinase activity . The A . nidulans gene encoding hexokinase was isolated by complementation of the frA1 mutation . The absence of hexokinase activity in the frA1 mutant did not interfere with glucose repression of the enzymes involved in alcohol and L-arabinose catabolism . This suggest that, unlike the situation in yeast where mutation of hexokinase PII abolishes glucose repression, the A . nidulans hexokinase might not be involved in glucose repression.

Nucleic Acids Res, 1996 Jun 1, 24(11), 1981 - 6
The transcription factors Sp1 and Oct-1 interact physically to regulate human U2 snRNA gene expression; Strom AC et al.; The expression of human small nuclear U2 RNA genes is controlled by the proximal sequence element (PSE), which determines the start site of transcription, and a distal sequence element (DSE) . The DSE contains an octamer element and three Sp1 binding sites . The octamer, like the PSE, is essential for U2 transcription . The Sp1 sites contribute to full promoter activity by distance-dependent cooperative interactions with the transcription factors Sp1 and Oct-1 . Here we show that purified recombinant Sp1 and Oct-1 bind cooperatively to the DSE and that they physically interact in vitro . Furthermore, we show that Sp1 and Oct-1 interact in vivo using a yeast two-hybrid system . The domain of Sp1 which interacts with Oct-1 is confined to the region necessary for transcriptional stimulation of U2 RNA transcription . This region contains the glutamine-rich activation domain B and a serine/threonine-rich part . The results demonstrate that Sp1, in addition to binding to a number of other factors, also interacts directly with transcription factor Oct-1.

J Pharmacol Exp Ther, 1996 Jun, 277(3), 1659 - 64
The biotransformation of clomipramine in vitro, identification of the cytochrome P450s responsible for the separate metabolic pathways; Nielsen KK et al.; The aim of the study was to identify the cytochrome P450s (CYPs) that catalyze the biotransformation of clomipramine in vitro . A high-performance liquid chromatography method was developed to assay N-desmethylclomipramine, 8-hydroxyclomipramine, 2-hydroxyclomipramine, 8-hydroxydesmethhylclomipramine, didesmethylclomipramine and 2-hydroxydesmethylclomipramine formed by microsomes prepared from human liver and yeast expressing human CYP1A1, 1A2, 2C8, 2C9, 2C18, 2C19, 2D6 and 3A4 . There was a statistically significant correlation between the formation rate of desmethylclomipramine and the immunoquantified concentration of CYP3A4 in 12 human liver microsome preparations (rs = 0.664, P = .028) . Ketoconazole was a very potent inhibitor of desmethylclomipramine formation (Ki = 0.054 microM) and microsomes from yeast expressing CYP3A4 were also active in forming the metabolite (formation rate: 25.6 nmol/nmol of CYP per hr) . Thus, the results are consistent with the assumption that the N-demethylation of clomipramine is catalyzed by CYP3A4 . As expected from in vivo panel studies, CYP2C19 in yeast was also very active in the N-demethylation (formation rate, 145 nmol/nmol of CYP per hr) . Fluvoxamine was a potent inhibitor of desmethylclomipramine formation (Ki, 0.15 microM), suggesting that CYP1A2 is a third CYP involved in the N-demethylation . CYP2D6 in yeast microsomes catalyzed the 8-hydroxylation of clomipramine and desmethylclomipramine (formation rates, 65 and 75 nmol/nmol of CYP per hr) and quinidine was a very potent inhibitor (Ki, 0.10 and 0.16 microM) . Both results confirm that CYP2D6 catalyzes the 8-hydroxylation in agreement with the results obtained in previous in vivo studies . Besides quinidine, paroxetine, fluoxetine and norfluoxetine, all were potent inhibitors of the 8-hydroxylations (Ki, 0.24-1.5 microM) and sertraline was a less potent inhibitor (Ki, 16 and 27 microM, respectively).

Gene, 1996 Jun 1, 171(2), 221 - 3
Drosophila melanogaster P1 genomic clone DS05563 contains the chaperonin-encoding gene Cctg; Walkley NA et al.; We report the sequence analysis of a Drosophila melanogaster (Dm) P1 genomic clone (DS05563) which contains the gamma-chaperonin-encoding gene, Cctg . The (Hs) Cctg orthologue was found to share strong sequence identity with a 1603-bp region of DS05563, suggesting that Dm Cctg is located within this region . Detailed analysis has shown that Dm Cctg comprises four exons and is interrupted by three introns of 55, 85 and 66 bp . Dm Cctg encodes a predicted peptide of 545 amino acids (aa) (approx . 60 kDa) . The predicted Dm CCT gamma aa sequence shares a high degree of sequence identity with gamma-orthologues from human (70%), mouse (70%), protozoa (60%) and yeast (60%), and also contains domains found in other chaperonins including bacterial GroEL, mitochondrial Hsp60 and plant Rubisco large subunit-binding protein . These data support the conclusion that the DS05563 clone contains the Dm Cctg gene.

Chromosoma, 1996 Jun, 104(8), 537 - 44
Region-specific YAC banding and painting probes for comparative genome mapping: implications for the evolution of human chromosome 2; Haaf T et al.; To date, several hundred nonchimeric yeast artificial chromosomes (YACs) from the Centre d'Etude du Polymorphisme Humain containing polymorphic sequence-tagged sites have been mapped by fluoresence in situ hybridization (FISH) on human metaphase chromosomes . Because they carry an average of 1 Mb of human genomic DNA, CEPH YACs generate high-intensity in situ hybridization signals . The available set of cytogenetically and genetically anchored YACs, approximately one every 5-10 cM evenly spaced over almost the entire human genome, provides complex region-specific probes for molecular cytogenetics . YAC probes can be adapted with unlimited flexibility to specific FISH applications such as the study of chromosomal evolution . We have generated representational probes for YAC banding and painting of human chromosome 2 and its great ape homologs . Convergent inversions were found in the pericentric region of the gorilla and orangutan homologs of chromosome 2p.

Mamm Genome, 1996 Jun, 7(6), 454 - 8
Co-localization of the ketohexokinase and glucokinase regulator genes to a 500-kb region of chromosome 2p23; Hayward BE et al.; The glucokinase regulator (GCKR) is a 65-kDa protein that inhibits glucokinase (hexokinase IV) in liver and pancreatic islet . The role of glucokinase (GCK) as pancreatic beta cell glucose sensor and the finding of GCK mutations in maturity onset diabetes of the young (MODY) suggest GCKR as a further candidate gene for type 2 diabetes . The inhibition of GCK by GCKR is relieved by the binding of fructose-1-phosphate (F-1-P) to GCKR . F-1-P is the end product of ketohexokinase (KHK, fructokinase), which, like GCK and GCKR, is present in both liver and pancreatic islet . KHK is the first enzyme of the specialized pathway that catabolizes dietary fructose . We have isolated genomic clones containing the human GCKR and KHK genes . By fluorescent in situ hybridization (FISH), KHK maps to Chromosome (Chr) 2p23.2-23.3, a new assignment corroborated by somatic cell hybrid analysis . The localization of GCKR, originally reported by others as 2p22.3, has been reassessed by high-resolution FISH, indicating that, like KHK, GCKR maps to 2p23.2-23.3 . The proximity of GCKR and KHK was further demonstrated both by two-color interphase FISH, which suggests that the two genes lie within 500 kb of each other, and by analysis of overlapping YAC and P1 clones spanning the interval between GCKR and KHK . A new microsatellite polymorphism was used to place the GCKR-KHK locus between D2S305 and D2S165 on the genetic map . The colocalization of these two metabolically connected genes has implications for the interpretation of linkage or allele association studies in type 2 diabetes . It also raises the possibility of coordinate regulation of GCKR and KHK by common cis-acting regulatory elements.

Genomics, 1996 Jun 1, 34(2), 236 - 40
A YAC contig of the human CC chemokine genes clustered on chromosome 17q11.2; Naruse K et al.; CC chemokines are cytokines that attract and activate leukocytes . The human genes for the CC chemokines are clustered on chromosome 17 . To elucidate the genomic organization of the CC chemokine genes, we constructed a YAC contig comprising 34 clones . The contig was shown to contain all 10 CC chemokine genes reported so far, except for one gene whose nucleotide sequence is not available . The contig also contains 4 CC chemokine-like genes, which were deposited in GenBank as ESTs and are here referred to as NCC-1, NCC-2, NCC-3, and NCC-4 . Within the contig, the CC chemokine genes were localized in two regions . In addition, the CC chemokine genes were more precisely mapped on chromosome 17q11.2 using a somatic cell hybrid cell DNA panel containing various portions of human chromosome 17 . Interestingly, a reciprocal translocation t(Y;17) breakpoint, contained in the hybrid cell line Y1741, lay between the two chromosome 17 chemokine gene regions covered by our YAC contig . From these results, the order and the orientation of CC chemokine genes on chromosome 17 were determined as follows: centromere-neurofibromatosis 1-(MCP-3, MCP-1, NCC-1, I-309)-Y1741 breakpoint-RANTES-(LD78gamma, AT744.2, LD78beta)-(NCC-3, NCC-2, AT744.1, LD78alpha)-NCC-4-retinoic acid receptor alpha- telomere.

Virology, 1996 Jun 1, 220(1), 208 - 12
Evidence for direct association of Vpr and matrix protein p17 within the HIV-1 virion; Sato A et al.; Vpr is one of the auxiliary proteins of HIV-1 and is selectively incorporated into the virion by a process involving the C-terminal p6 portion of the Gag precursor Pr55 . Vpr and the matrix protein p17 are the components of the viral preintegration complex and appear to play important roles in the nuclear transport of proviral DNA in nondividing cells . In the present study, we have demonstrated by coimmunoprecipitation experiments that Vpr associates with matrix protein p17 but not with capsid protein p24 within the HIV-1 virion . Experiments employing the yeast two-hybrid GAL4 assay for protein-protein interactions also demonstrated a direct association between Vpr and the C-terminal region of matrix protein p17 . Association of Vpr and the matrix protein p17 within the mature virion is consistent with their collaborative role in the nuclear transportation of the viral preintegration complex in nondividing cells such as macrophages.

J Bacteriol, 1996 Jun, 178(12), 3480 - 5
Gliding motility in slide cultures of Myxococcus xanthus in stable and steep chemical gradients; Tieman S et al.; A method was devised to construct stable and steep chemical gradients in slide cultures to study the movements of gliding cells . The movement of Myxococcus xanthus individual cells and small swarms was studied in these gradients . There was no response to gradients of Casitone and yeast extract that were previously reported to stimulate a positive chemotactic response with M . xanthus.

Biopolymers, 1996 Jun, 38(6), 769 - 79
RNA loop structure prediction via bond scaling and relaxation; Frederic T et al.; We have developed a method for predicting the structure of small RNA loops that can be used to augment already existing RNA modeling techniques . The method requires no input constraints on loop configuration other than end-to-end distance . Initial loop structures are generated by randomizing the torsion angles, beginning at one end of the polynucleotide chain and correlating each successive angle with the previous . The bond lengths of these structures are then scaled to fit within the known end constraints and the equilibrium bond lengths of the potential energy function are scaled accordingly . Through a series of rescaling and minimization steps the structures are allowed to relax to lower energy configurations with standard bond lengths and reduced van der Waals clashes . This algorithm has been tested on the variable loops of yeast tRNA-Asp and yeast tRNA-Phe, as well as the sarcin-ricin tetraloop and the anticodon loop of yeast tRNA-Phe . The results indicate good correlation between potential energy and the loop structure predictions that are closest to the variable loop crystal structures, but poorer correlation for the more isolated stem loops . The number of stacking interactions has proven to be a good objective measure of the best loop predictions . Selecting on the basis of energy and stacking, we obtain two structures with 0.65 and 0.75 A all-atom rms deviations (RMSD) from the crystal structure for the tRNA-Asp variable loop . The best structure prediction for the tRNA-Phe variable loop has an all-atom RMSD of 2.2 A and a backbone RMSD of 1.6 A, with a single base responsible for most of the deviation . For the sarcin-ricin loop from 28S ribosomal RNA, the predicted structure's all-atom RMSD from the nmr structure is 1.0 A . We obtain a 1.8 A RMSD structure for the tRNA-Phe anticodon loop.

Am J Hum Genet, 1996 Jun, 58(6), 1231 - 8
Molecular analysis of recombination in a family with Duchenne muscular dystrophy and a large pericentric X chromosome inversion; Shashi V et al.; It has been demonstrated in animal studies that, in animals heterozygous for pericentric chromosomal inversions, loop formation is greatly reduced during meiosis . This results in absence of recombination within the inverted segment, with recombination seen only outside the inversion . A recent study in yeast has shown that telomeres, rather than centromeres, lead in chromosome movement just prior to meiosis and may be involved in promoting recombination . We studied by cytogenetic analysis and DNA polymorphisms the nature of meiotic recombination in a three-generation family with a large pericentric X chromosome inversion, inv(X)(p21.1q26), in which Duchenne muscular dystrophy (DMD) was cosegregating with the inversion . On DNA analysis there was no evidence of meiotic recombination between the inverted and normal X chromosomes in the inverted segment . Recombination was seen at the telomeric regions, Xp22 and Xq27-28 . No deletion or point mutation was found on analysis of the DMD gene . On the basis of the FISH results, we believe that the X inversion is the mutation responsible for DMD in this family . Our results indicate that (1) pericentric X chromosome inversions result in reduction of recombination between the normal and inverted X chromosomes; (2) meiotic X chromosome pairing in these individuals is likely initiated at the telomeres; and (3) in this family DMD is caused by the pericentric inversion.

Am J Hum Genet, 1996 Jun, 58(6), 1223 - 30
Recombination hot spot in a 3.2-kb region of the Charcot-Marie-Tooth type 1A repeat sequences: new tools for molecular diagnosis of hereditary neuropathy with liability to pressure palsies and of Charcot-Marie-Tooth type 1A . French CMT Collaborative Research Group; Lopes J et al.; Charcot-Marie-Tooth type 1A (CMT1A) disease and hereditary neuropathy with liability to pressure palsies (HNPP) are autosomal dominant neuropathies, associated, respectively, with duplications and deletions of the same 1.5-Mb region on 17p11.2-p12 . These two rearrangements are the reciprocal products of an unequal meiotic crossover between the two chromosome 17 homologues, caused by the misalignment of the CMT1A repeat sequences (CMT1A-REPs), the homologous sequences flanking the 1.5-Mb CMT1A/HNPP monomer unit . In order to map recombination breakpoints within the CMT1A-REPs, a 12.9-kb restriction map was constructed from cloned EcoRI fragments of the proximal and distal CMT1A-REPs . Only 3 of the 17 tested restriction sites were present in the proximal CMT1A-REP but absent in the distal CMT1A-REP, indicating a high degree of homology between these sequences . The rearrangements were mapped in four regions of the CMT1A-REPs by analysis of 76 CMT1A index cases and 38 HNPP patients, who where unrelated . A hot spot of crossover breakpoints, located in a 3.2-kb region, accounted for three-quarters of the rearrangements, detected after EcoRI/SacI digestion, by the presence of 3.2-kb and 7.8-kb junction fragments in CMT1A and HNPP patients, respectively . These junction fragments, which can be detected on classical Southern blots, permit molecular diagnosis . Other rearrangements can also be detected by gene dosage on the same Southern blots.

Mol Cell Biol, 1996 Jun, 16(6), 3106 - 11
Sex-specific and non-sex-specific oligomerization domains in both of the doublesex transcription factors from Drosophila melanogaster; An W et al.; The doublesex gene of Drosophila melanogaster encodes the alternatively spliced, sex-specific transcription factors DSXM and DSXF . These factors regulate male- and female-specific transcription of many genes . For example, female-specific transcription of the yolk protein 1 gene is regulated by DSXM repression in males and DSXF activation in females . In this study we used in vitro interaction assays and the in vivo yeast two-hybrid method to identify and examine oligomerization domains of the DSX proteins . A 66-amino-acid segment common to both proteins (amino acids 39 to 104) contains a sequence-specific DNA binding domain and an oligomerization domain (OD1) . The OD1 domain oligomerizes up to at least a pentamer, but only dimers bound to a palindromic regulatory site in the yolk protein 1 gene are detected . Both subunits of the OD1 dimer are in contact with DNA . Another segment of each protein (amino acids 350 to 412 for DSXF and 350 to 427 for DSXM) contains a second oligomerization domain (OD2F and OD2M, respectively) . The OD2 domains have both sex-specific and non-sex-specific sequences which are necessary for oligomerization . On the basis of sequence analysis, we predict that OD2 oligomerizes through coiled-coil interactions . We speculate that the common function of OD1 and OD2 is to oligomerize the full-length proteins, whereas their specialized functions are to form a dimeric DNA binding unit and a sex-specific transcriptional activation or repression unit.

Hum Genet, 1996 Jun, 97(6), 784 - 93
Routine screening for microdeletions by FISH in 77 patients suspected of having Prader-Willi or Angelman syndromes using YAC clone 273A2 (D15S10); Erdel M et al.; About 70% of patients with Prader-Willi syndrome (PWS) and Angelman syndrome (AS) have a common interstitial de novo microdeletion encompassing paternal (PWS) or maternal (AS) loci D15S9 to D15S12 . Most of the non-deletion PWS patients and a small number of non-deletion AS patients have a maternal or paternal uniparental disomy (UPD) 15, respectively . Other chromosome 15 rearrangements and a few smaller atypical deletions, some of the latter being associated with an abnormal methylation pattern, are rarely found . Molecular and fluorescence in situ hybridization (FISH) analysis have both been used to diagnose PWS and AS . Here, we have evaluated, in a typical routine cytogenetic laboratory setting, the efficiency of a diagnostic strategy that starts with a FISH deletion assay using Alu-PCR (polymerase chain reaction)-amplified D15S10-positive yeast artificial chromosome (YAC) 273A2 . We performed FISH in 77 patients suspected of having PWS (n = 66) or AS (n = 11) and compared the results with those from classical cytogenetics and wherever possible with those from DNA analysis . A FISH deletion was found in 16/66 patients from the PWS group and in 3/11 patients from the AS group . One example of a centromere 15 co-hybridization performed in order to exclude cryptic translocations or inversions is given . Of the PWS patients, 14 fulfilled Holm's criteria, but two did not . DNA analysis confirmed the common deletion in all patients screened by the D15S63 methylation test and in restriction fragment length polymorphism dosage blots . In 3/58 non-deletion patients, other chromosomal aberrations were found . Of the non-deleted group, 27 subjects (24 PWS, 3 AS) were tested molecularly, and three patients with an uniparental methylation pattern were found in the PWS group . The other 24/27 subjects had neither a FISH deletion nor uniparental methylation, but two had other cytogenetic aberrations . Given that cytogenetic analysis is indispensable in most patients, we find that the FISH deletion assay with YAC 273A2 is an efficient first step for stepwise diagnostic testing and mutation-type analysis of patients suspected of having PWS or AS.

Hum Genet, 1996 Jun, 97(6), 770 - 6
Detection of a germline mutation and somatic homozygous loss of the von Hippel-Lindau tumor-suppressor gene in a family with a de novo mutation . A combined genetic study, including cytogenetics, PCR/SSCP, FISH, and CGH; Decker HJ et al.; von Hippel-Lindau (VHL) disease is a pleiotropic disorder featuring a variety of malignant and benign tumors of the eye, central nervous system, kidney, and adrenal gland . Recently the VHL gene has been identified in the chromosomal region 3p25-26 . Prognosis and successful management of VHL patients and their descendants depend on unambiguous diagnosis . Due to recurrent hemangioblastomas, a29-year-old patient without familial history of VHL disease was diagnosed to be at risk for the disease . Histopathological examination of a small renal mass identified a clear cell tumor with a G1 grading . Genetic characterization of the germline and of the renal tumor was performed . Polymerase chain reaction/single strand conformation polymorphism (PCR/SSCP) analysis with primers from the VHL gene identified a deletion of a single nucleotide in exon 2 in the patient's germline and in the tumor, but not in the DNA of his parents . This deletion therefore must be a de novo mutation . Comparative genome hybridization (CGH) and fluorescence in situ hybridization (FISH) analysis of the G1 tumor with differentially labelled yeast artifical chromosome (YAC) clones showed loss of 3p and of the 3p26 signals, respectively . In conclusion, we identified a de novo germline mutation in the VHL gene of a young patient and a somatic chromosome 3p loss at the homologous chromosome 3 in his renal tumor . Our results suggest a recessive mode of inactivation of the VHL gene, providing solid evidence for its tumor-suppressor gene characteristics . Our data show the diagnostic potential of genetic testing, especially in patients without VHL family history . Furthermore, the findings of homozygous inactivation of the VHL gene in a G1 tumor support the notion that the inactivation of the VHL gene is an early event in tumorigenesis of renal cell carcinoma.

Hum Genet, 1996 Jun, 97(6), 765 - 9
Isolation of the human chromosome 22q telomere and its application to detection of cryptic chromosomal abnormalities; Ning Y et al.; A number of human telomeres have been successfully cloned using a modified yeast artificial chromosome (YAC) vector (half-YAC) cloning strategy, but to date, human chromosome 22q has not been identified by this approach . We used an alternative approach of genomic walking, starting from a subtelomeric sequence, Tel-Bam3.4 . present on a number of human chromosomes including 22q . This approach was successful in the development of a cosmid contig representing the terminal 140 kb of human chromosome 22q, providing telomeric closure of the genetic and physical maps for 22q . The most distal region of the contig contains subtelomeric repeats which crosshybridize to a number of chromosomes, while the proximal sequences are unique for 22q . The unique sequence cosmid was used as a 22qter-specific probe for fluorescence in situ hybridization (FISH) analysis, which confirmed that this cosmid was distal to the most telomeric marker previously available for chromosome 22 . In addition, this cosmid was used to document a 22q terminal deletion that was not detectable by conventional cytogenetic analysis . Unique telomere-specific FISH probes such as this one will have significant diagnostic value in the detection of cryptic deletions and translocations in patients with unexplained mental retardation and other patient populations.

Nat Genet, 1996 Jun, 13(2), 227 - 9
A synaptobrevin-like gene in the Xq28 pseudoautosomal region undergoes X inactivation; D'Esposito M et al.; The X and Y chromosomes that maintain human dimorphism are thought to have descended from a single progenitor, with the Y chromosome becoming largely depleted of genes . A number of genes, however, retain copies on both X and Y chromosomes and escape the inactivation that affects most X-linked genes in somatic cells . Many of those genes are present in two pseudoautosomal regions (PARs) at the termini of the short (p) and long (q) arms of the sex chromosomes . For both PARs, pairing facilitates the exchange of information, ensuring the homogenisation of X and Y chromosomal material in these regions . We report here a strikingly different regulation of expression of a gene in Xq PAR . Unlike all Xp PAR genes studied so far, a synaptobrevin-like gene, tentatively named SYBL1, undergoes X inactivation . In addition, it is also inactive on the Y chromosome, thereby maintaining dosage compensation in an unprecedented way.

J Biol Chem, 1996 May 31, 271(22), 13250 - 7
Purification and characterization of a Src-related p57 protein-tyrosine kinase from Xenopus oocytes . Isolation of an inactive form of the enzyme and its activation and translocation upon fertilization; Sato K et al.; In the previous study (Fukami, Y., Sato, K.-I., Ikeda, K., Kamisango, K., Koizumi, K., and Matsuno, T . (1993) J . Biol . Chem . 268, 1132-1140), we found that an antibody termed anti-pepY antibody causes a severalfold activation of bovine brain c-Src . The anti-pepY antibody was raised against a synthetic peptide corresponding to residues 410-428 of chicken c-Src, one of the most conserved regions among the Src family protein-tyrosine kinases . In this study, we have used this antibody as an in vitro activator and purified a c-Src-related protein-tyrosine kinase from the particulate fraction of Xenopus laevis oocytes . A synthetic peptide corresponding to residues 7-26 of fission yeast Cdc2 was used as substrate . Immunoreactivity toward the antibody was also monitored during the purification . The purified kinase displayed a single polypeptide of 57 kDa on SDS-gel electrophoresis and showed a specific activity of 2.37 and 20.1 nmol/min/mg protein in the absence and the presence of the anti-pepY antibody, respectively . The purified enzyme underwent autophosphorylation and phosphorylated actin and the Cdc2 peptide exclusively on tyrosine residues . Specific antibodies against c-Src, Fyn, c-Yes, c-Fgr, Lck, Lyn, Hck, and Blk proteins did not recognize the p57 Xenopus tyrosine kinase . The kinase activity of the Xenopus enzyme was not affected by oocyte maturation but was found to be elevated severalfold upon fertilization . Fertilization also caused a translocation of the activated enzyme from the particulate fraction to the cytosolic fraction . The activation and translocation was observed within 1 min after fertilization . These results suggest a possible involvement of the p57 Xenopus tyrosine kinase in the signal transduction of fertilization.

Science, 1996 May 31, 272(5266), 1336 - 9
An orphan nuclear hormone receptor that lacks a DNA binding domain and heterodimerizes with other receptors; Seol W et al.; SHP is an orphan member of the nuclear hormone receptor superfamily that contains the dimerization and ligand-binding domain found in other family members but lacks the conserved DNA binding domain . In the yeast two-hybrid system, SHP interacted with several conventional and orphan members of the receptor superfamily, including retinoid receptors, the thyroid hormone receptor, and the orphan receptor MB67 . SHP also interacted directly with these receptors in vitro . In mammalian cells, SHP specifically inhibited transactivation by the superfamily members with which it interacted . These results suggest that SHP functions as a negative regulator of receptor-dependent signaling pathways.

Proc Natl Acad Sci U S A, 1996 May 28, 93(11), 5517 - 21
Cloning and characterization of a specific coactivator, ARA70, for the androgen receptor in human prostate cells; Yeh S et al.; The androgen receptor (AR) is a member of the steroid receptor superfamily that plays an important role in male sexual differentiation and prostate cell proliferation . Mutations or abnormal expression of AR in prostate cancer can play a key role in the process that changes prostate cancer from androgen-dependent to an androgen-independent stage . Using a yeast two-hybrid system, we were able to isolate a ligand-dependent AR-associated protein (ARA70), which functions as an activator to enhance AR transcriptional activity 10-fold in the presence of 10(-10) M dihydrotestosterone or 10(-9) M testosterone, but not 10(-6) M hydroxyflutamide in human prostate cancer DU145 cells . Our data further indicated that ARA70 Will only slightly induce the transcriptional activity of other steroid receptors such as estrogen receptor, glucocorticoid receptor, and progesterone receptor in DU145 cells . Together, these data suggest that AR may need a specific coactivator(s) such as ARA70 for optimal androgen activity.

J Biol Chem, 1996 May 24, 271(21), 12150 - 8
The Arabidopsis thaliana UBC7/13/14 genes encode a family of multiubiquitin chain-forming E2 enzymes; van Nocker S et al.; Covalent modification of proteins by attachment of multiubiquitin chains serves as an essential signal for selective protein degradation in eukaryotes . The specificity of ubiquitin-protein conjugation is controlled in part by a diverse group of ubiquitin-conjugating enzymes (E2s or UBCs) . We have previously reported that the product of the wheat TaUBC7 gene recognizes ubiquitin as a substrate for ubiquitination in vitro, catalyzing the condensation of free ubiquitin into multiubiquitin chains linked via lysine 48 (van Nocker, S., and vierstra, R . D . (1991) Proc . Natl . Acad . Sci . U . S . A . 88, 10297-10301) . Based on this activity, this E2 may play a central role in the ubiquitin proteolytic pathway by assembling chains in vivo . Here, we describe the cloning and characterization of a three-member gene family from Arabidopsis thaliana (designated AtUBC7/13/14) encoding structural homologs of TaUBC7 . Like TaUBC7, recombinant AtUBC7/13/14 proteins formed multiubiquitin chains in vitro . AtUBC7/13/14 mRNAs were found in all tissues examined, and unlike related UBCs from yeast, the levels of mRNA were not elevated by heat stress or cadmium exposure . Transgenic Arabidopsis were engineered to express increased levels of active AtUBC7, for the first time altering the level of an E2 in a higher eukaryote . Plants expressing high levels of AtUBC7 exhibited no phenotypic abnormalities and were not noticeably enriched in multiubiquitinated conjugates . These findings indicate that the in vivo synthesis of multiubiquitin chains is not rate-limited by the abundance of AtUC7 and/or involves other, yet undefined components.

J Biol Chem, 1996 May 17, 271(20), 11607 - 10
An affinity of human replication protein A for ultraviolet-damaged DNA; Burns JL et al.; Replication protein A (RPA), a heterotrimeric protein of 70-, 32-, and 14-kDa subunits, is an essential factor for DNA replication . Biochemical studies with human and yeast RPA have indicated that it is a DNA-binding protein that has higher affinity for single-stranded DNA . Interestingly, in vitro nucleotide excision repair studies with purified protein components have shown an absolute requirement for RPA in the incision of UV-damaged DNA . Here we use a mobility shift assay to demonstrate that human RPA binds a UV damaged duplex DNA fragment preferentially . Complex formation between RPA and the UV-irradiated DNA is not affected by prior enzymatic photo-reactivation of the DNA, suggesting an affinity of RPA for the (6-4) photoproduct . We also show that Mg2+ in the millimolar range is required for preferential binding of RPA to damaged DNA . These findings identify a novel property of RPA and implicate RPA in damage recognition during the incision of UV-damaged DNA.

J Biol Chem, 1996 May 17, 271(20), 11641 - 5
Interaction of insulin receptor substrate-2 (IRS-2) with the insulin and insulin-like growth factor I receptors . Evidence for two distinct phosphotyrosine-dependent interaction domains within IRS-2; He W et al.; Insulin receptor substrate 2 (IRS-2) has recently been shown to be a substrate of the insulin receptor (IR) . In this study we utilize the yeast two-hybrid system and assays of in vitro interaction to demonstrate that IRS-2 interacts directly with the IR and the insulin-like growth factor I receptor . We show that, like IRS-1, the region of IRS-2 that contains the putative phosphotyrosine binding and SAIN elements (188-591) is sufficient for receptor interaction and that this interaction is dependent upon the NPX(p)Y (where (p)Y is phosphotyrosine) motifs within the juxtamembrane domains of the receptors . In addition to this amino-terminal NPX(p)Y-binding domain, an additional domain of strong interaction was identified in the central region of IRS-2 and was localized between amino acids 591 and 733 . This interaction was found to be dependent upon receptor phosphorylation but was NPX(p)Y-independent . This region does not appear to have either an SH2 or a phosphotyrosine binding domain . Both of the interactions could also be demonstrated in vitro using IRS-2 glutathione S-transferase fusion proteins . We conclude that IRS-2, unlike IRS-1, can interact with tyrosine-phosphorylated receptors such as the IR and insulin-like growth factor I receptor via multiple independent binding motifs . Our findings suggest the existence of a previously unidentified phosphotyrosine-dependent binding domain within the central region of IRS-2.

J Biol Chem, 1996 May 17, 271(20), 12111 - 6
The ear of alpha-adaptin interacts with the COOH-terminal domain of the Eps 15 protein; Benmerah A et al.; The role of Eps15 in clathrin-mediated endocytosis is supported by two observations . First, it interacts specifically and constitutively with the plasma membrane adaptor AP-2 . Second, its NH2 terminus shows significant homology to the NH2 terminus of yeast End3p, necessary for endocytosis of alpha-factor . To gain further insight into the role of Eps15-AP-2 association, we have now delineated their sites of interactions . AP-2 binds to a domain of 72 amino acids (767-739) present in the COOH terminus of Eps15 . This domain contains 4 of the 15 DPF repeats characteristic of the COOH-terminal domain of Eps15 and shares no homology with known proteins, including the related Epsl5r protein . Precipitation of proteolytic fragments of AP-2 with Eps15-derived fusion proteins containing the binding site for AP-2 showed that Eps15 binds specifically to a 40-kDa fragment corresponding to the ear of alpha-adaptin, a result confirmed by precipitation of Eps15 by alpha-adaptin-derived fusion proteins . Our data indicate that this specific part of AP-2 binds to a cellular component and provide the tools for investigating the functions of the association between AP-2 and Eps15.

J Biol Chem, 1996 May 17, 271(20), 12088 - 94
Cloning, expression, and localization of 230-kDa phosphatidylinositol 4-kinase; Nakagawa T et al.; A phosphatidylinositol (PI) 4-kinase cDNA was cloned from a rat brain cDNA library . This cDNA encoded a protein of 2041 amino acids with a calculated molecular weight of 231,317 . The deduced amino acid sequence shared the identity of 52.3 and 34.4% in the presumed catalytic domain with two yeast PI 4-kinases, STT4 and PIK1, respectively, and showed 31.7% identity to p110alpha subunit of rat PI 3-kinase in the same domain . In addition, a 3' half coding region of the present cDNA was 89.6% identical to and its deduced amino acid sequence was 98.2% identical to the sequence for P14Kalpha, a recently reported human PI 4-kinase of type II, suggesting that P14Kalpha is an alternative form of the present PI 4-kinase molecule . The present cDNA contained sequences encoding the ankyrin repeat domain, lipid kinase unique domain, pleckstrin homology domain, presumed lipid kinase/protein kinase homology domain, proline-rich region, and SH3 domain . By examining PI kinase activity in transfected COS-7 cells using the epitope tag immunoprecipitation as well as the conventional way, the product phosphatidylinositol phosphate was identified as phosphatidylinositol 4-phosphate but not phosphatidylinositol 3-phosphate . This PI 4-kinase activity was markedly enhanced in the presence of Triton X-100 but relatively insensitive to inhibition by adenosine . By epitope tag immunohistochemistry, the immunoreactivity for this PI 4-kinase molecule was largely localized in close association with the membranes of the Golgi vesicles and vacuoles . By in situ hybridization analysis, the expression of mRNA for this PI 4-kinase was evident throughout the gray matter of entire brain with higher expression intensity in fetal brain . These data imply that this novel PI 4-kinase is involved in some processes essential to neuronal differentiation and maturation including the synaptogenesis and synaptic plasticity.

Science, 1996 May 17, 272(5264), 1008 - 10
Enhanced degradation of EGF receptors by a sorting nexin, SNX1; Kurten RC et al.; The vectorial movement of proteins requires specific recognition by components of the vesicular trafficking machinery . A protein, sorting nexin-1 (SNX1), was identified in a human cell line that bound to a region of the epidermal growth factor receptor (EGFR) containing the lysosomal targeting code . SNX1 contains a region of homology to a yeast vacuolar sorting protein, and overexpression of SNX1 decreased the amount of EGFR on the cell surface as a result of enhanced rates of constitutive and ligand-induced degradation . Thus, SNX1 is likely to play a role in sorting EGFR to lysosomes.

Oncogene, 1996 May 16, 12(10), 2171 - 6
Isolation and characterization of Nmi, a novel partner of Myc proteins; Bao J et al.; The Myc family of oncogenes is thought to play an important role in cell proliferation, differentiation, and neoplastic transformation . Although the structure and expression of Myc genes are well characterized, the function and biochemical properties of the Myc proteins are less well understood . Here, using a yeast genetic screen, we identified a novel gene, Nmi, that binds to N-myc and C-myc . It also interacts with other transcription factors in yeast . The carboxyl terminus of Nmi shows homology to an interferon-induced leucine zipper protein, IFP 35, whereas its amino terminus is homologous to a coiled-coil heptad repeat in the C . elegans protein, CEF59 . Co-precipitation studies of Nmi with N-myc and C-myc confirmed the interaction in mammalian cells . Nmi mRNA is expressed at low levels in all fetal and adult human tissues tested, except brain . Among several cancer cell lines, high expression of Nmi was found in myeloid leukemias, which also express high levels of C-myc . Nmi gene is localized on human chromosome 22q13.3 . Translocations of this region have been reported in some human leukemias.

Oncogene, 1996 May 16, 12(10), 2165 - 70
S phase specific formation of the human Rad51 protein nuclear foci in lymphocytes; Tashiro S et al.; The Rad51 protein, which is a homologue of the bacterial RecA protein, is involved in mitotic and meiotic recombination and in repair of double-strand breaks of DNA in yeast . The Rad51 homologue is conserved from yeast to human . In this study, the Rad51 protein was shown to be induced in peripheral blood lymphocytes (PBLs) 36 h after phytohemagglutinin (PHA) stimulation . Immunofluorescence study revealed that the distribution of the Rad51 protein in the nucleus was not uniform and focus-like staining was observed . Formation of the Rad51 foci was induced at 36 h after treatment of the cells with PHA . Twenty five percent of the cells had the foci at this time and the number of cells with foci declined thereafter . Cell cycle study using laser microscope by double staining method suggested that the appearance of the Rad51 nuclear foci was S phase specific . Furthermore, double staining study for the Rad51 protein and incorporated BrdU confirmed S phase specific appearance of the Rad51 nuclear foci . Formation of the Rad51 nuclear foci in PHA-stimulated lymphocytes might be involved in DNA recombination or DNA repair in S phase . The roles of RAD51 foci in S-phase will be discussed.

Oncogene, 1996 May 16, 12(10), 2101 - 8
Molecular and genetic studies on the region of translocation and duplication in the neuroblastoma cell line NGP at the 1p36.13-p36.32 chromosomal site; Casciano I et al.; Cytogenetic and molecular studies suggest that chromosome 1p might contain oncosuppressor genes involved in the pathogenesis of neuroblastoma and other adult tumors . The isolation of these genes by the 'positional cloning' approach will be facilitated by the characterization of cell lines with well defined chromosomal aberrations . In the present report we provide molecular data on the NGP neuroblastoma cell line which contains a reciprocal t(1;15) translocation . Two regions, possibly hosting oncosuppressor genes, have been identified: one is distal to the ENO1 locus, the other one is comprised between PND and A12M2 and corresponds to that of a constitutional t(1;17) translocation described in a neuroblastoma patient . Genetic data also suggest that the NGP cell line, despite the presence of two chromosomes 1, might be hemizygous for the subtelomeric 1p region.

Structure, 1996 May 15, 4(5), 599 - 611
The solution structure of the first zinc finger domain of SWI5: a novel structural extension to a common fold; Dutnall RN et al.; BACKGROUND: The 2Cys-2His (C2-H2) zinc finger is a protein domain commonly used for sequence-specific DNA recognition . The zinc fingers of the yeast transcription factors SWI5 and ACE2 share strong sequence homology, which extends into a region N-terminal to the first finger, suggesting that the DNA-binding domains of these two proteins include additional structural elements . RESULTS: Structural analysis of the zinc fingers of SWI5 reveals that a 15 residue region N-terminal to the finger motifs forms part of the structure of the first finger domain, adding a beta strand and a helix not previously observed in other zinc finger structures . Sequence analysis suggests that other zinc finger proteins may also have this structure . Biochemical studies show that this additional structure increases DNA-binding affinity . CONCLUSIONS: The structural analysis presented reveals a novel zinc finger structure in which additional structural elements have been added to the C2-H2 zinc finger fold . This additional structure may enhance stability and has implications for DNA recognition by extending the potential DNA-binding surface of a single zinc finger domain.

Genes Dev, 1996 May 15, 10(10), 1271 - 83
Intracellular receptors use a common mechanism to interpret signaling information at response elements; Starr DB et al.; The glucocorticoid receptor (GR) activates transcription in certain glucocorticoid response element (GRE) contexts, and represses or displays no activity in others . We isolated point mutations in one GRE, plfG, at which GR activated transcription under conditions in which the wild-type element was inactive or conferred repression, implying that GREs may carry signals that are interpreted by bound receptors . Consistent with this notion, we identified a mutant rat GR, K461A, which activated transcription in all GRE contexts tested, implying that this residue is important in interpretation of GRE signals . In a yeast screen of 60,000 GR mutants for strong activation from plfG, all 13 mutants isolated contained substitutions at K461 . This lysine residue is highly conserved in the zinc-binding region (ZBR) of the intracellular receptor (IR) superfamily; when it was mutated in MR and RARbeta, the resulting receptors similarly activated transcription at response elements that their wild-type counterparts repressed or were inactive . We suggest that IR response elements serve in part as signaling components, and that a critical lysine residue serves as an allosteric "lock" that restricts IRs to inactive or repressing configurations except in response element contexts that signal their conversion to transcriptional activators . Therefore, mutation of this residue produces altered receptors that activate in many or all response element contexts.

Genomics, 1996 May 15, 34(1), 128 - 33
A 1.5-Mb cosmid contig of the CMT1A duplication/HNPP deletion critical region in 17p11.2-p12; Murakami T et al.; Charcot-Marie-Tooth disease type 1A (CMT1A) is associated with a 1 . 5-Mb tandem duplication in chromosome 17p11.2-p12, and hereditary neuropathy with liability to pressure palsies (HNPP) is associated with a 1.5-Mb deletion at this locus . Both diseases appear to result from an altered copy number of the peripheral myelin protein-22 gene, PMP22, which maps within the critical region . To identify additional genes and characterize chromosomal elements, a 1.5-Mb cosmid contig of the CMT1A duplication/HNPP deletion critical region was assembled using a yeast artificial chromosome (YAC)-based isolation and binning strategy . Whole YAC probes were used for screening a high-density arrayed chromosome 17-specific cosmid library . Selected cosmids were spotted on dot blots and assigned to bins defined by YACs . This binning of cosmids facilitated the subsequent fingerprint analysis . The 1.5-Mb region was covered by 137 cosmids with a minimum overlap set of 52 cosmids assigned to 17 bins and 9 contigs.

Genomics, 1996 May 15, 34(1), 122 - 7
Physical and genetic mapping of the muscle phosphofructokinase gene (PFKM): reassignment to human chromosome 12q; Howard TD et al.; Phosphofructokinase (PFK) is a key rate-limiting enzyme in glycolysis and represents a major control point in the metabolism of glucose . There are at least three known isoforms of PFK in humans, referred to as the muscle, platelet, and liver forms, each of which is differentially expressed in various tissues . The gene for muscle phosphofructokinase, PFKM, is mutated in Tarui disease and conceivably contributes to non-insulin-dependent diabetes mellitus (NIDDM) . Based on physical and genetic mapping, we have found that the gene for PFKM does not map to chromosome 1 as previously described, but instead maps to chromosome 12 . PCR analysis with a somatic cell hybrid mapping panel using primers derived from intron 6 and exon 18 of the PFKM gene showed consistent amplification of cell lines containing chromosome 12 (concordance, 100%) . Fluorescence in situ hybridization analysis with CEPH YAC 762G4, isolated with exon 18 primers, indicated that this clone maps to 12q13, centromeric to the diacylglycerol kinase gene (DAGK) at 12q13 . 3 . A highly informative genetic marker isolated from YAC 762G4 was used to map PFKM genetically between the CHLC framework markers D12S1090 and D12S390 . This placement for 762G4 was significantly proximal to the recently reported locus for a third gene for maturity onset diabetes of the young (MODY) . The PFKM-associated microsatellite will be a valuable tool in the evaluation of PFKM in diabetic populations as well as in linkage analysis in families with Tarui disease.

Genomics, 1996 May 15, 34(1), 119 - 21
Physical mapping of the retinoblastoma interacting zinc finger gene RIZ to D1S228 on chromosome 1p36; Buyse IM et al.; The retinoblastoma interacting zinc finger gene RIZ is a member of the recently discovered PR domain family that includes the MDS1-EVI1 breakpoint gene involved in human leukemia . To help understand the role of RIZ in human diseases, we have determined the cytogenetic and physical localizations of the RIZ gene . Using fluorescence in situ hybridization, we determined that RIZ maps to 1p36 . On the physical map, RIZ is adjacent to the polymorphic marker D1S228 . We suggest that the RIZ gene may be a candidate target of 1p36 alterations that commonly occur in neuroendocrine, breast, liver, colon, and lymphoid tumors.

Genomics, 1996 May 15, 34(1), 55 - 62
YAC/STS map of 9 Mb of Xq26 at 100-kb resolution, localizing 6 ESTs, 6 genes, and 32 genetic markers; Pilia G et al.; To facilitate functional analysis of the Xq26 region, the physical map has been extended across 9 Mb with 192 YACs and markers including 90 STSs (sequence-tagged sites) and 50 hybridization probes . Six genes and six ESTs are localized . In addition, 32 markers that detect polymorphism permit an integration of physical with genetic linkage data . The localizations of eight uncloned disease genes are thereby delimited on the physical map . The data also suggest a possible gradient of recombination across the cytogenetic band, with little or no recombination reported in the centromeric 3.5-4 Mb.

Genomics, 1996 May 15, 34(1), 42 - 54
YAC/STS map across 12 Mb of Xq27 at 25-kb resolution, merging Xq26-qter; Zucchi I et al.; A 12-Mb YAC contig has been assembled spanning the Xq27 cytogenetic band with 203 YACs, 121 STSs, and >300 hybridization probes to a resolution of 25 kb . At its centromeric end, the contig is merged with a 9-Mb contig covering Xq26.1-q26.3 at a point 1 Mb telomeric to the factor IX gene; at its telomeric end, it is merged to 7.5 Mb of contigs from the IDS gene to the Xq28 telomere . Thus, the distal 29 Mb of the Xq arm is available cloned in long-range contiguity . The physical map has been integrated with current genetic data by the localization of 18 markers that detect polymorphism . Apparent recombination levels reach >4.5 cM/Mb near the centromeric border of Xq27 . The ratio of cM/Mb correspondingly delimits the location of several disease genes-including, for example, X-linked hypoparathyroidism in 3 Mb (6 cM) telomeric to Factor IX.

Cancer Res, 1996 May 15, 56(10), 2263 - 7
Distinct areas of allelic loss on chromosomal regions 10p and 10q in human prostate cancer; Trybus TM et al.; Utilizing tissue microdissection and PCR techniques, we have examined 35 prostate tumors paired with normal tissues from the same patients for allelic loss at 24 polymorphic loci spanning chromosome 10 . Twenty-five tumors (71%) were deleted for at least one chromosome 10 locus . Of the total 35 tumors, 6 (17%) were deleted for 10p loci only, 5 (14%) for 10q loci only, and 14 (40%) were deleted for both 10p and 10q loci . The common region of deletion on 10p included loci D10S211-D10S89-D10S111 . Fluorescence in situ hybridization of yeast artificial chromosome probes encompassing these loci demonstrated that the 10p region of deletion maps to 10p11.2 . Losses involving 10p loci alone were most common in localized (5/14, 36%) and least common in metastatic (0/8) tumors . The common region of deletion on 10q included loci D10S219-D10S215, consistent with the major region of deletion recently defined for prostate tumors on 10q . Losses involving 10q loci alone were lowest in localized and locally invasive tumors (1/14 and 2/12, respectively) and highest in tumors metastatic to regional lymph nodes (2/8) . These results suggest that 10p losses may define less invasive tumors, whereas 10q losses may play a role in the progression to more advanced tumor states in the prostate . Furthermore, this is the first report of allelic loss of a defined region on 10p potentially harboring tumor suppressor gene loci in human prostate cancer.

J Biol Chem, 1996 May 10, 271(19), 11284 - 90
The role of Phe-92 in the Ca(2+)-induced conformational transition in the C-terminal domain of calmodulin; Meyer DF et al.; Recent studies have shown that substitution of Ala for one or more Phe residues in calmodulin (CaM) imparts a temperature-sensitive phenotype to yeast (Ohya, Y., and Botstein, D . (1994) Science 263, 963-966) . The Phe residue immediately preceding the first Ca(2+) ligand in site III of CaM (Phe-92) was found to be of particular importance because the mutation at this position alone was sufficient to induce this phenotype . In the present work we have studied the functional and structural consequences of the Phe-92 --> Ala mutation in human liver calmodulin . We found that the mutant (CaMF92A) is incapable of activating phosphodiesterase, and the maximal activation of calcineurin is reduced by 40% as compared with the wild type CaM . Impaired regulatory properties of CaMF92A are accompanied by an increase in affinity for Ca(2+) at the C-terminal domain . To investigate the structural consequences of the F92A mutation, we constructed four recombinant C-terminal domain fragments (C-CaM) of calmodulin (residues 78-148): 1) wild type (C-CaMW); 2) Ala substituted for Phe-92 (C-CaMF92A); 3) cysteine residues introduced at position 85 and 112 to lock the domain with a disulfide bond in the Ca(2+)-free (closed) conformation (C-CaM85/112); and 4) mutations 2 and 3 combined (C-CaM85/112F92A) . The Cys-containing mutants readily form intramolecular disulfide bonds regardless whether Phe or Ala is present at position 92 . The F92A mutation causes a decrease in stability of the domain in the absence of Ca(2+) as indicated by an 11.8 degree C shift in the far UV circular dichroism thermal unfolding curve . This effect is reversed by the disulfide bond in the C-CaM85/112F92A mutant . The C-CaMW peptide shows a characteristic Ca(2+)-dependent increase in solvent-exposed hydrophobic surface which was monitored by an increase in the fluorescence of the hydrophobic probe 1,1'-bis(4-anilino)-naphthalene-5,5'-disulfonic acid . The fluorescence increase induced by C-CaMF92A is approximately 45% lower than that induced by C-CaMW suggesting that the F92A mutation causes a decrease in the accessibility of several hydrophobic side chains in the C-terminal domain of CaM in the presence of Ca(2+) . The Cys-85-Cys-112 disulfide bond causes a 10- or 5.9-fold decrease in Ca(2+) affinity depending on whether Phe or Ala is present at position 92, respectively, suggesting that coupling between Ca(2+) binding and the conformational transition is weaker in the absence of the phenyl ring at position 92 . Our results indicate that Phe-92 makes an important contribution to the Ca(2+)-induced transition in the C-terminal domain of CaM . This is most likely the reason for the severely impaired regulatory properties of the CaM mutants having Ala substituted for Phe-92.

J Biol Chem, 1996 May 3, 271(18), 10953 - 62
Wortmannin-sensitive trafficking pathways in Chinese hamster ovary cells . Differential effects on endocytosis and lysosomal sorting; Martys JL et al.; Phosphatidylinositol (PI) 3'-kinases are a family of lipid kinases implicated in the regulation of cell growth by oncogene products and tyrosine kinase growth factor receptors . The catalytic subunit of the p85/p110 PI 3'-kinase is homologous to VPS-34, a phosphatidylinositol-specific lipid kinase involved in the sorting of newly synthesized hydrolases to the yeast vacuole . This suggests that PI 3'-kinases may play analogous roles in mammalian cells . We have measured a number of secretory and endocytic trafficking events in Chinese hamster ovary cells in the presence of wortmannin, a potent inhibitor of PI 3'-kinase . Wortmannin caused a 40-50% down-regulation of surface transferrin receptors, with a dose dependence identical to that required for maximal inhibition of the p85/p110 PI 3'-kinase in intact cells . The redistribution of transferrin receptors reflected a 60% increase in the internalization rate and a 35% decrease in the recycling rate . Experiments with fluorescent transferrin showed that entry of transferrin receptors into the recycling compartment and efflux of receptors out of the compartment were slowed by wortmannin . Wortmannin altered the morphology of the recycling compartment, which was more vesiculated than in untreated cells . Using Semliki Forest virus as a probe, we also found that delivery of the endocytosed virus to its lysosomal site of degradation was slowed by wortmannin, whereas endosomal acidification was unaffected . In contrast to these effects on endocytosis and recycling, wortmannin did not affect intracellular processing of newly synthesized viral spike proteins . Wortmannin did induce missorting of the lysosomal enzyme cathepsin D to the secretory pathway, but only at a dose 20-fold greater than that required to inhibit p85/p110 PI 3'-kinase activity or to redistribute transferrin receptors . Our data demonstrate the presence of wortmannin-sensitive enzymes at three distinct steps of the endocytic cycle in Chinese hamster ovary cells: internalization, transit from early endosomes to the recycling and degradative compartments, and transit from the recycling compartment back to the cell surface . The wortmannin-sensitive enzymes critical for endocytosis and recycling are distinct from those involved in sorting newly synthesized lysosomal enzymes.

J Biol Chem, 1996 May 3, 271(18), 10821 - 6
Reconstitution of TFIIH and requirement of its DNA helicase subunits, Rad3 and Rad25, in the incision step of nucleotide excision repair; Sung P et al.; Yeast TFIIH is composed of six subunits: Rad3, Rad25, TFB1, SSL1, p55, and p38 . In addition to TFIIH, we have purified a subassembly of the factor that lacks Rad3 and Rad25 and which we refer to as TFIIHi . In the in vitro nucleotide excision repair (NER) system that consists entirely of purified proteins, we show that neither TFIIHi nor a mixture of purified Rad3 and Rad25 proteins is active in NER but that the combination of TFIIHi with Rad3 and Rad25 promotes the incision of UV-damaged DNA . These results provide the first evidence for a direct requirement of Rad3, Rad25, and of one or more of the TFIIHi subunits in the incision step of NER . The NER efficacy of TFIIH is greatly diminished or abolished upon substitution of Rad3 with the rad3 Arg-48 mutant protein or Rad25 with the rad25 Arg-392 mutant protein, respectively, thus indicating a role of the Rad3 and Rad25 DNA helicase functions in the incision of damaged DNA . Our results further indicate that the carboxyl-terminal domain kinase (CTD) TFIIK is dispensable for the incision of damaged DNA in vitro . These studies reveal the differential requirement of Rad3 DNA helicase and CTD kinase activities in damage-specific incision versus RNA polymerase II transcription.

J Biol Chem, 1996 May 3, 271(18), 10731 - 7
Proprotein-processing endoprotease furin decreases regulated secretory pathway-specific proteins in the pancreatic beta cell line MIN6; Kayo T et al.; Prohormone convertases PC2 and PC3, yeast Kex2-family endoproteases specific to the regulated secretory pathway, cleave proinsulin to insulin in the secretory granules of pancreatic beta cells . The well-differentiated beta cell line MIN6 expresses PC2 and PC3 and another regulated secretory pathway-specific protein chromogranin A . Furin, another yeast Kex2 endoprotease, exists in the trans-Golgi networks of many cell types . The beta cell line RINm5F (a cell line that is less differentiated than the MIN6 cell line) does not express the regulated pathway-specific proteins, but strongly expresses furin . We suspected that furin expression may cause the decrement of regulated secretory pathway-specific proteins . To test this hypothesis, we expressed a furin cDNA with a metallothionein promoter in MIN6 cells . With Zn2+ stimulation of furin expression, the messages of PC2, PC3, and chromogranin A decreased, and the processing of proinsulin to mature insulin became less efficient . The furin-expressing MIN6 cells exhibited less insulin content and weakened insulin secretion in response to a high glucose concentration . The conditioned medium from furin-expressing MIN6 cells also exerted a decrease of PC2 and PC3 expression in unaltered MIN6 cells . Thus, proteins cleaved by furin inside the cells or by truncated furin shed into the culture medium appear to cause decreased PC2 and PC3 expression, insulin content, and glucose-responsive insulin secretion in MIN6 cells.

Oncogene, 1996 May 2, 12(9), 1931 - 9
A gene from human chromosomal band 3p21.1 encodes a highly conserved arginine-rich protein and is mutated in renal cell carcinomas; Shridhar V et al.; We have identified a gene, called ARP for Arginine-rich protein, in human chromosomal band 3p21 . It is approximately 600 Kb telomeric to the ACY1 locus (Miller et al., 1989) and encodes a previously unidentified 234 amino acid long, highly basic protein . This gene is highly conserved at the DNA and RNA level . It is found in all species including hamster, rat, mouse, bovine and yeast . We have detected a point mutation (ATG50 to AGG) or deletion of ATG50 in 10 of 21 sporadic renal cell carcinomas . The mutable region is in an imperfect trinucleotide repeat in the coding region which is non-polymorphic among 50 normal individuals examined . The point mutation (ATG50 to AGG) or deletion of codon 50 removes a methionine and increases the stretch of arginines encoded by the AGG repeats in the ARP gene.

Nature, 1996 May 2, 381(6577), 86 - 9
A human RNA polymerase II complex associated with SRB and DNA-repair proteins; Maldonado E et al.; We report here the isolation of a human RNA polymerase II complex containing a subset of the basal transcription factors and the human homologues of the yeast SRB (for suppressors of RNA polymerase B) proteins . The complex contains transcriptional coactivators and increases the activation of transcription . In addition, some components of the RNA polymerase II complex participate in DNA repair.

Zentralbl Veterinarmed B, 1996 May, 43(3), 129 - 35
The immunomodulatory effect of the soluble fungal glucan (Pleurotus ostreatus) on delayed hypersensitivity and phagocytic ability of blood leucocytes in mice; Paulik S et al.; The effect of fungal and yeast glucan on different immune functions in mice was examined and compared . The simultaneous administration of glucan and a sensitizing dose of DNFB on the different sites significantly stimulated delayed-type hypersensitivity (DTH) response only when using fungal glucan . Both glucans tested, when administered before sensitization, significantly increased DTH response, but with a significantly higher level at the beginning of the investigation (on day 7) when using fungal glucan . The increase in phagocytic activity by the blood leucocytes started in the 1st week after fungal-glucan treatment, and in the 2nd week after yeast-glucan treatment, and took longer after administration of fungal glucan . The values of the phagocytic-activity index were significantly influenced only after fungal-glucan injection . The results of the study indicate that fungal glucan isolated from Pleurotus ostreatus could be a prospective immunomodulating substance.

DNA Cell Biol, 1996 May, 15(5), 387 - 99
Isolation and characterization of several members of the murine Hsd3b gene family; Clarke TR et al.; The enzyme 3 beta-hydroxysteroid dehydrogenase (3 betaHSD) is essential for all steroid hormone biosynthesis . Six distinct 3 betaHSD cDNAs in the mouse (3 betaHSD I-VI) have been isolated previously . This study reports the isolation of genes or partial genes encoding the 3 betaHSDI, II, III, and IV isoforms . Characterization of the genes revealed that they consist of four exons, the same structure that has been observed for characterized human 3 betaHSD genes . Primer extension and nuclease S1 analysis identified the start sites of transcription of Hsd3b -1 and -4 . The proximal promoter regions of Hsd3b-1 and -4 were sequenced and putative cis-acting sequences were determined . Previously, we reported that the then known 3 betaHSD genes (3 betaHSD I-IV) were located in a small region of mouse chromosome 3 . To analyze this locus further, six yeast artificial chromosome clones containing the 3 betaHSD sequence were identified . One clone appears to contain the complete 3 betaHSD locus within its 1,400-kbp insert . Further analysis of this YAC, along with analysis of mouse genomic DNA by pulsed-field gel electrophoresis, suggests all members of the 3 betaHSD gene family may be contained on a 400-kbp fragment.

Somat Cell Mol Genet, 1996 May, 22(3), 167 - 75
Molecular genetic characterization and comparative mapping of the human PCP4 gene; Cabin DE et al.; The mouse Pcp4 gene is highly expressed in brain, primarily in cerebellar Purkinje cells . It maps to chromosome 16 (Chr 16), in a region of conserved synteny with human chromosome 21 (Chr 21) . To further characterize PCP4 and its possible contribution to cerebellar hypoplasia in trisomy 21, or Down Syndrome (DS), we cloned and sequenced the full length human cDNA, isolated a YAC which carries the entire gene, determined the gene structure, and characterized its expression . The gene spans at least 55 kb and contains two introns, the placement of which is the same in mouse . Expression in the mouse brain during development was detected at embryonic day 10, and thereafter through development . The PCP4 YAC was placed on the human Chr 21 YAC contig by a link to a YAC carrying the markers D21S15 and D21S349 . This placement distal to ETS2 was confirmed by mapping on a somatic cell hybrid panel of Chr 21 translocations . This position caused an apparent break in gene order with mouse Chr 16 . However, mapping in the mouse was reassessed, and Pcp4 and a linked marker, D16Mit71, were both moved distal to Ets2, corresponding to the position of PCP4 on Chr 21.

Mycoses, 1996 May-Jun, 39(5-6), 225 - 31
Hydrolase activity of 150 Malassezia furfur isolates of different clinical origin; Mayser P et al.; Apart from pityriasis versicolor, Malassezia furfur is thought to play a significant role in the pathogenesis of seborrhoic eczema and Malassezia folliculitis . However, it has not been clarified whether in addition to host factors (e.g . immune status, greasy skin), yeast-dependent activities are responsible for manifestation of the disease . In this context interstrain variability of hydrolase activity of Malassezia isolates might be significant . For determination of hydrolase activity, washed yeast suspension was applied to selective agar for pathogenic fungi containing 8% (v/v) Tween 80 or Tween 60 and was incubated at 37 degrees C for 10 days . Growth was accompanied by formation of a dense white zone around the colony, in which free fatty acids corresponding to Tween 80 (C18:1) or Tween 60 (16:0, 18:0) were demonstrated by means of thin layer and gas chromatography . Thus, this phenomenon is a parameter for yeast-dependent hydrolysis of Tween 80 and Tween 60 . Considering different growth behaviour, a "hydrolase zone' (H2) was determined using the quotient colony diameter/(ring+colony) diameter in each of the 150 strains tested . Although no significant H2 variations were observed in strains of different clinical origin, the present study revealed that in addition to a known enzyme, which is located within the cell wall and/or membrane systems, these Malassezia isolates produce a very active hydrolase diffusing into the medium.

J Investig Allergol Clin Immunol, 1996 May-Jun, 6(3), 196 - 201
Fungal contamination of potential medical interest in Spanish grain stores; Mediavilla Molina A et al.; A one-year study was made of fungal spores detected in the air and in the grain of two silos and two seed stores near Cordoba, Spain . Gravimetric and volumetric methods were used simultaneously on culture mediums to sample the air . The dilution method was employed to analyze seed contamination . A total of 70 taxa were isolated, 67 of these from the air and 46 in seeds . The most abundant airborne taxa were: Aspergillus oryzae, Cladosporium cladosporioides and yeast, while yeast, A . niger and A . oryzae were the most common in seeds . The statistical test revealed differences between taxa found in the air and the wheat that could be of biological interest . Finally, it is worth noting among the species isolated the high percentage of species which have been cited in the references as potentially pathogenic or antigenic.

Biochem Mol Biol Int, 1996 May, 39(2), 421 - 9
Role of PM-ATPase, amino acid transport and free amino acid pool in the salt stress of, Candida membranefaciens; Khaware RK et al.; The salt tolerant yeast Candida membranefaciens exhibited a pleiotropic modification in response to high NaCl stress . The in vivo specific activity of Plasma Membrane-ATPase (PM-ATPase) of 1.35 M NaCl adapted cells was enhanced at the mid-log phase . The enhancement in the PM-ATPase activity was NaCl specific as cells stressed with identical concentration of KCl did not have any effect on PM-ATPase . The NaCl specific enhancement in the PM-ATPase activity was associated with decreased Km . Studies on H+ efflux correlated with the results of PM-ATPase . However, in vitro incubation of the enzyme with exogenously added salts like NaCl and KCl invariably inhibited enzyme activity by 70-90% in a dose dependent manner to suggest that in vivo effects of the salts on PM-ATPase were different from the in vitro effects . C . membranefaciens showed a higher intracellular levels of glutamate and aspartate in presence of 1.35 M NaCl which may impart osmoprotection to the stressed cells . It was interesting to observe that the transport activities of aspartate and glutamate were not enhanced according to their relative proportion in the total pool of free amino acids . Instead, transport of these and other amino acids (except lysine and arginine) showed a drastic reduction (upto 90%) in the 1.35 M NaCl grown cells.

Vet Immunol Immunopathol, 1996 May, 51(1-2), 201 - 10
The role of opsonization by antibody and complement in in vitro phagocytosis of microsporidian parasites by turbot spleen cells; Leiro J et al.; We investigated the role played by opsonization by antibody and complement in in vitro phagocytosis of microsporidian spores by turbot adherent phagocytes . Most turbot adherent cells displaying phagocytic activity are probably macrophages . Phagocytosis of yeast cells and polystyrene beads was greatly enhanced in the presence of both the Ig and the non-Ig (i.e . complement-containing) fractions of normal turbot serum, but phagocytosis of Glugea caulleryi or Tetramicra brevifilum spores was not affected by either fraction . Neither anti-G . caulleryi immune serum, nor anti-T . brevifilum immune serum (which cross-reacted considerably with G . caulleryi antigens), enhanced phagocytosis of G . caulleryi spores . Finally, spores treated with sodium m-periodate (to modify the structure of surface-borne sugars) were less effectively ingested than untreated spores, suggesting that phagocytosis of microsporidian spores involves recognition of such sugars by the phagocytic cell . The results of this study support the hypothesis that microsporidian parasites of fish in some way modulate the host phagocytic responses.

J Infect, 1996 May, 32(3), 197 - 204
An economic evaluation of universal vaccination against hepatitis B virus; Fenn P et al.; This report presents the results of an economic evaluation utilizing U.K . data, into a vaccination programme against Hepatitis B using a genetically engineered, yeast-derived vaccine, Engerix B . Cost-effectiveness ratios were calculated for four different programmes: an infant vaccination programme; a child vaccination programme; an adolescent vaccination programme; and a combined child and adolescent programme . For each programme, the number of annual cohorts vaccinated was varied from 1 to 25 . The outcome was defined as incremental life years gained, and the results are reported as costs per incremental life-year gained . Benefits were calculated in both undiscounted and discounted forms . All costs were discounted . The discount rate used was 6% . All major epidemiological and cost assumptions were subjected to a sensitivity analysis . Baseline results with benefits discounted range from pounds188015 to pounds301365 per life year gained, depending on the duration of programme and vaccination strategy . With benefits undiscounted, the comparable range is from pounds5234 to pounds13034.

Bone Marrow Transplant, 1996 May, 17 Suppl 3, S45 - 7
Very low level of major BCR-ABL expression in blood of some healthy individuals; Biernaux C et al.; The nested reverse transcriptase polymerase chain reaction (RT-PCR) provides a powerful tool for detection of minimal residual disease in CML . The RT-PCR used in the present study for detection of the major bcr-abl fusion gene, the hallmark and presumably the cause of CML, was optimized by: (a) increasing the amount of total RNA involved in the reverse transcription reaction to correspond to total RNA extracted from 10(8) cells; (b) using a specific abl primer in this reverse transcriptase reaction, and (c) reamplifying 10% of the RT-PCR product in a nested amplification . This optimized RT-PCR permitted to detect up to 1 copy of RNA bcr-abl synthesized in vitro, mixed with yeast RNA in a quantity equivalent to 10(8) white blood cells (WBC) . Using the highly sensitive RT-PCR, a systematic study of the possible expression of bcr-abl RNA in WBC of healthy adults, children and umbilical cord blood (UCB) revealed the presence of bcr-abl transcripts in blood cells of 22/73 adults, 1/22 children but not in 22 samples of UCB . The comparison of these three groups indicated a significant tendency for the anomaly to increase in frequency with age.

Plant Mol Biol, 1996 May, 31(2), 279 - 93
Small G proteins of two green algae are localized to exocytic compartments and to flagella; Huber H et al.; The Ypt/Rab proteins are small GTPases, which belong to the Ras superfamily and have been shown to be involved in endo- and exocytosis in mammalian cells and yeast . Using affinity-purified antibodies specific for four Ypt proteins, namely Ypt1p, Ypt4p, Ypt5p and Ypt6p, of the multicellular green alga Volvox carteri (YptVp) and its close unicellular relative Chlamydomonas reinhardtii (YptCp), we examined the abundance of the corresponding antigens during the asexual life cycle of Volvox, and their intracellular localization . The YptV proteins were found in all stages throughout the asexual life cycle and are tightly associated with intracellular membranes . Indirect immunofluorescence revealed that YptV4p, YptV5p and YptV6p are present in perinuclear regions of the cell, indicating an association with the Golgi region . Golgi localization of YptV4p and YptV6p in Volvox was confirmed by immunogold electron microscopy . In contrast, we found Ypt1p associated with the contractile vacuole in both V . carteri and C . reinhardtii . Furthermore, the YptV proteins were also detected along the entire length of the flagella of somatic Volvox cells . This flagellar location was substantiated by western blot analysis of extracts prepared from isolated flagella of both algae . While localization to exocytic compartments is in agreement with the established Ypt/Rab function in intracellular vesicle transport of eukaryotic cells, presence in the algal flagellum is the first hint of a possible role for small G proteins also in motility organelles.

Genome Res, 1996 May, 6(5), 431 - 8
Mapping of the OB receptor to 1p in a region of nonconserved gene order from mouse and rat to human; Chung WK et al.; As part of an effort to identify informative molecular markers for genetic analysis of human pedigrees segregating for obesity, we have developed a genetic map of human 1p in the region of the OB receptor (OBR), the gene that is defective in murine diabetes (Obrdb) and rat Zucker fatty (Obrfa) mutations located on mid-chromosome 4 and chromosome 5, respectively . OBR was mapped 0.9 cR centromeric to WI-9515 and 2.2 cR telomeric of WI-7249 by radiation hybrid (RH) mapping . Ten yeast artificial chromosomes (YACs) containing OBR were identified, confirming the location of OBR centromeric to WI-9515 and telomeric to WI-7249 . Additionally, five P1 artificial chromosomes (PACs) were identified that comprised a contiguous series of overlapping clones spanning the length of OBR . WI-5182 was contained within the two PACs that are 3' of OBR . Using a panel of 68 individuals from a single three-generation family and an additional nuclear family, we have mapped 18 polymorphic markers including phosphoglucomutase 1 (PGM1), which is centromeric to Obrdb / Obrfa, and D1S85, which is telomeric to Obrdb / Obrfa in the mouse and rat . The following composite map integrates these radiation hybrid, genetic, and physical maps: Centromere-@WI-7249-{OBR; WI-5182}-D1S198-{WI-9515; WI-6550; D1S2866}-D1S2825-{WI-3077; D1S2886}-{D1S515; DS1613; PGM1}-{D1S312; D1S473; D1S230; D1S246; D1S203}-D2S1643-{D1S1669; D1S1596;}UNCJ-D1S476- D1S85-D1S220-C8B-GTAT1A7 . Unresolvable markers are within brackets . A comparison of gene order on mouse chromosome 4, rat chromosome 5, and human 1p indicates that between rodents and humans, there has been a rearrangement of the gene order in the region surrounding OBR.

Genome Res, 1996 May, 6(5), 351 - 60
Genetic and physical mapping of the progressive epilepsy with mental retardation (EPMR) locus on chromosome 8p; Ranta S et al.; Progressive epilepsy with mental retardation (EPMR) is an autosomal recessive disorder discovered recently from an isolated region in Finland . The disorder is characterized by normal early development, generalized tonic-clonic seizures with onset at 5-10 years of age, and progressive mental retardation beginning 2-5 years after the onset of seizures . We recently mapped the EPMR locus to a 7-cM region on chromosome 8p between markers AFM185xb2 and D8S262 . A recombination detected with a new microsatellite marker AFMa054td9 narrows the region to 4 cM . A yeast artificial chromosome (YAC) contig containing 22 YACs was constructed across the disease gene region . The YAC contig is characterized by a collection of 19 YAC-end sequence-tag sites together with seven microsatellite markers . The entire YAC contig spans a minimum of 3 Mb . Moreover, the distal end of the contig contains a subtelomeric YAC yRM2205 that anchors the contig to the telomere . Construction of a YAC contig across the disease gene region is an essential step toward the isolation of the EPMR gene.

Brain Res Mol Brain Res, 1996 May, 38(1), 101 - 8
Changes in the phosphorylation of eukaryotic initiation factor 2 alpha, initiation factor 2B activity and translational rates in primary neuronal cultures under different physiological growing conditions; Alcazar A et al.; Phosphorylation of the alpha-subunit of eukaryotic initiation factor 2 (eIF-2) is one of the best known mechanisms regulating protein synthesis in a wide range of eukaryotic cells, from yeast to human . To determine whether this mechanism operates in primary neuronal cells, we have cultured primary neuronal cells for 7 days under two optimal growing conditions, complete medium (containing 15% serum) and serum-free medium, and determined the protein synthesis rate, eukaryotic initiation 2 and 2B (eIF-2B) activities, as well as the level of phosphorylation of eIF-2 . Cells cultured in serum-free medium exhibited a lower rate of protein synthesis (75%), concomitant to a decreased eIF-2 activity (71%), and slightly higher eIF-2(alpha P) levels (from 10 to 16% of total eIF-2) with respect to cells cultured in complete media . eIF-2B activity, as measured at saturating eIF-2 . GDP concentrations (assay independent on the presence of eIF-2(alpha P)) was similar under the two culture conditions . When neurons cultured in serum-free medium are exposed to complete medium for only 24 h, there is a clear decrease in the phosphorylation of eIF-2 alpha (16-3%) . This decrease correlates in time with an increase in the protein synthesis rate (154%), as well as eIF-2 activity (236%) . The increased levels of eIF-2(alpha P), a competitive inhibitor of eIF-2B in the guanine-exchange reaction, are responsible for the decreased eIF-2B activity found in the neurons cultured in serum-free medium . Additionally, eIF-2(alpha P) is accountable for the lower effect of exogenous eIF-2B in ternary complex formation from preformed eIF-2 . GDP in the serum-free media . These changes in phosphorylation of eIF-2 alpha in normal mammalian cells in response to changes in the extracellular medium are reported here for the first time.

Hum Mol Genet, 1996 May, 5(5), 571 - 9
Characterization of the split hand/split foot malformation locus SHFM1 at 7q21.3-q22.1 and analysis of a candidate gene for its expression during limb development; Crackower MA et al.; Split hand/split foot malformation (SHFM) is a heterogeneous limb developmental disorder, characterized by missing digits and fusion of remaining digits . An autosomal dominant form of this disorder (SHFM1) has been mapped to 7q21.3-q22.1 on the basis of SHFM-associated chromosomal rearrangements . Utilizing a YAC contig across this region, we have defined a critical interval of 1.5 Mb by the analysis of six interstitial deletion patients and mapped the translocation breakpoints of seven ectrodactyly patients within the interval . To delineate the basic molecular defect underlying SHFM, we have searched for candidate genes in a 500 kb region containing five of the translocation breakpoints . Three genes were identified, two genes of the Distal-less (dii) homeobox gene family, DLX5 and DLX6 and a novel gene, which we named DSS1 . DSS1 is predicted to encode a highly acidic polypeptide with no significant similarity to any known proteins but 100% amino acid sequence identify with its murine homolog (Dss1) . Using RNA in situ hybridization analysis, we detected a tissue-specific expression profile for Dss1 in limb bud, craniofacial primordia and skin . A deficiency in expression of Dss1, DLX5 and/or DLX6 during development may explain the SHFM phenotypes.

Hum Mol Genet, 1996 May, 5(5), 563 - 9
Construction of a mouse model of Charcot-Marie-Tooth disease type 1A by pronuclear injection of human YAC DNA; Huxley C et al.; Construction of animal models of human inherited diseases is particularly important for testing gene therapy approaches . Towards this end, we constructed a mouse model for Charcot-Marie-Tooth disease type 1A by pronuclear injection of a YAC containing the human PMP22 gene . In one transgenic line, the YAC DNA is integrated in about eight copies and the PMP22 gene is strongly expressed to give a peripheral neuropathy closely resembling the human pathology . The disorder is dominant, causes progressive weakness of the hind legs, and there is severe demyelination in the peripheral nervous system including the presence of onion bulb formations . This approach will be valuable for pathologies produced by over-expression of a gene including trisomy and amplification in cancer . Such models will be particularly useful for testing gene therapy approaches if the transgene is human.

J Med Genet, 1996 May, 33(5), 353 - 7
Localisation of two candidate genes for mental retardation using a YAC physical map of the Xq21.1-21.2 subbands; Colleaux L et al.; Genetic studies in families with X linked mental retardation have suggested the location of several MR genes in the human q21 region . Since the establishment of cloned resources is an essential step towards the cloning of genes involved in inherited diseases, we built a yeast artificial chromosome (YAC) contig and an STS map of this part of the X chromosome . The contig, which extends from PGK1 in Xq13.3 to DXS1002 in Xq21.2, consists of 30 YACs mapped with 21 markers and spans about 6 Mb . The YAC contig was used as a framework to localise several previously known genes and CEPH/Genethon polymorphic markers, as well as to construct a physical map of the region surrounding one of these genes . We recently localised a presumed MR locus to the region flanked by DXS233 (proximal) and CHM (distal) . In the present work, the zinc finger gene, ZNF6, has been shown to lie within this region and to be highly expressed in brain, making it a good candidate MR gene . Similarly the VDAC1 gene has been mapped between DXS986 and DXS72 and its candidate gene status for the Allan-Herndon-Dudley syndrome is discussed.

J Lipid Res, 1996 May, 37(5), 1144 - 52
Lipid transfer from insect fat body to lipophorin: comparison between a mosquito triacylglycerol-rich lipophorin and a sphinx moth diacylglycerol-rich lipophorin; Pennington JE et al.; Two insect lipoproteins, triacylglycerol-rich Aedes aegypti lipophorin and diacylglycerol-rich Manduca sexta lipophorin, were compared in their ability to load neutral lipid from fat body . When fat body of M . sexta was incubated in vitro with {3H}oleic acid, all radiolabeled fatty acids were esterified, predominantly to triacylglycerol . In A . aegypti fat body, however, half of the label remained as free fatty acids . When A . aegypti fat body was radiolabeled with {3H}glycerol, most of the radiolabel was incorporated in triacylglycerol . When either A . aegypti or M . sexta lipophorin was incubated with A . aegypti fat body, labeled with {3H}oleic acid, both lipophorins incorporated mainly radiolabeled free fatty acids, while almost no radiolabeled glycerides were transferred . When the same experiment was performed with A . aegypti fat body, radiolabeled with {3H}glycerol, very little transfer of radiolabeled glycerides was detected . In contrast, when either M . sexta or A . aegypti lipophorin was incubated with M . sexta fat body, both lipophorins incorporated neutral lipids, predominantly diacyglycerol . A . aegypti lipophorin incorporated half the amount of radiolabeled lipid, compared to M . sexta lipophorin . Lipophorins from both species were treated with triacylglycerol lipase of the yeast Candida cylindracea . Although this lipase readily delipidated M . sexta HDLp, it was not able to remove triacylglycerol from A . aegypti HDLp . The data presented suggest that, under the conditions used, lipid transfer from fat body to lipophorin in A . aegypti is not as efficient as in M . sexta.

Cytometry, 1996 May 1, 24(1), 39 - 48
Development of a monoclonal antibody to immuno-cytochemical analysis of the cellular localization of the peripheral benzodiazepine receptor; Dussossoy D et al.; Based on the amino acid sequence deduced from the cloned human peripheral benzodiazepine receptor (PBR) gene, monoclonal antibody (Mab 8D7) was produced against the C-terminal fragment of the receptor . Immunoblot experiments, performed against purified PBR, indicated that the antipeptide antibody recognized, under denaturing conditions, the corresponding amino acid sequence of the PBR . When mitochondrial membranes from PBR transfected yeast or from THP1 and U937 cells were used on immunoblot analysis, a high level of immunoreactivity was observed at 18 kDa, the PBR molecular mass deduced from cDNA, establishing the specificity of the antibody for the receptor . Moreover, binding experiments realized with intact mitochondria demonstrated that the immunogenic sequence was accessible to the antibody indicating that the C-terminal fragment of the PBR faces the cytosol . Using this Mab we developed a technique which allowed precise quantification of PBR density per cell . Furthermore, cellular localization studies by flow cytometric analysis and confocal microscopy on cell lines displaying different levels of PBR showed that Mab 8D7 was entirely colocalized with an antimitochondria Mab.

Clin Infect Dis, 1996 May, 22 Suppl 2, S102 - 11
Histoplasmosis and blastomycosis; Bradsher RW; Histoplasmosis and blastomycosis are caused by dimorphic fungi, can be epidemic or endemic, and can produce a spectrum of illness, from subclinical infection to progressive disseminated disease . Diagnosis of both is best made by visualization of yeast in tissue or by culture . Itraconazole is the drug of choice for treatment of both histoplasmosis and blastomycosis, except in cases of life-threatening infection, for which amphotericin B is indicated . A heavy inoculum of Histoplasma capsulatum may cause acute pulmonary infection in an otherwise healthy host, resulting in fever, hypoxia, and pulmonary infiltrates . Opportunistic histoplasmosis develops as chronic pulmonary histoplasmosis in those with a structural defect in the lung (emphysema) or as disseminated histoplasmosis in patients with cellular immune deficiency (due to immunosuppressants or AIDS) . Blastomyces dermatitidis causes both pulmonary and extrapulmonary disease . Lung involvement may mimic bacterial pneumonia, while chronic presentations mimic lung cancer or tuberculosis . Skin is the most common extrapulmonary site of disease, followed by bone, prostate, and central nervous system.

Genetics, 1996 May, 143(1), 237 - 47
The Caenorhabditis elegans sel-1 gene, a negative regulator of lin-12 and glp-1, encodes a predicted extracellular protein; Grant B et al.; The Caenorhabditis elegans lin-12 and glp-1 genes encode members of the LIN-12/NOTCH family of receptors . The sel-1 gene was identified as an extragenic suppressor of a lin-12 hypomorphic mutant . We show in this report that the sel-1 null phenotype is wild type, except for an apparent elevation in lin-12 and glp-1 activity in sensitized genetic backgrounds, and that this genetic interaction seems to be lin-12 and glp-1 specific . We also find that sel-1 encodes a predicted extracellular protein, with a domain sharing sequence similarity to predicted proteins from humans and yeast . SEL-1 may interact with the LIN-12 and GLP-1 receptors and/or their respective ligands to down-regulate signaling.

Genetics, 1996 May, 143(1), 129 - 36
Gene conversion alone accounts for more than 90% of recombination events at the am locus of Neurospora crassa; Bowring FJ et al.; We have used closely flanking molecular markers located approximately 4 kb distal and 6 kb proximal of the am locus to investigate the incidence of crossover events associated with the generation of prototrophic recombinants in a cross heteroallelic am1 am6 . Ninety-three percent of prototrophs were generated by events that did not recombine the molecular markers, indicating that simple conversion accounts for the formation of most prototrophs and that associated crossovers are much less frequent (approximately 0.07) than estimated previously using more distant flanking markers . This suggests that conversion and crossing over during meiosis may arise from distinct mechanisms or that if, as is widely supposed, conversion and crossing over result from alternate modes of resolution of Holliday junctions then, at least for the am locus of Neurospora, the mode of resolution is strongly biased in favor of retaining the parental association of flanking sequences . Because estimates of the association of conversion and crossing over based on more distant gene markers are similar for yeast and Neurospora (approximately 0.35), our observation may have general significance.

Mol Reprod Dev, 1996 May, 44(1), 56 - 62
YAC transgenesis in farm animals: rescue of albinism in rabbits; Brem G et al.; The generation of transgenic mice with mammalian genes cloned in yeast artificial chromosomes (YACs) has generated great interest in the field of gene transfer into livestock . Many of the problems associated with standard transgenesis-such as lack of crucial regulator elements and position effects related to the integration site, which lead to variation in expression levels irrespective of the dose of the transgene-have been practically overcome . The large size of YAC-derived gene constructs (in excess of 1 Mb) facilitates the presence and transfer of all elements required for the faithful regulation of a gene . With the experiments discussed in this report, we have addressed the possibility of applying the obvious advantages of YAC transgenesis to farm animals . We have generated transgenic rabbits carrying a 250 kb YAC covering the mouse tyrosinase gene by pronuclear microinjection, and thus rescued the albino phenotype of the transgenic individuals . To date, this is the first demonstration of a successful transfer of large genetic units into the germ line of farm animals . This development might improve the occurrence of transgene expression at physiological levels and specific sites in livestock . YAC transgenesis therefore will be applied in genetic engineering, for example, in the production of pharmacologically interesting proteins encoded by large gene units and generating transgenic donors for xenotransplantation.

J Microsc, 1996 May, 182 ( Pt 2), 79 - 83
Soft-X-ray damage to biological samples; Fujisaki H et al.; X-ray damage to biological samples was investigated in the wavelength region of 2.7-5 nm, which overlaps the so-called 'water-window', the wavelength range of 2.4-4.3 nm usually used in X-ray microscopy . Yeast cells and myofibrils were chosen as representatives of whole cell samples and motile protein systems, respectively . The samples were exposed to X-rays using an apparatus composed mainly of a laser-plasma X-ray source, a Wolter mirror condenser, and a sample cell . The yeast cells lost their dye exclusion ability when the X-ray flux was higher than 1 x 10(6) photons micron-2, while the myofibrils lost contractility when the X-ray flux was higher than 4 x 10(5) photons micron-2 . These X-ray fluxes are lower than the flux required for the X-ray microscope observation of biological samples at a resolution higher than that of light microscopes.

Insect Mol Biol, 1996 May, 5(2), 109 - 17
The polyubiquitin gene of the mosquito Anopheles gambiae: structure and expression; Beard CB et al.; The polyubiquitin gene from the mosquito Anopheles gambiae has been cloned and sequenced, and its structure is reported along with sequence analysis results . The gene consists of approximately seven tandem head-to-tail repeat units of the seventy-six amino acid-coding ubiquitin monomer . It is expressed constitutively in larvae, pupae and adults of An . gambiae, as well as in a cell line derived from this mosquito species . A probe made from a DNA fragment containing the coding region of the gene recognizes transcripts of approximately 3.6 kb and 4.4 kb in RNA isolated from all mosquito developmental stages and a unique transcript of approximately 3.0 kb in RNA from the cell line . Single monomeric units of the An . gambiae polyubiquitin gene shared from 75.9% to 85.5% identity at the DNA level with homologous sequences from other organisms ranging from yeast to man . A comparison of individual repeat units of the An . gambiae gene revealed that, in general, the 5' ends of the individual monomers are more highly conserved than the 3' ends . The gene mapped by in situ hybridization on ovarian nurse cell polytene chromosomes to a primary site at division 12C on chromosome 2R and to a secondary site at division 9C on the same chromosome.

Nat Genet, 1996 May, 13(1), 105 - 8
Identification of the gene FMR2, associated with FRAXE mental retardation; Gecz J et al.; Five folate-sensitive fragile sites have been characterized at the molecular level (FRAXA, FRAXE, FRAXF, FRA16A and FRA11B) . Three of them (FRAXA, FRAXE and FRA11B) are associated with clinical problems, and two of the genes (FMR1 in FRAXA and CBL2 in FRA11B) have been identified . All of these fragile sites are associated with (CCG)n/(CGG)n triplet expansions which are hypermethylated beyond a critical size . FRAXE is a rare folate sensitive fragile site only recently recognized . Its cytogenetic expression was found to involve the amplification of a (CCG)n repeat adjacent to a CpG island . Normal alleles vary from 6 to 25 copies . Expansions of greater than 200 copies were found in FRAXE expressing males and their FRAXE associated CpG island was fully methylated . An association of FRAXE expression with concurrent methylation of the CpG island and mild non-specific mental handicap in males has been reported by several groups . We now report the cloning and characterization of a gene (FMR2) adjacent to FRAXE . Elements of FMR2 were initially identified from sequences deleted from a developmentally delayed boy . We correlate loss of FMR2 expression with (CCG)n expansion at FRAXE, demonstrating that this is a gene associated with the CpG island adjacent to FRAXE and contributes for FRAXE-associated mild mental retardation.

Z Naturforsch {C}, 1996 May-Jun, 51(5-6), 404 - 8
Aspects of the cell growth of Candida guilliermondii in sugar cane bagasse hydrolysate; Molwitz M et al.; In this work the behavior of the growth of Candida guilliermondii FTI 20037 in sugar cane bagasse hemicellulosic hydrolysate on various oxygen transfer rates was investigated . The yeast was able to grow and produced xylitol at different performance levels . At 1.0 vvm (volume of air per volume of medium per minute) the highest growth with 24.4 g/l was observed, but no xylitol was produced . At aeration rate of 0.5 vvm the growth was lower, but therefore slight amounts of xylitol (xylitol yield factor-Yp/s = 0.15 g/g) were observed . The lowest cell concentration (10.7 g/l) and the highest xylitol yield (Yp/s = 0.46 g/g) was observed when aeration was changed from 0.5 vvm to 0.05 vvm after 14 h.

Curr Genet, 1996 May, 29(6), 537 - 48
The regulatory protein NIT4 that mediates nitrate induction in Neurospora crassa contains a complex tripartite activation domain with a novel leucine-rich, acidic motif; Feng B et al.; Expression of nit-3 and nit-6, the structural genes which encode nitrate reductase and nitrite reductase in Neurospora crassa, requires the global-acting NIT2 and the pathway specific NIT4 regulatory proteins . NIT4, which consists of 1090 amino-acid residues, possesses a Cys6/Zn2 zinc cluster DNA-binding-domain . NIT4 was dissected to localize transactivation domains by fusion of various segments of NIT4 to the DNA-binding domain of GAL4 for in vivo analysis in yeast . Three separate activation subdomains, and one negative-acting region, which function in yeast were located in the carboxyl-terminal region of NIT4 . The C-terminal tail of 28 amino-acid residues was identified as a minimal activation domain and consists of a novel leucine-rich, acidic region . Most deletions which removed even small segments of the NIT4 protein were found to lead to the loss of NIT4 function in vivo in N . crassa, implying that the central region of the protein which lies between the DNA-binding and activation domains is essential for function . The yeast two-hybrid system was employed to identify regions of NIT4 responsible for dimer formation . A short isoleucine-rich segment downstream from the zinc cluster, predicted to form a coiled coil, allowed dimerization in vivo; this same isoleucine-rich region also showed dimerization in vitro when examined via chemical cross linking . The enzyme nitrate reductase has been postulated to exert autogenous regulation by directly interacting with the NIT4 protein . This possible nitrate reductase-NIT4 interaction was investigated with the yeast two-hybrid system and by direct in vitro binding assays; both assays failed to identify such a protein-protein interaction.

Genomics, 1996 May 1, 33(3), 488 - 97
A 2-megabase physical contig incorporating 43 DNA markers on the human X chromosome at p11.23-p11.22 from ZNF21 to DXS255; Boycott KM et al.; A comprehensive physical contig of yeast artificial chromosomes (YACs) and cosmid clones between ZNF21 and DXS255 has been constructed, spanning 2 Mb within the region Xp11.23-p11.22 . As a portion of the region was found to be particularly unstable in yeast, the integrity of the contig is dependent on additional information provided by the sequence-tagged site (STS) content of cosmid clones and DNA marker retention in conventional and radiation hybrids . The contig was formatted with 43 DNA markers, including 19 new STSs from YAC insert ends and an internal Alu-PCR product . The density of STSs across the contig ranges from one marker every 20 kb to one every 60 kb, with an average density of one marker every 50 kb . The relative order of previously known genes and expressed sequence tags in this region is predicted to be Xpter-ZNF21-DXS7465E (MG66)-DXS7927E (MG81)-WASP, DXS1011E, DXS7467E (MG21)-DXS- 7466E (MG44)-GATA1-DXS7469E (Xp664)-TFE3-SYP (DXS1007E)-Xcen . This contig extends the coverage in Xp11 and provides a framework for the future identification and mapping of new genes, as well as the resources for developing DNA sequencing templates.

Genomics, 1996 May 1, 33(3), 473 - 9
Structure of the human type IV collagen COL4A6 gene, which is mutated in Alport syndrome-associated leiomyomatosis; Zhang X et al.; Basement membrane (type IV) collagen, a subfamily of the collagen protein family, is encoded by six distinct genes in mammals . Three of those, COL4A3, COL4A4, and COL4A5, are linked with Alport syndrome (hereditary nephritis) . Patients with leimoyomatosis associated with Alport syndrome have been shown to have deletions in the 5' end of the COL4A6 gene, in addition to having deletions in COL4A5 (Zhou et al., Science 261: 1167-1169, 1993) . The human COL4A6 gene is reported to be 425 kb as determined by mapping of overlapping YAC clones by probes for its 5' and 3' ends . In the present study we describe the complete exon/intron size pattern of the human COL4A6 gene . The 12 lambda phage clones characterized in the study spanned a total of 110 kb, including 85 kb of the actual gene and 25 kb of flanking sequences . The overlapping clones contained all 46 exons of the gene and all introns, except for intron 2 . Since the total size of the exons and all introns except for intron 2 is about 85 kb, intron 2 must be about 340 kb . All exons of the gene were assigned to EcoRI restriction fragments to facilitate analysis of the gene in patients with leiomyomatosis associated with Alport syndrome . The exon size pattern of COL4A6 is highly homologous with that of the human and mouse COL4A2 genes, with 27 of the 46 exons of COL4A6 being identical in size between the genes.

Hum Genet, 1996 May, 97(5), 655 - 8
Human chromosome 1 localization of the gene for a prostaglandin F2alpha receptor negative regulatory protein; Orlicky DJ et al.; A protein that copurifies with the bovine prostaglandin F2alpha (FP) receptor has been isolated and the corresponding rat cDNA has been cloned . Transfection experiments suggest that this protein inhibits binding of {3H}prostaglandin F2alpha ({3H}PGF2alpha) to FP . Histologically, this protein (FP regulatory protein or FPRP) shows a distribution coinciding well with those cells and tissues that respond to PGF2alpha . A portion of the 3' untranslated region of the human homolog to fprp was subcloned, sequenced, and oligonucleotide primers chosen that allow polymerase chain reaction (PCR) amplification specifically of the human fprp sequence . These primers were then used in a PCR-based mapping-protocol . The human fprp gene was first socalized through human/rodent somatic cell hybrids to human chromosome 1 (100% concordance), and further through yeast artificial chromosome (YAC) pools to region 1p13.1-q21.3 (level 1 mapping) . In view of the specific histologic localization of this negative regulator, possible pathological conditions are mentioned that may cosegrepate with this chromosomal region.

Hum Genet, 1996 May, 97(5), 604 - 10
Generation of sequence-tagged sites from Xp22.3 by isolating common Alu-PCR products of radiation hybrids retaining overlapping human X chromosome fragments; Glass IA et al.; Several human diseases have been mapped to Xp22.3 on the distal short arm of the human X chromosome, and many genes in this area have been found to be expressed from the inactive X chromosome . To facilitate physical mapping and characterization of this interesting region, we have constructed a battery of radiation hybrids containing human X chromosomal fragments, and isolated two hybrid clones A with overlapping fragments of Xp22.3 . Alu-PCR on these hybrids and identification of sequences common to both hybrids allowed the isolation of six sequences-tagged sites (STSs) from Xp22.3 . Five of the STSs were mapped+ to individual YACs comprising a recently constructed contig of this region . These novel STSs are useful markers for further physical characterization of this part of the genome.

Genes Dev, 1996 May 1, 10(9), 1084 - 95
p150Ship, a signal transduction molecule with inositol polyphosphate-5-phosphatase activity; Lioubin MN et al.; The production, survival, and function of monocytes and macrophages is regulated by the macrophage colony-stimulating factor (M-CSF or CSF-1) through its tyrosine kinase receptor Fms . Binding of M-CSF to Fms induces the tyrosine phosphorylation and association of a 150-kD protein with the phosphotyrosine-binding (PTB) domain of Shc . We have cloned p150 using a modified yeast two-hybrid screen . p150 contains one SH2 domain, two potential PTB-binding sites, an ATP/GTP-binding domain, several potential SH3-binding sites, and a domain with homology to inositol polyphosphate-5-phosphatases . p150 antibodies detect this protein in FDC-P1 myeloid cells, but the same protein is not detectable in fibroblasts . The antibodies immunoprecipitate a 150-kD protein from quiescent or M-CSF-stimulated FDC-P1 cells that hydrolyzes PtdIns(3,4,5)P3, to PtdIns(3,4)P2 . This activity is observed in Shc immunoprecipitates only after M-CSF stimulation . Retroviral expression of p15O in FD-Fms cells results in strong inhibition of cell growth in M-CSF and a lesser inhibition in IL-3 . Ectopic expression of p150 in fibroblasts does not inhibit growth . This novel protein, p150(ship) (SH2-containing inositol phosphatase), identifies a component of a new growth factor-receptor signaling pathway in hematopoietic cells.

Plant J, 1996 May, 9(5), 755 - 65
Detailed description of four YAC contigs representing 17 Mb of chromosome 4 of Arabidopsis thaliana ecotype Columbia; Schmidt R et al.; The detailed arrangement of 563 YAC clones comprising four contigs covering approximately 17 Mbp of chromosome 4 is presented . YAC clones were positioned relative to each other and to markers by taking into account marker and end fragment hybridization data and the sizes of all YAC clones . This analysis made it possible to estimate physical distances between the majority of chromosome 4 markers . It also identified a relatively large number of YAC clones containing chimaeric inserts . The YAC contig map of the Columbia ecotype presents an important resource for map-based cloning experiments, rapid mapping of DNA sequences and large-scale genomic sequencing programs.

Eur J Biochem, 1996 May 1, 237(3), 691 - 7
Characterization of DNA ligase from the fungus Coprinus cinereus; Matsuda S et al.; DNA ligase was highly purified from the fungus Coprinus cinereus at the miotic recombination stage, pachytene . The pachytene DNA ligase showed three polypeptides with molecular masses of 88, 84 and 80 kDa, as estimated by the {32P}AMP-labeling assay . These three polypeptides were susceptible to reaction with an mAb against a 16-amino-acid sequence in human DNA ligase I, which is conserved in C-terminal regions of mammalian, vaccinia virus and yeast DNA ligases . Since rapidly purified preparations from fresh pachytene cells exhibited a single polypeptide of DNA ligase with a molecular mass of 88 kDa, the smaller polypeptides seemed to be limited-degradation products of the 88-kDa polypeptide during the isolation and purification procedures . K(m) values for ATP and (dT)20 hybridized with (dA)n were 1.5 microM and 90 nM, respectively . This enzyme was capable of joining (dT)20.(rA)n and (rA)12-18 (dT)n as well as (dT)20.(dA)n and able to ligate blunt-ended DNA in the presence of poly(ethylene glycol) 6000 . DNA ligases were also partially purified from zygotene cells at the meiotic pairing stage and mitotic mycelium cells . In their molecular mass, immuno-reactivity, K(m) value and substrate specificity, they were indistinguishable from pachytene DNA ligase . These results suggest that the fungus C . cinereus at the pachytene stage contains DNA ligase with a molecular mass of 88 kDa as a main or a single species, which is quite similar to DNA ligases from the zygotene and mycelium cells in molecular and catalytic properties.

EMBO J, 1996 May 1, 15(9), 2217 - 26
RNA polymerase I associated factor 53 binds to the nucleolar transcription factor UBF and functions in specific rDNA transcription; Hanada K et al.; Mouse RNA polymerase I (Pol I) has, besides its 11 bona fide subunits, three polymerase associated factors, termed PAF53, 51 and 49 with respect to the size of each molecule . In order to analyze the function of PAFs, cDNA encoding PAF53 was isolated using an oligonucleotide probe derived from an oligopeptide sequence . The cDNA of PAF53 predicts a polypeptide of 434 amino acids with a sequence similarity to yeast Pol 1 49 kDa subunit . Anti-PAF53 antibody does not block the random transcription activity of Pol I, but blocks specific transcription from mouse ribosomal RNA promoter, demonstrating the requirement of PAF53 in the accurate initiation of Pol I transcription . Moreover, PAF53 interacted with mouse UBF in vitro, as revealed by Far-Western blotting and GST pull down assays . These results, together with the accumulation of PAF53 in the nucleolus of growing cells, suggest that PAF53 is involved in the formation of the initiation complex at the promoter by mediating the interaction between Pol I and UBF for the active rRNA synthesis.

J Cell Biol, 1996 May, 133(3), 647 - 55
Neuroglian-mediated cell adhesion induces assembly of the membrane skeleton at cell contact sites; Dubreuil RR et al.; The protein ankyrin links integral membrane proteins to the spectrin-based membrane skeleton . Ankyrin is often concentrated within restricted membrane domains of polarized epithelia and neurons, but the mechanisms responsible for membrane targeting and its segregation within a continuous lipid bilayer remain unexplained . We provide evidence that neuroglian, a cell adhesion molecule related to L1 and neurofascin, can transmit positional information directly to ankyrin and thereby polarize its distribution in Drosophila S2 tissue culture cells . Ankyrin was not normally associated with the plasma membrane of these cells . Upon expression of an inducible neuroglian minigene, however, cells aggregated into large clusters and ankyrin became concentrated at sites of cell-cell contact . Spectrin was also recruited to sites of cell contact in response to neuroglian expression . The accumulation of ankyrin at cell contacts required the presence of the cytoplasmic domain of neuroglian since a glycosyl phosphatidylinositol-linked form of neuroglian failed to recruit ankyrin to sites of cell-cell contact . Double-labeling experiments revealed that, whereas ankyrin was strictly associated with sites of cell-cell contact, neuroglian was more broadly distributed over the cell surface . A direct interaction between neuroglian and ankyrin was demonstrated using yeast two-hybrid analysis . Thus, neuroglian appears to be activated by extracellular adhesion so that ankyrin and the membrane skeleton selectively associate with sites of cell contact and not with other regions of the plasma membrane.

J Pharmacol Exp Ther, 1996 May, 277(2), 685 - 90
1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is N-demethylated by cytochromes P450 2D6, 1A2 and 3A4--implications for susceptibility to Parkinson's disease; Coleman T et al.; 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induces a Parkinson-like syndrome through biotransformation by monoamine oxidase B to the neurotoxic metabolite 1-methyl-4-phenylpyridine . Neuroprotection may be provided by parallel N-demethylation and N-oxidation pathways mediated by the microsomal cytochrome P450 and flavin monooxygenase systems, respectively . The aims of this study were to characterise the N-demethylation of MPTP by human liver microsomes over a wide range of concentrations, and to identify the cytochrome P450 enzymes involved in this reaction . The kinetics of the N-demethylation of MPTP (1 microM - 3 mM) by microsomes from the liver of an extensive metabolizer with respect to cytochrome P4502D6 (CYP2D6) activity were biphasic (apparent Km1 and Km2 values = 48 and 2882 microM) . The high affinity activity was abolished in the presence of quinidine (1 microM) and was absent in microsomes from a genotypically poor metabolizer with respect to CYP2D6 . Yeast microsomes containing heterologously expressed CYP2D6 N-demethylated MPTP (Km = 39 microM), and there was a high correlation between the quinidine-inhibitable N-demethylation of MPTP (50 microM) (0.7-91%, mean 44%, of total activity) and the alpha-hydroxylation of metoprolol in microsomes from 11 human livers (rs = 0.92; P < .001) . At 50 microM MPTP, N-demethylase activity in human liver microsomes was also inhibited by furafylline (10 microM) and ketoconazole (2 microM) (mean inhibition 39 and 13%, respectively; n = 11 livers) . Yeast microsomes containing heterologously expressed human CYP1A2 N-demethylated MPTP with a Km of 2246 microM . These findings indicate that CYP2D6, CYP1A2 and, to a lesser extent CYP3A4, may have a role in protecting against Parkinson's disease induced by MPTP and other potential environmental neurotoxins . The data provide some biochemical support for the proposition that genotypically poor metabolizers with respect to CYP2D6 are overrepresented in some populations of Parkinson's patients, and that smokers (induced CYP1A2?) are underrepresented.

J Histochem Cytochem, 1996 May, 44(5), 525 - 9
An evaluation of a new series of fluorescent dUTPs for fluorescence in situ hybridization; Wiegant J et al.; Synthesis of fluorochrome-modified deoxyribonucleotides has been carried out mostly by linking the fluorochrome molecule to the C-5 position of dUTP via an allylamine spacer, similar to the modification of allylamine-dUTP with the haptens biotin and digoxigenin . Recently, a new series of fluorescent nucleotides has been prepared by using an alkynyl bridge between the uracil moiety and the fluorochrome . Here we report the qualitative and quantitative analysis of fluorescence in situ hybridization results obtained on interphase cells and chromosomes with a variety of highly repetitive and single-copy DNA probes that were modified by nick translation with such alkynyl dUTPs . A qualitative comparison was made of the alkynyl dUTPs conjugated to the fluorochromes fluorescein, the cyanine dye Cy3, tetramethylrhodamine, Lissamine and Texas Red . With the exception of tetramethylrhodamine, all fluorochromes performed satisfactorily . The cyanine dye Cy3 provided the highest sensitivity, i.e., cosmid and YAC probes could easily be visualized by conventional fluorescence microscopy . In a quantitative assay, different nick translation conditions were tested using a human chromosome 1 satellite III probe (pUC1.77) and alkynyl dUTPs labeled with fluorescein and Cy3 . Using these two nucleotides, FISH signal intensities on interphase nuclei from human lymphocytes were quantitated by digital imaging microscopy . The strongest signals were obtained when during nick translation the ratio between dTTP and fluorescein-dUTP or Cy3-dUTP was 1:5.

Development, 1996 May, 122(5), 1489 - 98
Altering cell fates in sea urchin embryos by overexpressing SpOtx, an orthodenticle-related protein; Mao CA et al.; While many general features of cell fate specification in the sea urchin embryo are understood, specific factors associated with these events remain unidentified . SpOtx, an orthodenticle-related protein, has been implicated as a transcriptional activator of the aboral ectoderm-specific Spec2a gene . Here, we present evidence that SpOtx has the potential to alter cell fates . SpOtx was found in the cytoplasm of early cleavage stage embryos and was translocated into nuclei between the 60- and 120-cell stage, coincident with Spec gene activation . Eggs injected with SpOtx mRNA developed into epithelial balls of aboral ectoderm suggesting that SpOtx redirected nonaboral ectoderm cells to an aboral ectoderm fate . At least three distinct domains on SpOtx, the homeobox and regions in the N-terminal and C-terminal halves of the protein, were required for the morphological alterations . These same N-terminal and C-terminal regions were shown to be transactivation domains in a yeast transactivation assay, indicating that the biological effects of overexpressing SpOtx were due to its action as a transcription factor . Our results suggest that SpOtx is involved in aboral ectoderm differentiation by activating aboral ectoderm-specific genes and that modulating its expression can lead to changes in cell fate.

Virology, 1996 May 1, 219(1), 324 - 9
The two nonstructural proteins from wheat dwarf virus involved in viral gene expression and replication are retinoblastoma-binding proteins; Collin S et al.; Tumor-inducing viruses like simian virus 40 or the human adenovirus produce oncoproteins which interfere with the cellular retinoblastoma (Rb) tumor-suppressor protein to create an appropriate molecular environment in the nucleus for viral transcription and replication . Such a strategy has been considered to be restricted to animal viruses . Here we demonstrate that plant viruses may use similar mechanisms for recruiting host factors . Wheat dwarf virus (WDV) encodes two potential nonstructural proteins, C1 and C1:C2, both containing the consensus Rb-binding motif LeuXCysXGlu that allows the oncoproteins from animal viruses to inactivate Rb . C1:C2 is a key determinant of viral replication and V(virion)-sense expression . Using a yeast two-hybrid protein assay, we demonstrate for the first time that the C1:C2 protein from WDV interacts with a retinoblastoma protein, providing an explanation for the previously observed dependence of viral replication on an intact Rb-binding motif . We also show that C1, for which no function had been demonstrated, is required for V-sense gene expression . This suggests that V-sense expression might be dependent on the interaction of C1 with Rb . Our findings provide further evidence for the presence of transforming-like proteins in a plant virus and will help to explain the production of symptoms in a plant viral infection through a mechanism mediated by a key regulator of cell cycle and differentiation.

Diabetes, 1996 May, 45(5), 639 - 41
Mitochondrial FAD-glycerophosphate dehydrogenase and G-protein-coupled inwardly rectifying K+ channel: No evidence for linkage in maturity-onset diabetes of the young or NIDDM; Warren-Perry MG et al.; Two genes that have potentially important regulatory roles in insulin secretion are both located on chromosome 2q24.1 . G-protein-coupled muscarinic potassium channel (GIRK1) is an inwardly rectifying K+ channel that helps to maintain the resting potential and excitability of cells . Mitochondrial FAD-linked glycerophosphate dehydrogenase (m-GDH) catalyzes a rate-limiting step of the glycerol phosphate shuttle in pancreatic islets . Reduced m-GDH activity has been demonstrated in islets isolated from diabetic subjects compared with islets from nondiabetic control subjects and from the diabetic GK rat . To study the relationship between these candidate genes and NIDDM, we have examined a simple tandem-repeat polymorphism (STRP) close to both the KCN J3 (GIRK1) locus and the m-GDH locus . In a linkage study of three maturity-onset diabetes of the young (MODY) pedigrees, not linked to MODY1, MODY2, or MODY3, a cumulative score of - 9.6 at a recombination fraction of theta = 0 excluded linkage . In a population-association study, no linkage disequilibrium for the STRP was found between 190 unselected NIDDM patients and 60 geographically and age-matched white nondiabetic subjects (chi2 = 1.51 on 3 df, P = 0.68) . Thus, mutations involving the genes for GIRK1 or FAD-glycerophosphate dehydrogenase are unlikely to cause MODY, and a common mutation in either gene is unlikely to contribute to NIDDM in whites . These data do not exclude mutations in some families or other ethnic groups.

Radiat Res, 1996 May, 145(5), 632 - 5
DNA breakage upon K-shell excitation of phosphorus as a model for direct effects in radiation biology; Le Sech C et al.; Single-strand breaks (SSBs) and double-strand breaks (DSBs) induced in DNA under phosphorus K-shell resonant absorption have been studied using supercoiled plasmids . The kinetics of the production of SSBs and DSBs exhibits a linear and a quadratic dependence, respectively, on photon fluence . Cross sections and quantum yields have been measured . The resonant photoexcitation of the phosphorus atoms was found to increase the DSB/SSB ratio compared to the off-resonance excitation . This enhancement factor can be related to the measured enhancement of the rate of cellular death and gene mutation in yeast under similar experimental conditions reported previously in the literature . Such resonant excitation of a specific atom belonging to DNA turns out to be an elegant method to investigate pure direct effects.

Blood, 1996 May 1, 87(9), 3579 - 86
Molecular definition of a narrow interval at 7q22.1 associated with myelodysplasia; Johnson EJ et al.; Chromosome 7 translocations, deletions, or monosomy are associated with myelodysplasia (MDS) and acute myeloid leukemia both in children and adults . These chromosomal anomalies represent one of the most common cytogenetic abnormalities associated with these diseases and usually herald a poor prognosis . In this study two cosmid DNA probes that mapped to 7q22.1 and were known to be separated by approximately 500 kb were identified to flank the proximal inversion breakpoint in a patient carrying a constitutional inversion (7q22.1-34) associated with MDS . A yeast artificial chromosome (YAC) clone that encompassed the two cosmids was identified and shown to span the breakpoint . Fluorescence in situ hybridization was then used to analyze six additional patients with myelodysplasia and chromosomal rearrangements of the 7q22 region (three patients had translocations and three carried deletions) . The breakpoint in one of the patients was found to be contained within the same YAC clone that spanned the inversion breakpoint . Moreover, this same interval was determined to be absent in all three patients with chromosomal deletions . These results suggest that this segment of DNA on chromosome 7q22.1 may contain specific gene(s) that have a significant role in myeloid malignancies.

J Gen Virol, 1996 May, 77 ( Pt 5), 1019 - 23
Mapping the domains on the phosphoprotein of bovine respiratory syncytial virus required for N-P interaction using a two-hybrid system; Mallipeddi SK et al.; Specific interactions between the nucleocapsid protein (N) and the phosphoprotein (P) of bovine respiratory syncytial virus (BRSV) have been investigated using a yeast-based two-hybrid system . Plasmids encoding the yeast GAL4 DNA binding domain fused with the N gene and GAL4 activation domain fused with the P gene were cotransfected into competent yeast cells . The ability of the N and P proteins to interact in vivo was measured by activation of the lacZ reporter gene by the GAL4 transactivation region . Results indicated that the N and P proteins interact very strongly in vivo . When interactions between N and various deletion mutants of the P protein were examined, an internal region (aa 132-168) and the highly acidic C-terminal region (aa 236-241) of the P protein were found to be essential for N-P interaction . In addition, the highly basic N-terminal region (amino acids 1-40) was found to be involved in N-P interaction to a lesser extent.

Biochemistry, 1996 Apr 30, 35(17), 5385 - 94
Functional characterization of the ubiquitin variant encoded by the baculovirus Autographa californica; Haas AL et al.; The marked evolutionary conservation of ubiquitin is assumed to arise from constraints imposed by folding, stability, and interaction of the polypeptide with various components of the ATP, ubiquitin-dependent degradative pathway . The present studies characterize the most divergent (75% identity) of the species-specific ubiquitin isoforms encoded as a late gene product of the baculovirus Autographa californica {Guarino, L . A . (1990) Proc . Natl . Acad . Sci . U.S.A . 87, 409-413} . Viral ubiquitin supports 40% of the rate of ATP-dependent degradation exhibited by eukaryotic ubiquitin . Inhibition of proteolysis correlated with a lower steady-state concentration of ubiquitin-conjugated degradative intermediates . Rate studies revealed that viral ubiquitin exerts its effect at the step of isopeptide ligase-catalyzed (E3) ubiquitin conjugation since viral and eukaryotic polypeptides are identical in their abilities to support ATP-coupled activation by E1 and transthiolation to E2 carrier proteins . Other studies demonstrated viral ubiquitin severely attenuated the rate of K48-linked multiubiquitin chain formation in E3-independent conjugation catalyzed by recombination yeast CDC34 or rabbit reticulocyte E232K but not chain elongation of alternate linkages formed by yeast RAD6 or human E2EPF . The latter observations suggest nonconserved positions on viral ubiquitin constitute recognition signals for K48-linked chain formation . Sequence comparison of species-specific ubiquitin isoforms indicates that nonconserved positions localized to a defined region on the polypeptide surface distinct from the basic face required for E1 binding . These results suggest this novel ubiquitin isoform may function in baculoviral replication to block destruction of a short-lived protein(s) by the host degradative pathway, targeted through either E2-catalyzed K48-linked multibiquitin chain formation or general E3-mediated conjugation.

Mol Gen Genet, 1996 Apr 24, 251(1), 52 - 9
A 610 kb YAC clone harbors 7 cM of tomato (Lycopersicon esculentum) DNA that includes the male sterile 14 gene and a hotspot for recombination; Gorman SW et al.; Pollen development requires both sporophytic and gametophytic gene expression . We are using a map-based cloning technique to isolate sporophytic genes which, when mutant, cause pollen abortion and a male sterile (ms) phenotype in tomato (Lycopersicon esculentum) . We have genetically characterized one ms locus (ms14) using RFLP analysis and identified flanking markers . High-resolution genomic physical mapping indicates that the ms14 locus is located in a approximately 300 kb region . We have identified a YAC clone with an insert size of approximately 610 kb that contains the ms14-linked markers, reflects the organization of the physical map and therefore most probably contains the ms14 gene . In addition, we present evidence that the relationship between physical and genetic distance in this chromosomal region changes abruptly from approximately 105-140 kb/cM to less than 24kb/cM, and suggest that the TG393-TG104 region is a hotspot for recombination.

J Biol Chem, 1996 Apr 19, 271(16), 9816 - 22
PKN associates and phosphorylates the head-rod domain of neurofilament protein; Mukai H et al.; PKN is a fatty acid-activated serine/threonine kinase that has a catalytic domain highly homologous to that of protein kinase C in the carboxyl terminus and a unique regulatory region in the amino terminus . Recently, we reported that the small GTP-binding protein Rho binds to the amino-terminal region of PKN and activates PKN in a GTP-dependent manner, and we suggested that PKN is located on the downstream of Rho in the signal transduction pathway (Amano, M., Mukai, H., Ono, Y., Chihara, K., Matsui, T., Hamajima, Y., Okawa, K., Iwamatsu, A., and Kaibuchi, K . (1996) Science 271, 648-650; Watanabe, G., Saito, Y., Madaule, P., Ishizaki, T., Fujisawa, K., Morii, N., Mukai, H., Ono, Y . Kakizuka, A., and Narumiya, S . (1996) Science 271, 645-648) . To identify other components of the PKN pathway such as substrates and regulatory proteins of PKN, the yeast two-hybrid strategy was employed . By this screening, a clone encoding the neurofilament L protein, a subunit of neuron-specific intermediate filament, was isolated . The amino-terminal regulatory region of PKN was shown to associate with the head-rod domains of other subunits of neurofilament (neurofilament proteins M and H) as well as neurofilament L protein in yeast cells . The direct binding between PKN and each subunit of neurofilament was confirmed by using the in vitro translated amino-terminal region of PKN and glutathione S-transferase fusion protein containing the head-rod domain of each subunit of neurofilament . PKN purified from rat testis phosphorylated each subunit of the native neurofilament purified from bovine spinal cord and the bacterially synthesized head-rod domain of each subunit of neurofilament . Polymerization of neurofilament L protein in vitro was inhibited by phosphorylation of neurofilament L protein by PKN . The identification and characterization of the novel interaction with PKN may contribute toward the elucidation of mechanisms regulating the function of neurofilament.

J Biol Chem, 1996 Apr 19, 271(16), 9710 - 5
The N-terminal domain of tomato 3-hydroxy-3-methylglutaryl-CoA reductases . Sequence, microsomal targeting, and glycosylation; Denbow CJ et al.; The enzyme 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) catalyzes the conversion of 3-hydroxy-3-methylglutaryl-CoA to mevalonic acid, considered the rate-limiting step in isoprenoid biosynthesis . In plants, isoprenoid compounds play important roles in mediating plant growth and development, electron transport, photosynthesis, and disease resistance . Sequence comparisons of plant HMGR proteins with those from yeast and mammalian systems reveal high levels of sequence identity within the catalytic domain but significant divergence in the membrane domain . Mammalian HMGRs are integral membrane proteins of the endoplasmic reticulum with eight membrane-spanning regions . In contrast, the membrane domain of plant HMGRs is predicted to contain only one to two transmembrane spans . We have isolated and sequenced a clone (pCD4) encoding exon 1 of tomato hmg1 . The membrane domain structures of two differentially regulated tomato HMGR isoforms, HMG1 and HMG2, were analyzed using in vitro transcription and translation systems . Microsomal membrane insertion of the tomato HMGRs is co-translational and does not involve cleavage of an N-terminal targeting peptide . HMGR membrane topography was established by protease protection studies of the HMG1 membrane domain and an analogous region of HMG2 engineered to contain a c-myc epitope tag . The data indicate that both tomato HMGRs span the membrane two times with both the C and N termini located in the cytosol . Lumenal localization of the short peptide predicted to lie within the endoplasmic reticulum was further confirmed by in vitro glycosylation of an asparagine-linked glycosylation site present in HMG2.

J Biol Chem, 1996 Apr 19, 271(16), 9189 - 92
The orphan nuclear hormone receptor LXR alpha interacts with the peroxisome proliferator-activated receptor and inhibits peroxisome proliferator signaling; Miyata KS et al.; The yeast two-hybrid system was used to isolate novel cellular factors that interact with the mouse peroxisome proliferator-activated receptor alpha (PPARalpha) . One of the interacting clones isolated encoded LXRalpha, a recently described human orphan nuclear hormone receptor . LXRalpha bound directly to PPARalpha, as well as to the common heterodimerization partner 9-cis-retinoic acid receptor (RXRalpha) . LXRalpha did not form a DNA binding complex with PPARalpha on synthetic hormone response elements composed of direct repeats of the TGACCT consensus half-site or on naturally occurring peroxisome proliferator response elements (PPREs) or LXRalpha response elements . However, LXRalpha inhibited binding of PPARalpha/RXRalpha heterodimers to PPREs, and coexpression of LXRalpha in mammalian cells antagonized peroxisome proliferator signaling mediated by PPARalpha/RXRalpha in vivo . These findings identify a novel partner for PPARalpha and suggest that LXRalpha plays a role in modulating PPAR-signaling pathways in the cell.

Cancer Lett, 1996 Apr 19, 102(1-2), 85 - 90
A cDNA from the ovarian cancer critical region of deletion on chromosome 17p13.3; Phillips NJ et al.; Chromosome 17p13.3 is frequently deleted in human ovarian carcinoma, and the 15 kb critical region of deletion may contain a tumor suppressor gene . A 2.3 kb cDNA has been identified which spans 17 kb of genomic DNA, including 8.1 kb within the critical region, and thus is a candidate tumor suppressor gene . This highly conserved gene has significant sequence similarity to a yeast gene of unknown function and to one of the yeast enzymes in the diphthamide synthetic pathway, DPH2, that has a role in global protein synthesis regulation . This gene, named DPH2L (diphthamide biosynthesis protein 2-like), is expressed in multiple tissues and stages of development.

Oncogene, 1996 Apr 18, 12(8), 1803 - 8
Detailed physical and deletion mapping of 8p with isolation of YAC clones from tumour suppressor loci involved in colorectal cancer; Farrington SM et al.; Loss of heterozygosity (LOH) of markers at chromosome 8p is frequently noted in many different tumour types, including colorectal cancer . Numerous investigations indicate the presence of more than tumour suppressor gene (TSG) located on 8p . In this study, we describe a detailed LOH map in colorectal cancer and relate this to physical mapping data from reduced radiation 8p hybrids, yeast artificial chromosome (YAC) co-localisation of markers and fluorescence in situ hybridisation data . These data indicate the presence of two regions harbouring putative TSG's between the polymorphic markers for the LPL gene-D8S298 (approximately 4 Mb) and the markers D8S136-D8S137 (approximately 8 Mb) . Yeast Artificial Chromosomes (YAC) have been isolated from these regions of interest to aid the localisation of the putative TSG's.

Proc Natl Acad Sci U S A, 1996 Apr 16, 93(8), 3608 - 12
Stimulation of intrachromosomal homologous recombination in human cells by electroporation with site-specific endonucleases; Brenneman M et al.; In somatic mammalian cells, homologous recombination is a rare event . To study the effects of chromosomal breaks on frequency of homologous recombination, site-specific endonucleases were introduced into human cells by electroporation . Cell lines with a partial duplication within the HPRT (hypoxanthine phosphoribosyltransferase) gene were created through gene targeting . Homologous intrachromosomal recombination between the repeated regions of the gene can reconstruct a functioning, wild-type gene . Treatment of these cells with the restriction endonuclease Xba I, which has a recognition site within the repeated region of HPRT homology, increased the frequency or homologous recombination bv more than 10-fold . Recombination frequency was similarly increased by treatment with the rare-cutting yeast endonuclease PI-Sce I when a cleavage site was placed within the repeated region of HPRT . In contrast, four restriction enzymes that cut at positions either outside of the repeated regions or between them produced no change in recombination frequency . The results suggest that homologous recombination between intrachromosomal repeats can be specifically initiated by a double-strand break occurring within regions of homology, consistent with the predictions of a model.

J Neurosci, 1996 Apr 15, 16(8), 2488 - 94
CASK: a novel dlg/PSD95 homolog with an N-terminal calmodulin-dependent protein kinase domain identified by interaction with neurexins; Hata Y et al.; Neurexins are neuronal cell surface proteins with hundreds of isoforms . In yeast two-hybrid screens for intracellular molecules interacting with different neurexins, we identified a single interacting protein called CASK . CASK is composed of an N-terminal Ca2+, calmodulin-dependent protein kinase sequence and a C-terminal region that is similar to the intercellular junction proteins dlg-A, PSD95/SAP90, SAP97, Z01, and Z02 and that contains DHR-, SH3-, and guanylate kinase domains . CASK is enriched in brain in synaptic plasma membranes but is also detectable at low levels in all tissues tested . The cytoplasmic domains of all three neurexins bind CASK in a salt-labile interaction . In neurexin I, this interaction is dependent on the C-terminal three residues . Thus, CASK is a membrane-associated protein that combines domains found in Ca2+ - activated protein kinases and in proteins specific for intercellular junctions, suggesting that it may be a signaling molecule operating at the plasma membrane, possibly in conjunction with neurexins.

Genomics, 1996 Apr 15, 33(2), 309 - 12
Mapping of the gene for the p60 subunit of the human chromatin assembly factor (CAF1A) to the Down syndrome region of chromosome 21; Blouin JL et al.; Exon trapping was used to clone portions of genes from the Down syndrome critical region (DSCR) of human chromosome 21 . One trapped sequence showed complete homology with nucleotide sequence U20980 (GenBank), which corresponds to the gene for the p60 subunit of the human chromatin assembly factor-1 (CAF1A) . We mapped this gene to human chromosome 21 by fluorescence in situ hybridization, by the use of somatic cell hybrids, and by hybridization to chromosome 21-specific YACs and cosmids . The CAF1A gene localizes to YACs 745H11 and 230E8 of the Chumakov et al . (1992, Nature 359: 380) YAC contig, within the DSCR on 21q22 . This CAF1A, which belongs to the WD-motif family of genes and interacts with other polypeptide subunits to promote assembly of histones to replicating DNA, may contribute in a gene dosage-dependent manner to the phenotype of Down syndrome.

Genomics, 1996 Apr 15, 33(2), 289 - 91
Definition of the locus responsible for systemic carnitine deficiency within a 1.6-cM region of mouse chromosome 11 by detailed linkage analysis; Okita K et al.; Carnitine is an essential cofactor for oxidation of mitochondrial fatty acids . Carnitine deficiency results in failure of energy production by mitochondria and leads to metabolic encephalopathy, lipid-storage myopathy, and cardiomyopathy . The juvenile visceral steatosis (JVS) mouse, an animal model of systematic carnitine deficiency, inherits the JVS phenotype in autosomal recessive fashion, through a mutant allele mapped to mouse chromosome 11 . As a step toward identifying the gene responsible for JVS by positional cloning, we attempted to refine the jvs locus in the mouse by detailed linkage analysis with 13 microsatellite markers, using 190 backcross progeny . Among the 13 loci tested, 5 (defined by markers D11Mit24, D11Mit111, D11Nds9, D11Mit86, and D11Mit23) showed no recombination, with a maximum lod score of 52.38 . Our results implied that the jvs gene can be sought on mouse chromosome 11 within a genetic distance no greater than about 1.6 cM.

Genomics, 1996 Apr 15, 33(2), 258 - 70
A 9.75-Mb map across the centromere of human chromosome 10; Jackson MS et al.; We present a yeast artificial chromosome (YAC) and pulsed-field gel electrophoresis (PFGE) map across the centromere of human chromosome 10 that links expressed sequences in 10p11 to expressed sequences in 10q11.2 . This map is the first of its kind to link genes across a human centromere . It consists of a 2.5-Mb YAC contig extending from 10p11 to our previously published 5.35-Mb PFGE map of the centromeric satellite arrays, and a 2.65-Mb YAC contig extending from these satellite arrays to 10q11.2 . This map covers approximately 6.5-7% of the total DNA of chromosome 10 . Two Genethon genetic markers, D10S578 and D10S604, are included . These markers are only 1 cM apart but are separated by a physical distance of more than 9.2 Mb, including the centromere . This gives a ratio of genetic to physical distance of 0.11 cM/Mb, 9-11 times lower than average estimates for the human genome and chromosome 10 . Markers linked to the centromere include the duplicated zinc finger genes ZNF11A, ZNF33A, and ZNF37A (which map to 10p11) and ZNF11B, ZNF33B, and ZNF37B (which map to 10q11.2) . Restriction mapping confirms that the genes on each arm lie in opposite orientation with respect to the centromere, consistent with the hypothesis that a pericentric inversion has occurred in this region during primate evolution.

Genomics, 1996 Apr 15, 33(2), 153 - 8
A 4.5-megabase YAC contig and physical map over the hemochromatosis gene region; Burt MJ et al.; We have constructed a yeast artificial chromosome (YAC) contig over the candidate hemochromatosis gene region . This contig comprises 16 YACs from the CEPH, Washington University, and ICI YAC libraries and covers 4.5 Mb at 6p21.3-6p22 . The complete contig has been restriction mapped, enabling the precise relationship between the YACs to be determined and the mapping of a total of 12 STSs . Nine of these are highly polymorphic STSs that are closely linked to hemochromatosis; this series includes D6S265 and D6S1260, which comprise the most proximal and distal markers linked to HC . This is the first YAC contig that spans the hemochromatosis candidate region, and it provides valuable resource material for the cloning of this and other genes in the region distal to the MHC class I complex.

Eur J Biochem, 1996 Apr 15, 237(2), 496 - 504
Characterization of the functional gene encoding mouse class III alcohol dehydrogenase (glutathione-dependent formaldehyde dehydrogenase) and an unexpressed processed pseudogene with an intact open reading frame; Foglio MH et al.; Multiple forms of vertebrate alcohol dehydrogenase (ADH) have been identified, but only one form, class III ADH, has been conserved in all organisms studied . Class III ADH functions in vitro as a glutathione-dependent formaldehyde dehydrogenase, which suggests that this was the original function that drove the evolution of ADH . Genetic analysis of class III ADH in yeast supports this view, but such studies are lacking in higher eukaryotes . The mouse ADH family has been previously analyzed and it contains three forms of ADH including the class III enzyme . We have initiated a molecular genetic analysis of the mouse class III ADH gene (Adh-2) by screening a genomic library with a full-length cDNA . Two overlapping clones contained the complete Adh-2 gene composed of nine exons in a 12-kb region, with the placement of introns matching that observed in other mammalian ADH genes . In this screening, we also isolated a clone (psi Adh-2) that lacks introns and which resembles a processed pseudogene . psi Adh-2 contained 25 point mutations relative to the previously analyzed Adh-2 cDNA, but still retained an intact open reading frame . Northern blot analysis using gene-specific probes provided evidence that psi Adh-2 does not produce a mRNA in either liver or kidney, whereas Adh-2 does . The functionality of the two genes was also compared by fusion of their 5'-flanking regions to a lacZ reporter gene . Reporter gene expression following transfection into mouse F9 embryonal carcinoma cells indicated that only Adh-2 possesses promoter activity.

Genes Dev, 1996 Apr 15, 10(8), 997 - 1007
A human RNA helicase-like protein, HRH1, facilitates nuclear export of spliced mRNA by releasing the RNA from the spliceosome; Ohno M et al.; Because the nuclear export of mRNA occurs only after the splicing reaction is completed, intron-containing pre-mRNA does not normally appear in the cytoplasm . As a mechanism to secure this, intron-containing RNA is retained in the nucleus via formation of the spliceosome . Therefore, the process of releasing spliced mRNA from the spliceosome after completion of splicing is an essential step for triggering the nuclear export of the spliced mRNA . In budding yeast, RNA helicase-like protein Prp22 is implicated in this process . Here we demonstrate the function of HRH1, a human protein homologous to Prp22, in mammalian cells using dominant-negative HRH1++ mutants (dn-HRH1) . dn-HRH1 protein stalls on the spliceosome and prevents release of the spliced RNA from the spliceosome in vitro . Expression of dn-HRH1 in mammalian cells leads to inhibition of splicing and to extensive nuclear export of unspliced pre-mRNA, probably because of the incapability of recycling spliceosome components that normally retain the pre-mRNA in the nucleus . The arginine/serine-rich domain (RS domain) of HRH1, which is missing in Prp22, confers a nuclear localization signal, and appears to facilitate the interaction of HRH1 with the spliceosome . This is the first report on a bona fide mammalian homolog of yeast Prp splicing factor, and also on a mammalian RNA helicase-like splicing factor.

Genes Dev, 1996 Apr 15, 10(8), 963 - 73
TANK, a co-inducer with TRAF2 of TNF- and CD 40L-mediated NF-kappaB activation; Cheng G et al.; We describe a new signal mediator of NF-kappaB activation, TANK, that acts in a pathway common to two surface receptors CD40 and TNFR II . TRAF family members interact directly with these receptors . Using the yeast two-hybrid system, TANK was identified as an intracellular protein without previous homologs that interacts with all three known TRAF family members . In cotransfection experiments, TANK and TRAF2 activate NF-kappaB synergistically, requiring both the amino-terminal portion of TANK and the ring finger domain of TRAF2 . TANK has a negatively acting carboxyl terminus and is constitutively inactive, but TRAF2 binding overcomes the internal inhibitory influence . We propose that ligand binding to CD40 or TNFR II leads to the formation of a TRAF2/TANK complex, mediating NF-kappaB activation.

J Biol Chem, 1996 Apr 12, 271(15), 8971 - 6
Interaction between c-Rel and the mitogen-activated protein kinase kinase kinase 1 signaling cascade in mediating kappaB enhancer activation; Meyer CF et al.; The Rel family of transcription factors are important mediators of various cytokine stimuli such as interleukin (IL)-1, tumor necrosis factor (TNF)-alpha, and CD28 costimulation in T cell effector responses . These stimuli induce Rel family DNA-binding activity to the kappaB enhancer and CD28 response elements of many cytokine gene promoters leading to cytokine production . Consistent with the importance of Rel family induction during immune responses, c-Rel knockout mice exhibit profound defects in T cell functions including IL-2 secretion and T cell proliferative responses to CD28 plus T cell receptor costimulation . The novel protein kinases, c-Jun NH2-terminal kinases (JNKs)/stress-activated protein kinases, are also activated by TNF-alpha, IL-1, and CD28 costimulation . Because of the common regulation of c-Rel and JNK1 by these agents in T cells, we investigated the role of JNK1 in c-Rel activation . We found that MAP kinase kinase kinase (MEKK) 1, a JNK1 activator, induced transcription from the human immunodeficiency virus-1 long terminal repeat and IL-2R alpha promoters in a kappaB-dependent manner . Coexpression of IkappaBalpha, a c-Rel inhibitor, inhibited the MEKK1-induced transcriptional activity . JNK1 synergized with MEKK1 in activating transcription from a kappaB-driven heterologous promoter . Furthermore, JNK1 associated with c-Rel in vivo in Jurkat T cells by coimmunoprecipitation assays and bound directly to c-Rel in a yeast two-hybrid assay . c-Rel also competed with c-Jun in in vitro kinase assays . However, JNK1 did not phosphorylate c-Rel, NF-kappaB, and IkappaB alpha in vitro, indicating that c-Rel may serve as a docking molecule to allow JNK1 phosphorylation of certain Rel-associated proteins . Transactivation of the IL-2Ralpha and HIV-kappaB-driven promoters by c-Rel was augmented by coexpression of MEKK1 . These results demonstrate the first significant role for the MEKK1 kinase cascade module in c-Rel-mediated transcription.

J Biol Chem, 1996 Apr 12, 271(15), 8882 - 6
Interaction between the Grb10 SH2 domain and the insulin receptor carboxyl terminus; Hansen H et al.; Grb10 is a member of a recently identified family of adapter proteins that are thought to play a role in receptor tyrosine kinase-mediated signal transduction . We identified and isolated the Grb10 SH2 domain based on its interaction with the intracellular domain of the insulin receptor beta-subunit using the yeast two-hybrid system . The interaction was specific for the insulin receptor and the insulin-like growth factor-1 receptor, and it required a catalytically active receptor kinase domain and an intact Grb10 SH2 domain . Glutathione S-transferase fusion proteins containing the Grb10 SH2 domain associated in an insulin-dependent manner with insulin receptors from cell lysates and with purified insulin receptors . Co-precipitation experiments revealed the association of cellular Grb10 with hormone-stimulated insulin receptors in cell extracts . The Grb10 SH2 domain did not bind to an insulin receptor lacking 43 amino acids at the carboxyl terminus, and it exhibited highest affinity for a phosphopeptide containing Tyr(P)-1322 . Unlike p85 and Syp, which also bind to Tyr(P)-1322, Grb10 was not found to associate with insulin receptor substrate-1 . These results suggest that Grb10 is a novel insulin receptor interactive protein and provide direct evidence for an insulin receptor substrate-1-independent function of the insulin receptor carboxyl terminus in protein binding.

J Biol Chem, 1996 Apr 12, 271(15), 8627 - 32
Interactions of cellular polypeptides with the cytoplasmic domain of the mouse Fas antigen; Orlinick JR et al.; The mouse Fas/APO-1 antigen represents a 45-kilodalton transmembrane receptor that initiates apoptosis by a poorly defined signaling mechanism . The cytoplasmic domain of Fas does not display any known enzymatic activities but is capable of interacting with a number of proteins that were identified recently using the yeast interactive cloning method . To investigate direct biochemical interactions from cellular lysates prepared from Fas-responsive cells, a series of recombinant glutathione S-transferase-mouse Fas fusion proteins representing different regions of the mouse Fas cytoplasmic domain was used . Polypeptides of 25, 50, and 70 kilodaltons were found to associate with the Fas intracellular domain, and this binding was stable in the presence of 1 M NaCl . These interactions were also detected using a mouse Fas fusion protein containing an Ile to Asn mutation, which is responsible for a lymphoproliferative disorder in certain strains of mice (lprcg) . Furthermore, the binding of cellular proteins to Fas could be blocked upon incubation with a polyclonal antibody directed against the cytoplasmic domain of Fas . The strong association of cellular proteins with the cytoplasmic region implies that constitutive interactions may exist to regulate apoptotic signaling through the Fas antigen.

J Biol Chem, 1996 Apr 12, 271(15), 8564 - 9
Sos, Vav, and C3G participate in B cell receptor-induced signaling pathways and differentially associate with Shc-Grb2, Crk, and Crk-L adaptors; Smit L et al.; B cell antigen receptor (BCR)-mediated signal transduction controls B cell proliferation and differentiation . The BCR activates Ras, presumably by the formation of a Shc-Grb2 adaptor complex, which recruits the Grb2-associated guanine nucleotide exchange factor Sos to the plasma membrane . In order to reveal additional BCR-induced signaling events involving the Grb2 adaptor, we undertook the isolation of Grb2-binding proteins . Using the yeast two-hybrid system and bacterial fusion proteins, Vav and C3G were identified as Grb2 binders . Vav is a putative nucleotide exchange factor and a target for BCR-induced tyrosine phosphorylation . C3G exerts nucleotide exchange activity on the Ras-related Rap1 protein . While Sos binds to both Grb2 Src homology-3 (SH3) domains, Vav was found to associate selectively with the carboxyl-terminal SH3 domain, while C3G bound selectively to the amino-terminal SH3 domain of bacterially expressed Grb2 . Despite the association of Vav with Grb2 in vitro, we could not demonstrate an interaction between endogenous Vav and Grb2 molecules in primary B cells . Instead, Vav was found to inducibly associate with the Grb2-related adaptor protein Crk upon BCR stimulation . C3G did not bind to either Grb2, Shc, or Crk in vivo . Instead, C3G was found in association with the Crk-L adaptor, both before and after BCR stimulation . We show that Crk-L also participates in BCR signaling, since it inducibly interacts with tyrosine-phosphorylated Cbl . We conclude that, in addition to Sos, Vav and C3G play a role in BCR-mediated signal transduction . These guanine nucleotide exchange factors selectively associate with Grb2, Crk, and Crk-L, respectively, which may serve to direct them to different target molecules . Since Cbl binds to Grb2, Crk, as well as Crk-L, we hypothesize that Cbl may affect the function of all three exchangers.

Nature, 1996 Apr 11, 380(6574), 544 - 7
A human peptidyl-prolyl isomerase essential for regulation of mitosis; Lu KP et al.; The NIMA kinase is essential for progression through mitosis in Aspergillus nidulans, and there is evidence for a similar pathway in other eukaryotic cells . Here we describe the human protein Pin1, a peptidyl-prolyl cis/trans isomerase (PPIase) that interacts with NIMA . PPIases are important in protein folding, assembly and/or transport, but none has so far been shown to be required for cell viability . Pin1 is nuclear PPIase containing a WW protein interaction domain, and is structurally and functionally related to Ess1/Ptf1, an essential protein in budding yeast . PPIase activity is necessary for Ess1/Pin1 function in yeast . Depletion of Pin1/Ess1 from yeast or HeLa cells induces mitotic arrest, whereas HeLa cells overexpressing Pin1 arrest in the G2 phase of the cell cycle . Pin1 is thus an essential PPIase that regulates mitosis presumably by interacting with NIMA and attenuating its mitosis-promoting activity.

Mol Gen Genet, 1996 Apr 10, 250(6), 681 - 91
Molecular analysis of the Arabidopsis pattern formation of gene GNOM: gene structure and intragenic complementation; Busch M et al.; The GNOM gene is required for pattern formation along the main body axis of the embryo in the flowering plant Arabidopsis thaliana . Mutations in the GNOM gene alter the asymmetric division of the zygote and interfere with the formation of distinct apical-basal regions in the developing embryo . We have isolated the GNOM gene by positional cloning, characterised its structure and determined the molecular lesions in mutant alleles . Although the predicted 163 kDa GNOM protein has a conserved domain in common with the yeast secretory protein Sec7p, it is most closely related in size and overall similarity to the product of the yeast YEC2 gene, which is not essential for cell viability . Four fully complementing gnom alleles carry missense mutations in conserved regions, seven partially complementing alleles have premature stop codon mutations and two non-complementing alleles have splice-site lesions . Our results suggest that the GNOM protein acts as a complex of identical subunits and that partial complementation may involve low levels of full-length protein generated by inefficient translational read-through.

Biochim Biophys Acta, 1996 Apr 10, 1306(1), 5 - 8
The HIR protein family: isolation and characterization of a complete murine cDNA; Scamps C et al.; A full-length cDNA has been isolated for the murine homolog of the human HIRA protein, a member of the HIR family of nuclear proteins that is encoded from the chromosome 22 region critical for the DiGeorge syndrome . This family also contains Hir1p and Hir2p, two proteins identified as regulators of histone gene transcription in yeast . The murine and human amino acid sequences are 95.3% identical, with a striking 99.2% identity in the N-terminal WD repeat domain that is characteristic of the family . The two cDNAs are highly conserved within the coding regions, but also in the entire 5' untranslated region and in a strikingly long stretch of nucleotides in the 3' untranslated region.

FEBS Lett, 1996 Apr 8, 384(1), 61 - 4
A20, an inhibitor of cell death, self-associates by its zinc finger domain; De Valck D et al.; A20 is a primary response gene which is induced after monocyte adherence or cytokine stimulation of a variety of cells . The A20 protein belongs to a novel class of Cys2/Cys2 zinc finger proteins, and has been characterized as an inhibitor of both apoptotic and necrotic cell death . In order to clarify its molecular mechanism of action, we used the yeast-based two-hybrid system to screen for A20-associated proteins . Here we report that A20 is able to self-associate, and demonstrate that the latter interaction is mediated by its zinc finger domain.

J Biol Chem, 1996 Apr 5, 271(14), 8488 - 92
Identification of mitogen-activated protein (MAP) kinase-activated protein kinase-3, a novel substrate of CSBP p38 MAP kinase; McLaughlin MM et al.; CSBP p38 is a mitogen-activated protein kinase that is activated in response to stress, endotoxin, interleukin 1, and tumor necrosis factor . Using a catalytically inactive mutant (D168A) of human CSBP2 as the bait in a yeast two-hybrid screen, we have identified and cloned a novel kinase which shares approximately 70% amino acid identity to mitogen-activated protein kinase-activated protein kinase (MAPKAP kinase)-2, and thus was designated MAPKAP kinase-3 . The binding of CSBP to MAPKAP kinase-3 was confirmed in vitro by the precipitation of epitope-tagged CSBP1, CSBP2, and CSBP2(D168A) and endogenous CSBP from mammalian cells by a bacterially expressed GST-MAPKAP kinase-3 fusion protein and in vivo by co-precipitation of the epitope-tagged proteins co-expressed in HeLa cells . MAPKAP kinase-3 was phosphorylated by both CSBP1 and CSBP2 and was then able to phosphorylate HSP27 in vitro . Treatment of HeLa cells with sorbitol or TNF resulted in activation of CSBP and MAPKAP kinase-3 and activation of MAPKAP kinase-3 could be blocked by preincubation of cells with SB203580, a specific inhibitor of CSBP kinase activity . These data suggest that MAPKAP kinase-3 is activated by stress and cytokines and is a novel substrate of CSBP both in vitro and in vivo.

Cell, 1996 Apr 5, 85(1), 49 - 60
MafB is an interaction partner and repressor of Ets-1 that inhibits erythroid differentiation; Sieweke MH et al.; Using a yeast one-hybrid screen with a DNA-bound Ets-1 protein, we have identified MafB, an AP-1 like protein, as a direct interaction partner . MafB is specifically expressed in myelomonocytic cells and binds to the DNA-binding domain of Ets-1 via its basic region or leucine-zipper domain . Furthermore, it represses Ets-1 transactivation of synthetic promoters containing Ets binding sites and inhibits Ets-1-mediated transactivation of the transferrin receptor, which is known to be essential for erythroid differentiation . Accordingly, overexpression of MafB in an erythroblast cell line down-regulates the endogenous transferrin receptor gene and inhibits differentiation without affecting cell proliferation . These results highlight the importance of inhibitory interactions between transcription factors in regulating lineage-specific gene expression.

Cell, 1996 Apr 5, 85(1), 115 - 24
RNase III cleaves eukaryotic preribosomal RNA at a U3 snoRNP-dependent site; Elela SA et al.; A yeast gene homologous to bacterial RNase III (RNT1) encodes a double-strand-specific endoribonuclease essential for ribosome synthesis . Two rRNA processing events are blocked in cells temperature sensitive for RNT1: cleavage at the snoRNA-dependent AO site in the 5' ETS and cleavage in the 3' ETS . Recombinant RNT1 protein accurately cleaves a synthetic 5' ETS RNA at AO site in vitro, in the absence of snoRNA or other factors . A synthetic 3' ETS substrate is specifically cleaved at a site 21 nt downstream of the 3' end 28S rRNA . These observations show that a protein endonuclease collaborates with snoRNAs in eukaryotic rRNA processing and exclude a catalytic role for snoRNAs at certain pre-rRNA cleavage.

Biochim Biophys Acta, 1996 Apr 3, 1280(1), 155 - 60
Vanadate inhibits vacuolar H(+)-ATPase-mediated proton transport in chicken kidney microsomes by an ADP-dependent mechanism; David P et al.; Recent reports indicate that vacuolar-type proton ATPases from chicken osteoclasts (Chatterjee et al . (1992) Proc . Natl . Acad . Sci . USA 89, 6257-6261), yeast vacuoles and chromaffin granules (Beltran and Nelson (1992) Acta Physiol . Scand . Suppl . 607, 41-47) can be inhibited by vanadate, albeit at a concentration much higher than that required to inhibit P-type ATPases . We have characterized the mechanism by which vanadate inhibits vacuolar-type ATPase-mediated proton transport by chicken kidney microsomes . The initial rate of proton transport is somewhat less sensitive to vanadate than the total acidification, with IC50 values of 1.58 mM and 0.78 mM vanadate, respectively . The inhibition of both the initial rate and total acidification is noncompetitive with respect to ATP . The inhibition is abolished when ADP is removed by an ATP-regenerating system, and the addition of exogenous ADP increases the vanadate inhibition of proton transport in a synergistic manner, thus demonstrating that inhibition by vanadate is dependent on the presence of ADP and explaining the lower effect of vanadate on the initial rate of acidification . Phosphate protects proton transport activity from inhibition by vanadate . These effects of ADP and phosphate suggest that inhibition by vanadate may involve the formation of a complex with ADP at a nucleotide binding site, possibly at the catalytic site of the enzyme.

Proc Natl Acad Sci U S A, 1996 Apr 2, 93(7), 3149 - 54
A high-resolution physical map of human chromosome 11; Qin S et al.; The development of a highly reliable physical map with landmark sites spaced an average of 100 kbp apart has been a central goal of the Human Genome Project . We have approached the physical mapping of human chromosome 11 with this goal as a primary target . We have focused on strategies that would utilize yeast artificial chromosome (YAC) technology, thus permitting long-range coverage of hundreds of kilobases of genomic DNA, yet we sought to minimize the ambiguities inherent in the use of this technology, particularly the occurrence of chimeric genomic DNA clones . This was achieved through the development of a chromosome 11-specific YAC library from a human somatic cell hybrid line that has retained chromosome 11 as its sole human component.To maximize the efficiency of YAC contig assembly and extension, we have employed an Alu-PCR-based hybridization screening system . This system eliminates many of the more costly and time-consuming steps associated with sequence tagged site content mapping such as sequencing, primer production, and hierarchical screening, resulting in greater efficiency with increased throughput and reduced cost . Using these approaches, we have achieved YAC coverage for >90% of human chromosome 11, with an average intermarker distance of <100 kbp . Cytogenetic localization has been determined for each contig by fluorescent in situ hybridization and/or sequence tagged site content . The YAC contigs that we have generated should provide a robust framework to move forward to sequence-ready templates for the sequencing efforts of the Human Genome Project as well as more focused positional cloning on chromosome 11.

Genes Cells, 1996 Apr, 1(4), 409 - 19
A membrane cofactor protein transgenic mouse model for the study of discordant xenograft rejection; Yannoutsos N et al.; BACKGROUND: In recent years, interest has been revived in the possibility of transplanting organs into humans from a phylogenetically disparate species such as the pig (xenotransplantation) . Such discordant xenografts, however, are subject to hyperacute rejection (HAR) and activation of host complement plays a major role in this rejection . This problem may be solved through the use of transgenic technology by providing the grafted tissue with molecules that down-regulate the action of host complement . RESULTS: Transgenesis with a yeast artificial chromosome (YAC) was used to produce transgenic mice with the complete genomic gene of the human complement regulator membrane cofactor protein (MCP) . Transgenic mice were obtained that exhibit full regulation of MCP as normally observed in humans . Hearts from these mice were shown to be significantly protected from HAR caused by human serum in an in vivo experimental procedure . CONCLUSIONS: We conclude that MCP can protect discordant xenografts from HAR caused by human serum and that transgenic mice can be used effectively as in vivo models for the study of the role of human complement regulatory molecules in xenotransplantation.

Antibiot Khimioter, 1996 Apr, 41(4), 16 - 22
{Induction of elastase biosynthesis in Streptomyces sp.82}; Vlakhov S et al.; The effect of 6 nonspecific substrates such as tryptose, caseic acid, Hamerstene casein, tryptone, yeast extract and fish meal on the production of extracellular elastase was studied . Tryptone, yeast extract and fish meal were shown to have the inducing effect . The highest effect was observed with the use of fish meal . The effect of yeast extract was slightly lower . The optimal concentrations of the inductors were: 1 per cent for fish meal and 0.5 per cent for yeast extract . Two fractions were isolated from fish meal: protein concentrate with the molecular weight of more than 10,000 and amino acid concentrate with the molecular weight of 200-500 . The induction of the biosynthesis of streptomycete elastase was observed only in the presence of the first fraction . The enzymatic hydrolysis of the protein fraction resulted in the formation of three new fractions: fraction A with the molecular weight of 3000, fraction B with the molecular weight of 1500 and fraction C with the molecular weight of 1000 . Fraction A was the only one with the inducing effect.

Indian J Med Res, 1996 Apr, 103, 222 - 6
Cellular immune response to retinal S-antigen & interphotoreceptor retinoid binding protein fragments in idiopathic human uveitis; Rajasingh J et al.; The role of retinal antigens in idiopathic human uveitis has been studied in 38 patients of uveitis, and 30 patients of systemic connective tissue disease (CTD) and 30 healthy volunteers . Lymphocyte proliferative responses were tested in vitro against native S-antigen, its uveitopathogenic peptides (peptide M, peptide G), yeast histone H3 peptide and uveitopathogenic fragment of interphotoreceptor retinoid binding protein (IRBP: R16) to establish their role in pathogenesis of human uveitis . Seven patients with uveitis, and none among CTD patients and healthy volunteers, responded (stimulation index > 3) to at least one retinal antigen used . One uveitis patient showed response to native S-antigen, peptide M and yeast histone H3 . One responded to both S-antigen and peptide M and another responded to both peptide G and R16 peptide . Two responded to S-antigen only, one to peptide M and one to peptide G . In addition, one uveitis patient responded to yeast histone H3 only . These results suggest that retinal antigens may play a role in the etiopathogenesis of a subset of idiopathic human uveitis.

Nippon Rinsho, 1996 Apr, 54(4), 986 - 91
{Detailed deletion map of chromosomal arm 9q in esophageal squamous cell carcinoma}; Miura K et al.; We examined loss of heterozygosity (LOH) in 37 esophageal squamous cell carcinomas using microsatellite markers mapped to 9q31-34.1 . Partial or interstitial deletions were detected in 13 of them and the detailed deletion map defined a commonly deleted region between the D9S262 and D9S154 loci at 9q31-q32 . The genetic distance was estimated to be approximately 4 cM . To narrowly define the commonly deleted region, we isolated six microsatellite markers from YAC (yeast artificial chromosome) clones covering the deleted region . As the distal 9q region also has been implicated as the site of a tumor suppressor gene(s) related to squamous cell carcinomas of other tissues, our map provides useful information for attempts to identify a common gene for carcinomas of this cell type.

Appl Environ Microbiol, 1996 Apr, 62(4), 1471 - 4
Styrene metabolism in Exophiala jeanselmei and involvement of a cytochrome P-450-dependent styrene monooxygenase; Cox HH et al.; The yeast-like fungus Exophiala jeanselmei degrades styrene via initial oxidation of the vinyl side chain to phenylacetic acid, which is subsequently hydroxylated to homogentisic acid . The initial reactions are catalyzed by a NADPH- and flavin adenine dinucleotide-dependent styrene monooxygenase, a styrene oxide isomerase, and a NAD(+)-dependent phenylacetaldehyde dehydrogenase . The reduced CO-difference spectrum of microsomal preparations of styrene-grown cells shows a characteristic absorption maximum at 450 nm, which strongly suggests the involvement of a cytochrome P-450-dependent styrene monooxygenase . Inhibition of styrene monooxygenase activity in cell extracts by cytochrome P-450 inhibitors SKF-525-A, metyrapone, and CO confirms this assumption.

Genetics, 1996 Apr, 142(4), 1225 - 35
Interallelic complementation at the suppressor of forked locus of Drosophila reveals complementation between suppressor of forked proteins mutated in different regions; Simonelig M et al.; The Su(f) protein of Drosophila melanogaster shares extensive homologies with proteins from yeast (RNA14) and man (77 kD subunit of cleavage stimulation factor) that are required for 3' end processing of mRNA . These homologies suggest that su(f) is involved in mRNA 3' end formation and that some aspects of this process are conserved throughout eukaryotes . We have investigated the genetic and molecular complexity of the su(f) locus . The su(f) gene is transcribed to produce three RNAs and could encode two proteins . Using constructs that contain different parts of the locus, we show that only the larger predicted gene product of 84 kD is required for the wild-type function of su(f) . Some lethal alleles of su(f) complement to produce viable combinations . The structures of complementing and noncomplementing su(f) alleles indicate that 84-kD Su(f) proteins mutated in different domains can act in combination for partial su(f) function . Our results suggest protein-protein interaction between or within wild-type Su(f) molecules.

Hum Mol Genet, 1996 Apr, 5(4), 451 - 9
Xist expression from an Xist YAC transgene carried on the mouse Y chromosome; Matsuura S et al.; We have constructed mouse transgenic lines carrying a YAC clone encompassing the Xist gene in order to investigate the factors influencing Xist expression and the initiation of X-inactivation . Two transgenic lines were derived, one carrying four copies integrated at an autosomal site and a second line carrying four copies integrated at a single site on the Y chromosome . Xist expression was not observed in mice carrying the autosomal insertion . However, Xist expression from the Y-inserted transgenes was observed and at levels commensurate with that found in normal female mice . Methylation sites in the autosomal transgene both 5' and 3' of the Xist gene are hypermethylated and appear to reflect methylation patterns observed on the active X chromosome . For the Y-linked transgene, methylation sites 5' and 3' of the Xist gene are hypomethylated reflecting patterns found on the inactive X chromosome . However, the 5' and 3' methylation levels have been decoupled at the active transgenic locus . The data suggest that sequences in the vicinity of Xist can initiate some of the features that are associated with the initiation process of X-inactivation.

Hum Mol Genet, 1996 Apr, 5(4), 441 - 50
Transgenic mice carrying an Xist-containing YAC; Heard E et al.; The initiation of X-chromosome inactivation in female mammals is controlled by a key locus, the X-inactivation centre (Xic) . The Xist gene, which maps to the candidate region for Xic and is expressed exclusively from the inactive X chromosome, is thought to be an essential component of the Xic . To test whether sequences spanning several hundred kilobases and including Xist from the Xic region are capable of initiating inactivation, we have created a series of transgenic mice using a 460 kb yeast artificial chromosome (YAC) . Analysis in these mice of the expression of Xist, of a LacZ reporter gene and of two genes in the region that are normally silent on the inactive X chromosome, suggests that essential sequences for Xist expression and X-inactivation may be absent in these transgenic animals.

Hum Genet, 1996 Apr, 97(4), 462 - 7
Identification of three new microsatellite markers in the spinocerebellar ataxia type 2 (SCA2) region and 1.2 Mb physical map; Nechiporuk T et al.; Spinocerebellar ataxia type 2 (SCA2) is a neurodegenerative disease recently mapped to chromosome 12q close to the locus D12S84 by genetic linkage analysis . To generate additional genetic markers in the SCA2 region, we constructed a physical map of the region using yeast artificial chomosome (YAC), P1 artificial chromosome (PAC) and cosmid clones . The physical map was found to agree well with the genetic map . Three novel microsatellite markers were isolated and physically mapped . A novel approach to isolate CAG repeats directly from YAC DNAs is described.

Hum Genet, 1996 Apr, 97(4), 441 - 52
Identification and characterization of three genes and two pseudogenes on chromosome 13; de Fatima Bonaldo M et al.; A study was conducted on the feasibility of isolating genes and pseudogenes that map to chromosome 13 by a hybridization-based approach using a 13-specific library and pools of repeat-free cDNA clones . Five pairs of cDNA and chromosome 13 genomic clones were identified and characterized . Partial or full-length sequence was derived from all cDNAs, and database searches were performed for putative gene identification . Partial sequence was also obtained from the chromosome 13 genomic clones for comparison with those of the hybridizing cDNAs . As a result of these analyses we identified three genes, a putative homologue of a porcine mRNA encoding an unidentified hepatic protein, a putative homologue of a yeast integral membrane protein, and a gene for a translationally controlled tumor protein, and two processed pseudogenes, ribosomal proteins L23a and S3a . The latter was formerly identified as the v-fos transformation effector gene, Fte-1, and recently cited as a possible candidate for the BRCA2 gene on chromosome 13 . All genes and pseudogenes were localized to cytogenetic bands by in situ hybridization of metaphase chromosomes with probes derived from the chromosome 13 genomic clones.

J Clin Microbiol, 1996 Apr, 34(4), 816 - 7
Quality control guidelines for National Committee for Clinical Laboratory Standards--recommended broth macrodilution testing of ketoconazole and itraconazole; Rex JH et al.; Ketoconazole and itraconazole were tested in a multilaboratory study to establish quality control (QC) guidelines for yeast antifungal susceptibility testing . Two isolates that had been previously identified as QC isolates for amphotericin B, fluconazole, and flucytosine (Candida parapsilosis ATCC 22019 and Candida krusei ATCC 6258) were tested in accordance with the National Committee for Clinical Laboratory Standards M27-P guidelines . Each isolate was tested 20 times with the two antifungal agents in the five laboratories by using a lot of RPMI 1640 unique to each laboratory as well as a lot common to all five laboratories, thus generating 200 MICs per drug per organism . Overall, 96 to 99% of the MICs for each drug fell within the desired 3-log2 dilution range (mode +/- 1 log2 dilution) . By using these data, 3-log2 dilution QC ranges encompassing 98% of the observed MICs for three of the organism-drug combinations and 94% of the observed MICs for the fourth combination were established . These QC ranges are 0.064 to 0.25 micrograms/ml for both ketoconazole and itraconazole against C . parapsilosis ATCC 22019 and 0.125 to 0.5 micrograms/ml for both ketoconazole and itraconazole against C . krusei ATCC 6258.

Indian J Biochem Biophys, 1996 Apr, 33(2), 93 - 102
RNA polymerase II dependent genes that do not code for protein; Lakhotia SC; In recent years more and more examples of RNA polymerase II dependent non-coding transcripts have been described . Although these have frequently been ignored as "selfish DNA elements", it is becoming increasingly clear that many, if not all, of them have very important biological roles . Examples of such "genes" from Drosophila, mammals, other vertebrates, yeast etc . are considered . Although the specific mechanisms through which these non-coding transcripts function in the cell are not clear, comparisons reveal certain common themes, particularly the importance of secondary structures, rather than the primary base sequence of these transcripts . While some of these transcripts may function as ribozymes or as anti-sense regulators, most others may function more directly through their specific protein-binding properties . Since RNA is believed to be the first "living" molecule, it is very likely that some genes even today function only through this class of molecules . It is expected that instead of being ignored as examples of "selfish DNA", a more positive search for their functions will help unravel the significance of this novel class of genes.

Antonie Van Leeuwenhoek, 1996 Apr, 69(3), 211 - 5
A new type of growth exhibited by Trimmatostroma abietis; Yoshida S et al.; Trimmatostroma abietis initially grew as hyphae when grown in various media containing yeast extract or bactopeptone . It grew as segmented elements (lumbricoid elements) characterized by bidirectional growth, when grown in Czapek-Dox broth or yeast nitrogen base supplemented with 1% glucose . A lumbricoid element usually was 10-70 microns in length, with transverse septation only and contained 3 to 15 cells . Growth and propagation, as revealed by time-lapse photomicrography occurred as follows . Elements usually grew by apical elongation without widening; after simple apical elongation adjacent parts of two central cells eventually started to grow, resulting in the separation of the element into two.

Brain Res Mol Brain Res, 1996 Apr, 37(1-2), 249 - 58
Analysis of interaction sites in homo- and heteromeric complexes containing Bcl-2 family members and the cellular prion protein; Kurschner C et al.; The cellular prion protein (PrP) binds to the C-terminus of Bcl-2 but not Bax . Therefore, we examined whether the C-terminus of Bcl-2 was important for other homomeric and heteromeric protein-protein interactions . Using the yeast two hybrid system and co-immunoprecipitation, three sites of homomeric interactions were identified within Bcl-2 . The carboxy terminal 37 amino acids selectively homodimerized . Two additional regions of Bcl-2 (residues 1-129 and 126-200) interacted with each other, but not themselves permitting both intra- and intermolecular association . In addition, we analyzed heteromeric interactions of Bcl-2 with PrP and two Bcl-2 related proteins, Bax and A1 . The domain requirements for binding of those three proteins to Bcl-2 were different from one another . Bax binding required almost the entire Bcl-2 molecule, while A1 bound to the amino terminal region (residues 1-82) . PrP associated with the carboxy terminus of Bcl-2 (amino acids 200-236) . These data suggest configurational models for Bcl-2 containing complexes . First, Bcl-2 may exist as both heterodimers and heteromultimers . Second, molecules such as Bax and A1 may serve to cap chains of Bcl-2 homodimers by interacting with dimerization domains in the extramembrane region . PrP may disrupt chains of Bcl-2 molecules at the homomeric association site in the transmembrane region.

Braz J Med Biol Res, 1996 Apr, 29(4), 413 - 30
Protein targeting to the plant vacuole--a historical perspective; Dombrowski JE et al.; Although many properties of the targeting of plant endomembrane proteins are similar to mammalian and yeast systems, several clear differences are found that will be stressed in this review . In the past few years, we have seen an advancement in our understanding of the signals for vacuolar protein targeting and some insights into the mechanisms of transport to the vacuole in the plant cell . This work will form the basis for elucidation of the fundamental principles that govern protein trafficking through the secretory system to the vacuole.

Steroids, 1996 Apr, 61(4), 257 - 62
Interaction of the Ubc9 human homologue with c-Jun and with the glucocorticoid receptor; Gottlicher M et al.; Glucocorticoid hormones convert the glucocorticoid receptor (GR) from an inactive cytosolic complex to a nuclear form that regulates transcription . Binding of GR to palindromic DNA-recognition sites (hormone response elements) leads to activated target gene transcription . GR also exerts negative actions on transcription, e.g., by interfering with the function of several other transcription factors such as AP-1, NK-kappa B, CREB, and Oct-1 . Physical interactions of GR with AP-1 subunits are readily detectable but do not seem sufficient since nonrepressing GR mutants still interact in vitro, so that specific conformational changes and/or interactions with additional partner proteins may be required for negative action . In an attempt to find such partner proteins, we defined regions of c-Jun and GR essential for mutual interference and used in those a yeast two-hybrid screen for interacting proteins . Repeatedly we isolated overlapping cDNA sequences of one protein interaction with both c-Jun and GR . This protein does not interact with c-Fos or a non-repressing GR mutant and expressed in mammalian cells does not substantially affect AP-1 or GR activity . Interestingly, however, the protein rescues yeast cells from the toxic effects of the GR fragment used for screening . The protein represents the human homologue of the yeast E2 ubiquitin-conjugating enzyme, Ubc9; its specific interactions with both GR and c-Jun, but not mutant GR, suggest that it may exert physiologic regulatory functions.

J Rheumatol, 1996 Apr, 23(4), 665 - 71
Inorganic pyrophosphate generation from adenosine triphosphate by cell-free human synovial fluid; Park W et al.; OBJECTIVE . To quantify inorganic pyrophosphate (PPi) production from extracellular adenosine triphosphate (ATP) by human synovial fluids (SF) . METHODS . Serial measurements of ATP hydrolysis rate (t1/2) were performed by the luciferase method from a starting concentration of 500 nM in 21 pathologic and one normal cell-free SF samples incubated under physiologic conditions . ATP was then pumped into a sample of each fluid, using the rate constant derived from the t1/2 of that fluid, to provide steady state levels simulating those reported in SF . Trace {32P} gamma ATP was added at the start of the infusion; conversion to {32P} Pi and to {32P} PPi was determined by precipitation of Pi as reduced phosphomolybdate before and after treatment with yeast inorganic pyrophosphatase . Finally, the pumping experiment was repeated and PPi production was calculated from direct measurement of PPi at time zero and at 60 min . PPi hydrolysis was measured in each fluid by {32P} Pi precipitation from {32P} PPi tracer added at time zero . RESULTS . ATP was hydrolyzed by all SF . The mean t1/2 (seconds) in 8 osteoarthritis (OA) samples was 72 s, in 5 calcium pyrophosphate dihydrate (CPPD) 30 s (p < 0.02), in 3 rheumatoid arthritis (RA) 1160 s, in normal 86 s, in 3 olecranon bursal (OB) 54 s, and in 2 total knee replacement fluid samples 17 and 121 s . The major product of ATP hydrolysis was PPi in all but 2 fluids (1 RA, 1 OB), even at lower than steady state levels . At simulated in vivo steady state ATP levels, mean conversion of APT to PPi was stoichiometric in OA and CPPD fluids . PPi hydrolysis was < 4% in all noninflammatory fluids . CONCLUSION . PPi is the major product of extracellular ATP catabolism in most SF . Hydrolysis rates were significantly faster in SF containing CPPD crystals . Mean PPi production by these fluids at simulated in vivo steady state levels was 6-fold that of OA SF (p < 0.01) . Hydrolysis of extracellular ATP by ectonucleotide pyrophosphohydrolases can account for all PPi produced by joint issues previously estimated from {32P} PPi pool and turnover studies in human knee joints.

Genome Res, 1996 Apr, 6(4), 290 - 9
Toward the construction of integrated physical and genetic maps of the mouse genome using interspersed repetitive sequence PCR (IRS-PCR) genomics; Hunter KW et al.; Using two recently developed techniques, IRS-PCR YAC walking and IRS-PCR genotyping, a framework-integrated physical and genetic map of the mouse genome was constructed . The map consists of 821 contigs, containing 7746 YAC clones originating from three different YAC libraries . Three hundred eighty of the contigs have been anchored to the genetic map . Approximately 16% of the physical length of the mouse genome is estimated to be represented.

Genome Res, 1996 Apr, 6(4), 255 - 66
Mapping the RP10 locus for autosomal dominant retinitis pigmentosa on 7q: refined genetic positioning and localization within a well-defined YAC contig; McGuire RE et al.; Retinitis pigmentosa is a genetically heterogeneous disease that has autosomal dominant, autosomal recessive and X-linked forms . Autosomal dominant retinitis pigmentosa (adRP) has thus far been associated with eight distinct loci, including the rhodopsin and peripherin/RDS genes as well as unidentified genes on chromosomes 7p, 7q, 8q, 17p, 17q, and 19q . The RP10 locus for adRP on chromosome 7q was first mapped in a Spanish family; later, an unrelated American family was identified that also showed linkage to 7q . By combining the linkage results from both families, we are able to assign the disease gene to a 5-cM interval on 7q . Based on extensive physical mapping of this region, the genetic interval is now fully contained within a approximately 5-Mb segment on a well-defined YAC contig . These studies significantly reduce the size of the RP10 critical region, exclude a number of possible candidate genes, and provide the necessary cloned DNA for the positional cloning of the RP10 gene.

Curr Opin Genet Dev, 1996 Apr, 6(2), 203 - 7
Replication origins in eukaroytes; Donovan S et al.; Recent experiments in budding yeast and Xenopus have provided new insights into the regulation of eukaroytic DNA replication . The multi-subunit origin recognition complex plays a key role in initiation, remaining bound at origins of replication during most of the cell cycle . Early in the cell cycle, Cdc6 and the Mcm proteins 'reset' chromatin for another round of DNA replication . Cyclin-dependent kinases appear to play a dual role, both in activating replication origins and blocking the formation of new pre-replicative complexes; thus limiting replication to once per cell cycle.

Curr Opin Genet Dev, 1996 Apr, 6(2), 146 - 50
DNA repair and transcription; Bhatia PK et al.; The transcription factor TFIIH continues to be a subject of interest . In addition to its function as a repair and transcription factor, TFIIH includes a cyclin-dependent kinase and a cyclin, which raises the possibility that nucleotide excision repair (NER), RNA polymerase II transcription and cell cycle control are connected . Progress in mechanistic studies of NER include the identification of dual incision activities operating on either side of base damage and the isolation of a repairosome supercomplex in yeast . Additionally, NER has been demonstrated in reconstituted human and yeast systems, both of which include TFIIH.

Mol Immunol, 1996 Apr, 33(6), 561 - 72
Biochemical analysis of the interaction of fibronectin with IgG and localization of the respective binding sites; Rostagno A et al.; Fibronectin (Fn), a mosaic protein composed of multiple copies of three different module types (Fl, F2 and F3), has been found associated with circulating immune complexes (ICs) and immunoglobulin (Ig) aggregates in a variety of IC diseases and myeloproliferative disorders . We have previously shown that a proteolytic fragment of Mr = 25,900 Da, from the NH2-terminal domain of Fn, composed of five type 1 modules (1Fl -5Fl) binds to the major Ig classes under physiologic conditions, suggesting that the presence of Fn in ICs and cryoglobulins results from a physicochemical binding interaction between these two molecules . Using an ELISA, we now show that the interaction between Fn and IgG is: (1) not influenced by any other constituent of plasma; (2) unaffected by temperature; and (3) has an estimated Kd of 3.77 x 10(-9) M . In addition, we have further delineated the respective sites involved in the interaction between Fn and IgG . Recombinant type l module pairs (1Fl.2Fl and 4Fl.5Fl) from the NH2-terminus of Fn, expressed in yeast, were employed in an ELISA and affinity chromatography and compared with the 25.9 kDa (1Fl - 5Fl) fragment and intact Fn for binding to IgG . The 4Fl.5Fl and the 25.9 kDa fragment bound to immobilized IgG and inhibited Fn binding to IgG to nearly the same extent as the intact molecule (IC50: Fn = 6.77 x 1O(-9) M; 25.9 kDa fragment = 5 x 10(-9) M; 4Fl.5Fl = 7.6 x 10(-9) M) . Thus, the binding site for IgG on the Fn molecule is localized to and completely conferred by the 4Fl.5Fl module pair (residues 151-244) . Similar experiments using papain-generated Fab and Fc fragments of IgG localized the Fn binding site on IgG to the Fe region of the IgG molecule . Fn bound to the Fc fragment with a nearly identical Kd of 3.69 x 10(-9) M, as to intact IgG (3.77 x 10(-9) M) . These studies support the hypothesis that the interaction between Fn and Ig may contribute to the pathophysiology of immune complex related disorders.

Kidney Int, 1996 Apr, 49(4), 947 - 52
Structural and functional aspects of the phosphate carrier from mitochondria; Kramer R; The paper reviews the major structural and functional aspects of the phosphate carrier from the inner mitochondrial membrane in comparison to other mitochondrial carrier proteins . The mitochondrial phosphate carrier catalyzes the transport of inorganic phosphate from the cytosol into the mitochondrial matrix and is thus essential for the energy metabolism of the cell . The phosphate carrier from beef and pig heart, from rat liver and from yeast mitochondria has been purified by chromatographic methods and functionally reconstituted in proteoliposomes . The primary sequence of the phosphate carrier from several different species has been determined . The carrier protein of Mr 33 to 34 kDa most likely acts as a dimer in the membrane . The phosphate carrier has been characterized with respect to transport kinetics, energy dependence and carrier mechanism mainly after functional reconstitution into artificial bilayers (liposomes) . Three different modes of action were elucidated, namely homologous phosphate/phosphate antiport, heterologous phosphate/proton symport or phosphate/hydroxyl antiport, respectively, as well as unphysiological uniport (efflux) after modification of essential SH-groups . Both with respect to its primary structure and its functional (kinetic) properties, the phosphate carrier is a member of the well-defined mitochondrial carrier protein family.

J Pathol, 1996 Apr, 178(4), 410 - 4
Interphase fluorescence in situ hybridization detection of t(2;13)(q35;q14) in alveolar rhabdomyosarcoma--a diagnostic tool in minimally invasive biopsies; McManus AP et al.; The identification of t(2;13)(q35;q14) is a useful aid in the accurate diagnosis of rhabdomyosarcoma, distinguishing it from other small round cell tumours and supporting the distinction between alveolar and embryonal forms . Cytogenetic analysis is difficult and with the increased use of minimally invasive biopsy methods and primary chemotherapy, adequate tumour material is not always available . To overcome these difficulties, two-colour interphase fluorescence in situ hybridization (FISH) to detect t(2;13)(q35;q14) was developed and its utility in assessing minimally invasive biopsies was investigated . Two cosmid clones which mapped proximal or distal to the breakpoint region 13q14 and one yeast artificial chromosome clone that mapped distal to the 2q35 breakpoint were identified . In interphase cells containing t(2;13)(q35;q14), the configuration of cosmid and yeast artificial chromosome signals demonstrated the presence of the translocation . Five cases of rhabdomyosarcoma were analysed by interphase FISH . The t(2;13)(q35;q14) was detected in all four alveolar tumours and was confirmed by cytogenetics in two cases, but was absent in one embryonal tumour . This sensitive detection method is applicable to minimal amounts of fresh or frozen tumour.

Plant Cell Physiol, 1996 Apr, 37(3), 369 - 76
Molecular cloning and characterization of a cDNA clone that encodes a Cdc2 homolog from Nicotiana tabacum; Setiady YY et al.; We have isolated a cDNA clone (cdc2Nt1) that encodes a homolog of p34(cdc2/CDC28) kinase from tobacco (Nicotiana tabacum) . The cdc2Nt1 protein showed extensive similarity to other homologs of Cdc2 from plants . Complementation studies showed that the cdc2Nt1 gene was able to overcome cell cycle arrest at both the G1/S and the G2/M transitions of cdc28ts mutants of budding yeast, demonstrating that the cdc2Nt1 protein was able to replace the Cdc28 kinase at both the G1/S and the G2/M transitions . Analysis of gene expression demonstrated that the cdc2Nt1 gene was transcribed constitutively throughout the cell cycle but that it was preferentially expressed in activity dividing tobacco BY-2 cells.

Mamm Genome, 1996 Apr, 7(4), 303 - 11
A long-range physical map of human chromosome 21q22.1 band from the YAC continuum; Eki T et al.; The human Chromosome (Chr) 21q22.1 region contains several genes for cytokines and neurotransmitters and the gene for superoxide dismutase (mutant forms of which can cause familial amyotrophic lateral sclerosis) . A region of approximately 5.8 Mb encompassing D21S82 and the glycinamide ribonucleotide transformylase (GART) loci was covered by overlapping YAC clones, which were contiguously ordered by clone walking with sequence-tagged site (STSs) . A total of 76 markers, including 29 YAC end-specific STSs, were unambiguously ordered in this 5.8-Mb region, and the average interval between markers was 76 kb . Restriction maps of the YAC clones with rare-cutting enzymes were simultaneously prepared, and the restriction sites were aligned to obtain a consensus restriction map of the proximal region of the 21q22.1 band . The restriction map made from 44 overlapping YACs contains 54 physically assigned STSs . By integrating the consensus map of the adjacent 1.8-Mb region, we obtained a fine physical map spanning 6.5 Mb of human Chr 21q22.1 . This map contains 24 precisely positioned end-specific STSs and 12 NotI-linking markers . More than 39 potential CpG islands were identified in this region and were found to be unevenly distributed . This physical map and the YACs should be useful as a reference map and as a resource for further structural analysis of the Giemsa-negative band (R-band) of Chr 21q22.1.

Mamm Genome, 1996 Apr, 7(4), 253 - 61
YAC clone contigs covering 5 Mb of a repeat sequence island on the mouse X chromosome; Mileham P et al.; We have initiated work towards the construction of YAC clone contigs across a repeat sequence island region on the mouse X Chromosome (Chr) . The repeat sequence island region-the 141 island-located at band A3 contains 50 copies of a localized long complex repeat unit (LCRU) . We have isolated 87 YAC clones from the 141 island and have used a dual faceted approach towards the construction of contigs across the repeat sequence island . First, we have identified YAC clones originating from the same region of the island by the identification of commonly held LCRU restriction site variants . Second, we have constructed rare cutter restriction maps of each YAC clone . Taken together, we have been able to assemble one large contig of 2.8 Mb and a number of smaller contigs . In total, contigs covering 5Mb of the island region have been identified . The island region would appear to represent a major component of the A3 Giemsa dark band on the mouse X Chr.

Planta Med, 1996 Apr, 62(2), 137 - 40
Analgesic and antipyretic activities of an aqueous extract and of the flavone linarin of Buddleia cordata; Martinez-Vazquez M et al.; The dried aqueous extract of leaves of Buddleia cordata (loganiaceae) and its main flavonoid glycoside, linarin, have been evaluated for analgesic and antipyretic effects in mice and rats, respectively . Both the extract and linarin exerted significant and dose-dependent analgesic and antipyretic activities, the first being obtained against a chemical stimulus (writhing a test in mice) and the second being obtained by a pyretogenic stimulus (yeast-induced hyperthermia test) . Furthermore, the response of the animals in the hot plate test was modified by linarin and an aqueous extract . These activities were similar to that showed by morphine sulfate (MS) and they were inhibited by naxolone pretreatment, a specific morphinic antagonist compound . These findings lead to the conclusion that the aqueous extract and linarin exert central analgesic properties . On the other hand, linarin was shown to be responsible for the antipyretic activity of this species.

Mol Cell Biol, 1996 Apr, 16(4), 1714 - 21
An activation domain of the helix-loop-helix transcription factor E2A shows cell type preference in vivo in microinjected zebra fish embryos; Argenton F et al.; The E2A protein is a mammalian transcription factor of the helix-loop-helix family which is implicated in cell-specific gene expression in several cell lineages . Mouse E2A contains two independent transcription activation domains, ADI and ADII; whereas ADI functions effectively in a variety of cultured cell lines, ADII shows preferential activity in pancreatic beta cells . To analyze this preferential activity in an in vivo setting, we adapted a system involving transient gene expression in microinjected zebra fish embryos . Fertilized one- to four-cell embryos were coinjected with an expression plasmid and a reporter plasmid . The expression plasmids used encode the yeast Gal4 DNA-binding domain (DBD) alone, or Gal4 DBD fused to ADI, ADII, or VP16 . The reporter plasmid includes the luciferase gene linked to a promoter containing repeats of UASg, the Gal4-binding site . Embryo extracts prepared 24 h after injection showed significant luciferase activity in response to each of the three activation domains . To determine the cell types in which the activation domains were functioning, a reporter plasmid encoding beta-galactosidase and then in situ staining of whole embryos were used . Expression of ADI led to activation in all major groups of cell types of the embryo (skin, sclerotome, myotome, notochord, and nervous system) . On the other hand, ADII led to negligible expression in the sclerotome, notochord, and nervous system and much more frequent expression in the myotome . Parallel experiments conducted with transfected mammalian cells have confirmed that ADII shows significant activity in myoblast cells but little or no activity in neuronal precursor cells, consistent with our observations in zebra fish . This transient-expression approach permits rapid in vivo analysis of the properties of transcription activation domains: the data show that ADII functions preferentially in cells of muscle lineage, consistent with the notion that certain activation domains contribute to selective gene activation in vivo.

Mol Cell Biol, 1996 Apr, 16(4), 1706 - 13
cDNA cloning and tissue-specific expression of a novel basic helix-loop-helix/PAS factor (Arnt2) with close sequence similarity to the aryl hydrocarbon receptor nuclear translocator (Arnt); Hirose K et al.; We isolated mouse cDNA clones (Arnt2) that are highly similar to but distinct from the aryl hydrocarbon receptor (AhR) nuclear translocator (Arnt) . The composite cDNA covered a 2,443-bp sequence consisting of a putative 2,136-bp open reading frame encoding a polypeptide of 712 amino acids . The predicted Arnt2 polypeptide carries a characteristic basic helix-loop-helix (bHLH)/PAS motif in its N-terminal region with close similarity (81% identity) to that of mouse Arnt and has an overall sequence identity of 57% with Arnt . Biochemical properties and interaction of Arnt2 with other bHLH/PAS proteins were investigated by coimmunoprecipitation assays, gel mobility shift assays, and the yeast two-hybrid system . Arnt2 interacted with AhR and mouse Sim as efficiently as Arnt, and the Arnt2-AhR complex recognized and bound specifically the xenobiotic responsive element (XRE) sequence . Expression of Arnt2 successfully rescued XRE-driven reporter gene activity in the Arnt-defective c4 mutant of Hepa-1 cells . RNA blot analysis revealed that expression of Arnt2 mRNA was restricted to the brains and kidneys of adult mice, while Arnt mRNA was expressed ubiquitously . In addition, whole-mount in situ hybridization of 9.5-day mouse embryos showed that Arnt2 mRNA was expressed in the dorsal neural tube and branchial arch 1, while Arnt transcripts were detected broadly in various tissues of mesodermal and endodermal origins . These results suggest that Arnt2 may play different roles from Arnt both in adult mice and in developing embryos . Finally, sequence comparison of the currently known bHLH/PAS proteins indicates a division into two phylogenetic groups: the Arnt group, containing Arnt, Arnt2, and Per, and the AhR group, consisting of AhR, Sim, and Hif-1alpha.

Mol Cell Biol, 1996 Apr, 16(4), 1576 - 83
Functional interactions between the hBRM/hBRG1 transcriptional activators and the pRB family of proteins; Strober BE et al.; hBRG1 and hBRM are mammalian homologs of the SNF2/SW12 yeast transcriptional activator . These proteins exist in a large multisubunit complex that likely serves to remodel chromatin and, in so doing, facilitates the function of specific transcription factors . The retinoblastoma protein (pRB) inhibits cell cycle progression by repressing transcription of specific growth-related genes . Using the yeast two-hybrid system, we demonstrate that the members of the hBRG1/hBRM family of proteins interact with the pRB family of proteins, which includes pRB, p107, and p130 . Interaction between the hBRG1/hBRM family with the pRB family likely influences cellular proliferation, as both hBRG1 and hBRM, but not mutants of these proteins unable to bind to pRB family members, inhibit the formation of drug-resistant colonies when transfected into the SW13 human adenocarcinoma cell line, which lacks endogenous hBRG1 or hBRM . Further, hBRM and two isoforms of hBRG1 induce the formation of flat, growth-arrested cells in a pRB family-dependent manner when introduced into SW13 cells . This flat-cell inducing activity is severely reduced by cotransfection of the wild-type E1A protein and variably reduced by the cotransfection of mutants of E1A that lack the ability to bind to some or all members of the pRB family.

Mol Cell Biol, 1996 Apr, 16(4), 1391 - 400
Characterization of the intron-encoded U19 RNA, a new mammalian small nucleolar RNA that is not associated with fibrillarin; Kiss T et al.; We have characterized a new member (U19) of a group of mammalian small nuclear RNAs that are not precipitable with antibodies against fibrillarin, a conserved nucleolar protein associated with most of the small nucleolar RNAs characterized to date . Human U19 RNA is 200 nucleotides long and possesses 5'-monophosphate and 3'-hydroxyl termini . It lacks functional boxes C and D, sequence motifs required for fibrillarin binding in many other snoRNAs . Human and mouse RNA are 86% homologous and can be folded into similar secondary structures, a finding supported by the results of nuclease probing of the RNA . In the human genome, U19 RNA is encoded in the intron of an as yet not fully characterized gene and could be faithfully processed from a longer precursor RNA in HeLa cell extracts . During fractionation of HeLa cell nucleolar extracts on glycerol gradients, U19 RNA was associated with higher-order structures of approximately 65S, cosedimenting with complexes containing 7-2/MRP RNA, a conserved nucleolar RNA shown to be involved in 5.8S rRNA processing in yeast cells.

Nat Genet, 1996 Apr, 12(4), 368 - 75
Karyotyping human chromosomes by combinatorial multi-fluor FISH; Speicher MR et al.; We have developed epifluorescence filter sets and computer software for the detection and discrimination of 27 different DNA probes hybridized simultaneously . For karyotype analysis, a pool of human chromosome painting probes, each labelled with a different fluor combination, was hybridized to metaphase chromosomes prepared from normal cells, clinical specimens, and neoplastic cell lines . Both simple and complex chromosomal rearrangements could be detected rapidly and unequivocally; many of the more complex chromosomal abnormalities could not be delineated by conventional cytogenetic banding techniques . Our data suggest that multiplex-fluorescence in situ hybridization (M-FISH) could have wide clinical utility and complement standard cytogenetics, particularly for the characterization of complex karyotypes.

Cancer Genet Cytogenet, 1996 Apr, 87(2), 95 - 102
Fluorescence in situ hybridization-based approaches for detection of 12p overrepresentation, in particular i(12p), in cell lines of human testicular germ cell tumors of adults; Mostert MM et al.; Overrepresentation of the short arm of chromosome 12 is frequently detected in human testicular germ cell tumors of adolescents and adults (TGCT) . This overrepresentation mostly results from the formation of an isochromosome: i(12p) . Whether the overrepresentation consistently involves the complete 12p arm including the centromere is still unclear . We studied five TGCT-derived cell lines (NT2, 2102Ep, H12.1, NCCIT, and S2), combining conventional chromosome banding, fluorescence in situ hybridization (FISH), and comparative genomic hybridization (CGH) to investigate the suitability of each of these techniques to detect aberrations involving chromosome 12 . Karyotyping showed one or more i(12p)s in NT2, 2102Ep, H12.1, and S2 . However, FISH with a centromere-specific probe (p alpha 12H8), a 12p "paint" and a 12p11.2--p12.1 region-specific probe yeast artificial chromosome (YAC)#5 and CGH could not confirm the presence of an i(12p) in S2 . Additional randomly distributed 12p sequences were detected by FISH in H12.1, NCCIT, and S2 . In most of these cases, (a part of) the centromere was included . All overrepresented 12p regions, except for those in S2, showed hybridization with YAC#5 . CGH showed increased copy numbers of the complete 12p arm in the cell lines with one or more i(12p)s but no overrepresentation was noted in the cell lines without i(12p) . In metaphase spreads, the centromeric block of the i(12p)s differed in size as compared with those of normal chromosomes 12 . This was rarely noted in interphase nuclei . A decrease in size of the centromeric block in 2102Ep and H12.1 caused a weak FISH signal, which was difficult to detect, especially in interphase nuclei . The ratio between p alpha 12H8- and YAC#5-derived signals reflected the presence or absence of one or more i(12p)s . Our results indicate that double FISH with a centromere- and a 12p-specific probe can be used to detect 12p overrepresentation {including i(12p)} in TGCT both in metaphase spreads and interphase nuclei . CGH confirmed the relative overrepresentation of 12p sequences as detected by FISH and showed that in these cell lines the complete 12p was involved.

Cancer Genet Cytogenet, 1996 Apr, 87(2), 103 - 6
More detailed characterization of some of the HL60 karyotypic features by fluorescence in situ hybridization; Volpi EV et al.; We performed a focused chromosome analysis on the HL60 cell line by multicolor fluorescence in situ hybridization (FISH), using probes for the unequivocal identification of specific chromosome regions and subregions . The purpose of this karyotypic re-evaluation was to confirm and to characterize in more detail chromosome rearrangements already identified by means of classic cytogenetic approaches and recurrently detected from the initial establishment of the cell line . The observations reported may help reassess the potential of the HL60 cell line in understanding the molecular events underlying the non-random karyotype alterations associated with acute myeloid leukemias (AML).

Plant J, 1996 Apr, 9(4), 537 - 48
Coordinate transcriptional induction of myo-inositol metabolism during environmental stress; Ishitani M et al.; The pathway from glucose 6-phosphate (G 6-P) to myoinositol 1-phosphate (Ins 1-P) and myo-inositol (Ins) is essential for the synthesis of various metabolites . In the halophyte Mesembryanthemum crystallinum (common ice plant), two enzymes, myo-inositol O-methyltransferase (IMT1) and ononitol epimerase (OEP1), extend this pathway and lead to the accumulation of methylated inositols, D-ononitol and D-pinitol, which serve as osmoprotectants . This paper describes transcripts for the enzyme, Inps1, encoding myo-inositol 1-phosphate synthase (INPS1), from the ice plant . Two Inps-like sequences are present in the genome . The deduced amino acid sequences of the cloned transcript are 49.5% and 87-90%, respectively, identical to those of yeast and other higher plant sequences . Inps1 RNA amounts are upregulated at least fivefold and amounts of free Ins accumulate approximately 10-fold during salinity stress . Inps1 induction is by transcription, similar to the induction of Imt1 . In contrast, Arabidopsis thaliana does not show upregulation of Inps1 or increased amounts of Ins when salt-stressed . The lack of Inps1 induction in Arabidopsis exemplifies differences in glycophytic and halophytic regulation of gene expression at the point of entry into a pathway that leads to osmoprotection . The stress-induced coordinate upregulation of this pathway and its extension by novel enzymes in the ice plant also highlights biochemical differences.

FASEB J, 1996 Apr, 10(5), 631 - 6
Mechanical stretch activates the stress-activated protein kinases in cardiac myocytes; Komuro I et al.; We have recently shown that mechanical stress activates a phosphorylation cascade of protein kinases including Raf-1 and the extracellular signal-regulated kinases (ERKs) in cultured cardiac myocytes partially through the enhanced secretion of angiotensin II . Osmotic stress in budding yeast has been shown to activate similar signaling molecules including Hog-1, a distant relative of the ERK family . In the present study, we examined whether mechanical stretch of cardiac myocytes activates the stress-activated protein kinases (SAPKs)/c-Jun NH2-terminal kinase, the mammalian homologs of yeast Hog-1 that regulate gene expression through activation of the transcription factor, AP-1 . When cardiac myocytes of neonatal rats cultured on a deformable silicone dish were stretched, activity of SAPKs was increased from 10 min, peaked at 30 min, and gradually decreased thereafter . The increase in activity of SAPKs was proportional to the stretch . Unlike ERKs, the activation of SAPKs by stretching cardiac myocytes was not dependent on the secreted angiotensin II . The chelation of extracellular Ca2+ or down-regulation of protein kinase C did not attenuate activation of SAPKs by stretch . Transfection experiments using an AP-1 binding site-containing reporter gene revealed that stretch increases AP-1 activity in cardiac myocytes . In conclusion, like osmotic stress in yeast, mechanical stretch activates SAPKs in cardiac myocytes without the participation of angiotensin II . These results suggest that the activation of SAPKs may regulate gene expression during mechanical stress-induced cardiac hypertrophy.

Proc Soc Exp Biol Med, 1996 Apr, 211(4), 323 - 31
A selenium supplement associated or not with vitamin E delays early renal lesions in experimental diabetes in rats; Douillet C et al.; Seventy rats were separated into five groups: one group of 12 was used as a control and received a purified diet, and four groups of streptozotocin-induced diabetic rats, totalling 58, were fed the same diet without or with selenium (Se) supplementation . Of the noncontrol rats, 14 were without supplementation (Group D), 14 were fed a Se-rich yeast diet (i.e., selenion) (Group DSel), 14 received selenomethionine (Group DSm), and 16 received selenomethionine + tocopherol acetate (Group DSmE) . Supplementation with Se in all groups was 0.99 micromole/100g of diet and with tocopherol acetate was 0.145 micromole/100 g . All diabetic rats were mildly balanced by insulin . After 24 weeks of diet, plasma glucose tended to decrease in diabetic Se-supplemented groups DSmE > DSm > DSel versus Group D . In DSm and DSmE groups, plasma lipid peroxides also decreased compared with Group D, but this decrease reached significance only for DSmE (P < 0.01 for both TBARS and conjugated dienes) . Plasma triglycerides also decreased in DSm and DSmE groups versus Group D (P < 0.01; P < 0.05, respectively) . At the same time, Se increased significantly in kidneys of Groups DSel and DSm versus D and more weakly in Group DSmE, but in this case was associated with a large increase of vitamin E . These beneficial effects of selenium supplement and more so of selenium combined with vitamin E were associated with a protection of kidneys in diabetic rats which found expression in a significant correction of renal hyperfiltration (P < 0.05) and in a diminution of the number and severity of glomerular lesions (P < 0.0005).

J Invest Dermatol, 1996 Apr, 106(4), 753 - 8
Assignment of psoriasin to human chromosomal band 1q21: coordinate overexpression of clustered genes in psoriasis; Hardas BD et al.; Psoriasin is an abundant low molecular weight protein in keratinocytes from psoriatic lesions . Because of similarities in gene structure and expression to other genes on human chromosomal band 1q21, we hypothesized that psoriasin might also map to this region . To test this hypothesis, we identified and used a genomic lambda clone (lambda 9.2) as a probe for fluorescent in situ hybridization . lambda 9.2 detected the 1q21 region in 81% of 52 chromosomes 1 examined, although it also hybridized to acrocentric chromosomes . lambda 9.2 DNA yielded polymerase chain reaction amplification of 121-bp sequence colinear with psoriasin cDNA, as did genomic DNA from hybrid cell lines containing all or part of chromosome 1 . The psoriasin gene was present on a 380-kb yeast artificial chromosome clone that was previously mapped to 1q21 and shown to contain calcyclin; here it is also shown to contain MRP8 and CaN19 . Psoriasin and several other tightly linked 1q21 genes were markedly overexpressed in psoriatic lesions, suggesting a role for these clustered genes in the regulation of epidermal proliferation.

Genomics, 1996 Apr 1, 33(1), 65 - 74
A 1.6-Mb P1-based physical map of the Down syndrome region on chromosome 21; Ohira M et al.; The Down syndrome (DS) region on chromosome 21, which is responsible for the main features of DS such as characteristic facial features, a congenital heart defect, and mental retardation, has been defined by molecular analysis of DS patients with partial trisomy 21 . The 2 . 5-Mb region around the marker D21S55 between D21S17 and ERG in 21q22 is thought to be important, although contributions of other regions cannot be excluded . In this region, we focused on a 1.6-Mb region between a NotI site, LA68 (D21S396, which is mapped distal to D21S17) and ERG, because analysis of a Japanese DS family with partial trisomy 21 revealed that the proximal border of its triplicated region was distal to LA68 . We constructed P1 contigs with 46 P1 clones covering more than 95% of the 1.6-Mb region . A high-resolution restriction map using BamHI was also constructed for more detailed analysis . Our P1 contig map supplements other physical maps previously reported and provides useful materials for further analysis including gene isolation and sequencing of the DS region.

Genomics, 1996 Apr 1, 33(1), 46 - 52
Genetic and physical mapping at the limb-girdle muscular dystrophy locus (LGMD2B) on chromosome 2p; Bashir R et al.; The limb-girdle muscular dystrophies (LGMD) are a genetically heterogeneous group of disorders, different forms of which have been mapped to at least six distinct genetic loci . We have mapped an autosomal recessive form of LGMD (LGMD2B) to chromosome 2p13 . Two other conditions have been shown to map to this region or to the homologous region in mouse: a gene for a form of autosomal recessive distal muscular dystrophy, Miyoshi myopathy, shows linkage to the same markers on chromosome 2p as LGMD2B, and an autosomal recessive mouse mutation mnd2, in which there is rapidly progressive paralysis and muscle atrophy, has been mapped to mouse chromosome 6 to a region showing conserved synteny with human chromosome 2p12-p13 . We have assembled a 6-cM YAC contig spanning the LGMD2B locus and have mapped seven genes and 13 anonymous polymorphic microsatellites to it . Using haplotype analysis in the linked families, we have narrowed our region of interest to a 0-cM interval between D2S2113 and D2S2112/D2S145, which does not overlap with the critical region for mnd2 in mouse . Use of these most closely linked markers will help to determine the relationship between LGMD2B and Miyoshi myopathy . YACs selected from our contig will be the starting point for the cloning of the LGMD2B gene and thereby establish the biological basis for this form of muscular dystrophy and its relationship with the other limb-girdle muscular dystrophies.

Cancer Res, 1996 Apr 1, 56(7), 1629 - 34
Detailed deletion mapping in squamous cell carcinomas of the esophagus narrows a region containing a putative tumor suppressor gene to about 200 kilobases on distal chromosome 9q; Miura K et al.; We previously reported definition of a region containing a putative tumor suppressor gene for esophageal squamous cell carcinoma within an approximately 4-cM genomic segment at 9q31-q32 . We have investigated this region further using six new microsatellite markers isolated from yeast artificial chromosome clones covering the deleted region and have narrowly defined the commonly deleted region to a segment between two loci, KM9.1 and D9S177 . On the basis of the contig map of cosmid and yeast artificial chromosome clones, we estimate the physical size of the region of interest to be about 200 kb . Because the distal 9q region also has been implicated as the site of a tumor suppressor gene(s) related to squamous cell carcinomas of other tissues, our map provides useful information for attempts to identify a common gene for carcinomas of this cell type.

J Neurosci, 1996 Apr 1, 16(7), 2157 - 63
Interaction between the C terminus of NMDA receptor subunits and multiple members of the PSD-95 family of membrane-associated guanylate kinases; Niethammer M et al.; Selective concentration and anchoring of ionotropic receptors at the synapse is essential for neuronal signaling . Little is known about the molecules that mediate receptor clustering in the CNS . With use of the yeast two-hybrid system to screen a rat brain cDNA library and by in vitro binding assays, we have identified an interaction between NMDA receptor subunits 2A and 2B (NR2A and NR2B) and three distinct members of the PSD-95/SAP90 family of membrane-associated putative guanylate kinases . The interaction is mediated by binding of the C terminus of the NMDA receptor subunits to the first two PDZ (also known as GLGF or DHR) domains of PSD-95/SAP90, an abundant synaptic protein associated with the membrane cytoskeleton . PSD-95 is also known to bind and cluster Shaker-type voltage-gated K+ channels . Similarities between the C-termini of NR2 subunits and K+ channels suggest a common C-terminal binding motif for PDZ domains . These data suggest that PDZ domains can function as modules for protein-protein interactions . Members of the PSD-95 family might serve to anchor NMDA receptors to the submembrane cytoskeleton and aid in the assembly of signal transduction complexes at postsynaptic sites.

Radiat Res, 1996 Apr, 145(4), 408 - 18
Evidence for activities inhibiting in trans initiation of DNA replication in extract prepared from irradiated cells; Wang Y et al.; We have previously shown that replication in vitro of plasmids containing the Simian virus 40 (SV40) origin of replication is reduced when an extract of irradiated cells is used (Wang et al., Radiat . Res . 142, 169-175, 1995) . We proposed that the observed reduction in the overall replication activity is due to a reduction in the efficiency of initiation events, and that it is caused by the induction or activation by ionizing radiation of a factor(s) that inhibits DNA replication in trans . Here, we extend these studies and provide evidence that the reduced replication activity of an extract prepared from irradiated cells is not the result of a nonspecific inactivation of proteins or of an increase in the requirement for SV40 large tumor antigen (TAg), the only noncellular protein required for in vitro DNA replication . Mixing experiments demonstrate the presence of a dominant inhibitory activity(ies) in the extract of irradiated cells that efficiently stalls replication in reactions assembled using extract of nonirradiated cells . The inhibitory activity is a stable, nondialyzable molecule . Studies of kinetics suggest that the inhibitory activity(ies) affects the initiation steps of DNA replication and acts, at least partly, by modifying TAg, the key initiation protein of SV40 ori DNA replication . It is likely that the same inhibitory activity(ies) regulates cellular DNA replication by modifying the cellular homologues of TAg . Purification and characterization of this inhibitory activity(ies) will contribute to our understanding of the mechanism developed by the cell to regulate DNA replication after exposure to ionizing radiation and will define a checkpoint operating in S phase . Genetic evidence for a checkpoint in S phase distinct from the checkpoints operating in G1 and G2 phase has been reported in yeast.

Biochem Biophys Res Commun, 1996 Mar 27, 220(3), 802 - 6
A human protective protein gene partially overlaps the gene encoding phospholipid transfer protein on the complementary strand of DNA; Shimmoto M et al.; The entire human protective protein gene has been cloned, and structural analysis revealed that the gene spans 7.5kb and comprises 15 exons . Furthermore, it partially overlaps on the opposite strand with the gene encoding phospholipid transfer protein . This region of DNA on chromosome 20 appears to encode two distinct mRNAs expressing defined functional products, and the mRNAs overlap by 58 nucleotides at their 3'-untranslated ends.

Biochemistry, 1996 Mar 26, 35(12), 3764 - 71
An amphiphilic lipid-binding domain influences the topology of a signal-anchor sequence in the mitochondrial outer membrane; Steenaart NA et al.; Mas70p is targeted and inserted into the mitochondrial outer membrane in the N(in)-C(cyto) orientation, via an NH2-terminal signal-anchor sequence . The signal-anchor is comprised of two domains: an NH2-terminal hydrophilic region which is positively charged (amino acids 1-10), followed by the predicted transmembrane segment (amino acids 11-29) . Substitution of the NH2-terminal hydrophilic domain with a matrix-targeting signal caused the signal-anchor to adopt the reverse orientation in the membrane (N(cyto)-C(in)) . This substitution resulted in an increase in the net positive charge of the hydrophilic region, from +4 to +8 . In contrast to the endoplasmic reticulum and the bacterial inner membrane, where the net positive charge is an important determinant in conferring protein topology in the lipid bilayer, we show here that the reversal of the Mas70p signal-anchor was not due to differences in the number and positions of basic amino acids in the hydrophilic domain . However, a reduction in the hydrophobic moment of predicted amphiphilic helices containing an arginine, obtained by converting the apolar amino acids flanking the arginine to polar residues, caused the otherwise N(cyto)-C(in) signal-anchor to re-adopt the original N(in)-C(cyto) orientation of Mas70p . The reduced hydrophobic moment at the NH2-terminus significantly reduced the ability of this domain to bind to synthetic liposomes whose lipid composition reflected that of the outer membrane . These results identify amphiphilicity as an important determinant in causing retention of the NH2-terminus of a mitochondrial signal-anchor on the cytosolic side of the outer membrane . In addition to potential interactions between this domain and cytosolic-exposed components of the import machinery, this retention may result as well from interaction of the NH2-terminus with the surrounding membrane surface.

Biochemistry, 1996 Mar 26, 35(12), 3670 - 6
Mutational analysis of the role of hydrophobic residues in the 338-348 helix on actin in actomyosin interactions; Miller CJ et al.; Yeast actin mutants with alanines replacing I341 and I345 were studied to assess the role of hydrophobic residues in the alpha-helix 338-348 in interactions with myosin . In structural models of the actomyosin complex, this helix on actin was assigned a prominent role in the strong binding of myosin to actin . Substitution of I341 with alanine reduced the strong binding of actin to myosin subfragment-1 (S1) 9-fold compared to wild-type actin . In addition, the Vmax of the actin-activated S1 ATPase was reduced 4-fold with no change in the Km . In contrast, substitution of I345 with alanine had no significant effect on either the strong binding to S1 or the actin activation of S1 ATPase . The I341A actin filaments were found to slide in the in vitro motility assays at a lower mean velocity (1.6 +/- 0.4 microns/s) than wild-type actin filaments (2.6 +/- 0.3 microns/s) . Only 65% of the mutant actin filaments moved in such assays in comparison to 95% of the wild-type filaments . However, addition of 2.0 mM MgADP to the motility assay buffer induced movement of all the I341A filaments at a velocity (1.6 +/- 0.1 microns/s) similar to that of wild-type actin (1.7 +/- 0.1 microns/s) . The decrease in motility of the I341A actin filaments in the absence of ADP was attributed to a negative load slowing the mutant filaments and the smaller force produced by the heavy meromyosin and I341A actin system . The latter conclusion was confirmed by showing that a greater percentage of NEM-modified heavy meromyosin (external load) was required for arresting the motion of wild-type actin in the in vitro motility assay than that needed for stopping the I341A filaments.

Chem Biol Interact, 1996 Mar 25, 100(2), 177 - 85
Signal transduction mechanism in response to aflatoxin B1 exposure: protein kinase C activity; Mistry KJ et al.; A single dose of the carcinogen aflatoxin B1 (7 mg/kg body weight) to male Wistar rats significantly enhanced the hepatic activity of protein kinase C in the particulate and nuclear fractions . The particulate fraction showed stimulation at 4 and 7 h, while the nuclear activity was increased at 17 h following administration of aflatoxin B1 . The enzyme activity in cytosol revealed a significant decline corresponding to stimulation in particulate fraction . The carcinogen-activated protein kinase C stimulated autophosphorylation, and was found to accelerate in vitro phosphorylation of two model DNA synthesizing enzymes--the Klenow fragment of replicative DNA polymerase of E . Coli and a DNA primase-polymerase complex of yeast mitochondrial origin . Prior phosphorylation of these enzymes led to significant enhancement of their activities . The results imply that activation of protein kinase C and consequently the activation of DNA synthesizing enzymes may play an important role in the initiation of carcinogenesis.

J Biol Chem, 1996 Mar 22, 271(12), 7120 - 7
The sequence of the dictyostelium myo J heavy chain gene predicts a novel, dimeric, unconventional myosin with a heavy chain molecular mass of 258 kDa; Hammer JA 3rd et al.; The complete sequence of the Dictyostelium myo J heavy chain gene has been determined from overlapping genomic clones . The gene spans approximately 7400 base pairs, is split by two small introns, and encodes a 2241-residue, 258-kDa heavy chain polypeptide that that is composed of an N-terminal 944-residue myosin head domain, a central 863-residue domain that is predicted to form an alpha helical coiled-coil containing six hinges, and a C-terminal 434-residue globular domain . The head domain is notable in that it contains a approximately 30 residue insert near the nucleotide binding pocket, and five potential calmodulin/myosin light chain binding sites at the head/tail junction . The existence within the Myo J tail domain of both an extensive coiled-coil structure and a large globular domain suggests that this myosin is dimeric and incapable of self-assembly into filaments . While these properties, as well as the overall predicted structure of the Myo J protein, are reminiscent of class V myosins, the sequence of the 434-residue globular tail piece of Myo J shows no similarity to that of either yeast or vertebrate myosins V . Consistent with this, phylogenetic analyses based on myosin head sequence comparisons do not classify Myo J as a type V myosin . These and other sequence comparisons indicate that Myo J and two as-yet-unclassified unconventional myosins from Arabidopsis represent members of the newest class within the myosin superfamily (class XI) . Northern blots analyses suggest that Myo J may function predominantly in vegetative Dictyostelium cells . Finally, Southern blot analyses suggest that Dictyostelium possesses another myosin that is very closely related to Myo J.

J Biol Chem, 1996 Mar 22, 271(12), 6575 - 8
The histone folds in transcription factor TFIID; Nakatani Y et al.; The transcription factor TFIID is a multimeric protein complex containing the TATA box-binding polypeptide (TBP) and TBP-associated factors . We have previously reported that the N-terminal regions of dTAFII62 and dTAFII42 have sequence similarities with histones H4 and H3 . Here, we demonstrate that the histone-homologous regions of dTAFII62 and dTAFII42 form a heteromeric complex both in vitro and in a yeast two-hybrid system . Neither dTAFII62 nor dTAFII42 forms a homomeric complex, in agreement with a nucleosomal histone character . Moreover, circular dichroism measurements show that the heteromeric complex is dominated by alpha-helical secondary structure . These results strongly suggest the existence of a histone-like surface on TFIID.

Oncogene, 1996 Mar 21, 12(6), 1289 - 97
Distinct 3p21.3 deletions in lung cancer and identification of a new human semaphorin; Roche J et al.; Loss of chromosome 3p is a critical event in the pathogenesis of lung cancer . Overlapping homozygous 3p21.3 deletions in lung cancer cell lines involving GNAI2 were characterized and found to involve a region of genomic instability . A new widely expressed Semaphorin, H.SemaIV, was isolated from the GNAI2 deletion region . Reduced H.SemaIV expression allowed identification of additional cell lines with submicroscopic or larger deletions of the locus which occurred in a heterogeneous manner . We also demonstrate the presence of a distinct 3p21.3 homozygous deletion region, adjacent to the DNA mismatch repair gene, hMLH1, and identified deletions in direct tumors . This appears to represent one of the first demonstrations of homozygous deletions affecting 3p in direct lung tumors.

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