Jpn J Hum Genet, 1997 Dec, 42(4), 525 - 32 Fish mapping of a translocation breakpoint at 6q21 (or q22) in a patient with heterotaxia; Kato R et al.; Heterotaxia is a congenital lateralization defect of visceral organs . As several single-genes that act on the formation of left-right asymmetry during embryogenesis have been identified in animals, a defect in the similar system may play a role in heterotaxia in man . We previously reported a Japanese girl with heterotaxia associated with a de novo balanced translocation (6;18)(q21 or q22;q21.3 or q22) . In the present study, based on a hypothesis that one of the putative situs-determining genes is disrupted at a breakpoint of the translocation, we first isolated a yeast artificial chromosome (YAC) clone covering a breakpoint, 6q21 (or q22) of the translocation . Then, using STSs mapped on the YAC, we isolated bacterial artificial chromosome (BAC) clones spanning the breakpoint . FISH analysis using the BAC clones as probes revealed that the breakpoint is confined to a segment between two STS loci, WI-4066 and the CHLC.GATA6B06.192, within a genetic distance of 1.4 cM . The human connexin43 gene was not disrupted in our patient, although mutations of this gene have been reported in patients with complex heart disease and heterotaxia . The molecular localization of the translocation breakpoint in our patient may contribute to the positional cloning of a putative heterotaxia gene. Genetics, 1998 Apr, 148(4), 1829 - 32 High frequency recombination during the sexual cycle of Dictyostelium discoideum; Francis D; Analysis of Dictyostelium development and cell biology has suffered from the lack of an ordinary genetic system whereby genes can be arranged in new combinations . Genetic exchange between two long ignored strains, A2Cycr and WS205 is here reexamined . Alleles which differ in size or restriction sites between these two strains were found for seven genes . Six of these are in two clusters on chromosome 2 . Frequencies of recombinant progeny indicate that the genetic map of the two mating strains is colinear with the physical map recently worked out for the standard nonsexual strain, NC4 . The rate of recombination is high, about 0.1% per kilobase in three different regions of chromosome 2 . This value is comparable to rates found in yeast, and will permit fine dissection of the genome. J Bioenerg Biomembr, 1997 Dec, 29(6), 549 - 59 Localized firefly luciferase probes ATP at the surface of mitochondria; Aflalo C; The concentration of ATP generated by yeast mitochondria and consumed by yeast hexokinase was monitored using native firefly luciferase in solution, or recombinant luciferase localized at the surface of mitochondria . In the absence of hexokinase, both probes perform similarly in detecting exogenous or mitochondrially-generated ATP . The steady-state concentrations of ATP can be reduced in a dose-dependent manner by hexokinase . With hexokinase added in large excess, the localized probe reports substantial ATP concentrations while none is detectable by soluble luciferase . Thus, ATP accumulates near the membrane where it appears, relatively to solution, and vice versa for ADP . The extent of nucleotide gradients is shown to be correlated with the specific activity of oxidative phosphorylation and with the viscosity of the medium, but independent of the concentration of the organelles . A simple model involving diffusional restrictions is presented to describe this behavior . The metabolic and evolutionary implications of cellular catalysis limitation by physical processes are discussed. FEBS Lett, 1998 Mar 27, 425(2), 259 - 62 Two high conductance channels of the mitochondrial inner membrane are independent of the human mitochondrial genome; Murphy RC et al.; Patch-clamp techniques were used to characterize the channel activity of mitochondrial inner membranes of two human osteosarcoma cell lines: a mitochondrial genome-deficient (rho0) line and its corresponding parental (rho+) line . Previously, two high conductance channels, mitochondrial Centum picoSiemen (mCS) and multiple conductance channels (MCC), were detected in murine mitochondria . While MCC was assigned to the protein import in yeast mitochondria, the role of mCS is unknown . This study demonstrates that mCs and MCC activities from mouse mitochondria are indistinguishable from those of human mitochondria . The channel activities and their functional expression levels are not altered in cells lacking mtDNA . Hence, rho0 cells may provide a model system for elucidating the role of mitochondrial channels in disease processes and apoptosis. Curr Opin Lipidol, 1998 Apr, 9(2), 103 - 11 Expression of large genomic clones in transgenic mice: new insights into apolipoprotein B structure, function and regulation; McCormick SP et al.; Extensive manipulation of the apolipoprotein B gene in yeast and bacterial artificial chromosome clones and subsequent expression of these clones in transgenic mice have provided fresh insights into several aspects of apolipoprotein B biology, including the identification of sequences important for lipoprotein (a) assembly, the demonstration that intestinal expression of apolipoprotein B is controlled by DNA sequences > 50 kb from the gene, and the extraordinary finding that apolipoprotein B is expressed in the heart. Curr Opin Lipidol, 1998 Apr, 9(2), 93 - 7 Genetic analysis of hydroxymethylglutaryl-coenzyme A reductase regulated degradation; Hampton RY; Hydroxymethylglutaryl-coenzyme A reductase degradation occurs in the endoplasmic reticulum, and is regulated by the mevalonate pathway . In order to discover the molecules that mediate the degradation process and its control, we conducted a genetic analysis of the degradation of the yeast Hmg2p isozyme of hydroxymethylglutaryl-coenzyme A reductase . Hmg2p degradation occurs by the action of HRD genes that direct Hmg2p to the ubiquitin-proteasome pathway . Regulation of HRD-dependent Hmg2p degradation appears to occur by the action of a separate set of CRD genes. J Virol, 1998 May, 72(5), 4297 - 307 A chimeric Ty3/Moloney murine leukemia virus integrase protein is active in vivo; Dildine SL et al.; This report describes the results of experiments to determine whether chimeras between a retrovirus and portions of Ty3 are active in vivo . A chimera between Ty3 and a Neo(r)-marked Moloney murine leukemia virus (M-MuLV) was constructed . The C-terminal domain of M-MuLV integrase (IN) was replaced with the C-terminal domain of Ty3 IN . The chimeric retroviruses were expressed from an amphotrophic envelope packaging cell line . The virus generated was used to infect the human fibrosarcoma cell line HT1080, and cells in which integration had occurred were selected by G418 resistance . Three independently integrated viruses were rescued . In each case, the C-terminal Ty3 IN sequences were maintained and short direct repeats of the genomic DNA flanked the integration site . Sequence analysis of the genomic DNA flanking the insertion did not identify a tRNA gene; therefore, these integration events did not have Ty3 position specificity . This study showed that IN sequences from the yeast retrovirus-like element Ty3 can substitute for M-MuLV IN sequences in the C-terminal domain and contribute to IN function in vivo . It is also one of the first in vivo demonstrations of activity of a retrovirus encoding an integrase chimera . Studies of chimeras between IN species with distinctive integration patterns should complement previous work by expanding our understanding of the roles of nonconserved domains. Z Ernahrungswiss, 1998 Mar, 37(1), 38 - 46 {Influence of lifestyle on the use of supplements in the Brandenburg nutrition and cancer study}; Klipstein-Grobusch K et al.; Differences in dietary habits and lifestyle factors associated with a high dietary intake of fruit and vegetables are discussed and used to explain the disparity between results of observational epidemiologic studies consistently showing antioxidative vitamins to exert a protective effect on chronic diseases, and intervention studies so far not confirming this association . Within the scope of the "Brandenburger Ernahrungs- und Krebsstudie", the East German contribution to the European Prospective Investigation into Cancer and Nutrition (EPIC), we examined whether study participants using supplements on a regular basis--minerals, vitamins, protein formulation, bran/linseed, fiber, yeast or garlic pills--differed from those who did not report use of supplements according to selected lifestyle factors and dietary intake of vitamins, minerals, fiber, cholesterol, and fat from food . The study sample consisted of 10,522 participants (4,500 men and 6,022 women) aged 35-65 years enrolled in the cohort from January 1995 to July 1996 . Regular intake of one or more supplements during the past year was reported by 32.6% of women and 25.5% of men . Vitamin supplements were used by 18.8% of the women and 15.8% of the men . Figures for minerals were 14.2% for women and 8.6% for men, respectively . Garlic pills were taken regularly by 9.7% of men and 9.3% of women . Prevalence of supplement use was generally higher in women and was more pronounced in elderly participants . The most frequently used combinations were vitamin and mineral supplements, followed by a combination of garlic and either vitamin or mineral supplements . Increased use of supplements was significantly associated with higher level of education attained, regular engagement in sporting activities, health complaints, and dietary change during the previous year . No association between use of supplements and smoking status nor elevated alcohol consumption was observed . Body mass index above 30 was significantly related to increased intake of garlic pills, and in women to significantly increased use of vitamin and mineral supplements . For both men and women, age-adjusted consumption of fruit and vegetables and intake of vitamins, minerals, and fiber from food was higher for participants using mineral but also vitamin supplements compared to those who did not use these supplements . For the cohort of the "Brandenburger Ernahrungs- und Krebsstudie" we observed on the one hand that age, gender, and health-conscious lifestyle factors were related to supplement use . On the other hand presence of subjective health complaints was related to supplement use, especially for use of vitamins and minerals . Participants, who regularly consumed minerals and vitamins were also shown to have a higher intake of foods and nutrients considered to exert an antioxidative effect. Clin Nucl Med, 1998 Apr, 23(4), 226 - 8 Acute cholecystitis in AIDS patients: correlation of Tc-99m hepatobiliary scintigraphy with histopathologic laboratory findings and CD4 counts; Cacciarelli AG et al.; BACKGROUND: AIDS patients are susceptible to opportunistic gastrointestinal infections including ascending cholangitis and cholecystitis, especially if CD4 count is < 200 . Incidence of acalculous cholecystitis has not been reported previously . PURPOSE: We aim to evaluate the incidence of acalculous cholecystitis in AIDS patients and to identify causative organisms and mortality rate following cholecystectomy . MATERIALS AND METHODS: We reviewed the files of 46 patients in order to meet the objectives of this study . RESULTS: CD4 counts were < 200 in 31 patients and > 200 in 15 patients . HIDA imaging was performed in 31 patients; in 8, the CD4 count was > 200 and all had calculous cholecystitis . The gallbladder was visualized in 3 patients for a sensitivity of 63% and no organisms were found in the gallbladder specimens . In 23 patients, the CD4 count was < 200; the gallbladder was visualized in 5 patients for a HIDA sensitivity of 78%; 16 (52%) had acalculous cholecystitis; and 15 had calculous cholecystitis . In acalculous cholecystitis, Cryptosporidium was found in six cases, cytomegalovirus (CMV) in six cases, and fungus, yeast, tuberculosis, and mycobacterium avium intracellular each in one case . The thirty day mortality rate was 18%; 5 of 28 who underwent open cholecystectomy died within 30 days, 4 of them with a CD4 count < 200 . There was no mortality in the 26 patients who underwent laparoscopic cholecystectomy . CONCLUSION AND RECOMMENDATIONS: (1) Because of the high incidence of 52% of acalculous cholecystitis in AIDS patients with a CD4 count < 200, we recommend using intravenous cholecystokinin if the gallbladder is visualized on hepatobiliary scintigraphy in order to determine gallbladder ejection fraction and exclude acalculous cholecystitis . (2) Laparoscopic rather than open cholecystectomy should be the surgical procedure of choice in AIDS patients especially if the CD4 count is < 200. Prog Cell Cycle Res, 1996, 2, 187 - 95 Telomeres, telomerase, and the cell cycle; Buchkovich KJ; Telomeres protect the ends of chromosomes from degradation and fusion . In most eukaryotes telomeres are replicated by a specialised polymerase, telomerase . Telomerase synthesises one strand of the telomere; while conventional DNA polymerases synthesise the complementary strand . Additional processing of telomeres occurs in ciliates and yeast during each cell cycle . Telomerase activity and RNA levels change as cells enter and exit the cell cycle . Gradual telomere shortening in the absence of telomerase does not immediately affect cell cycling; however, "critically" short telomeres are hypothesised to play a role in senescence and the triggering of DNA damage checkpoints. Prog Cell Cycle Res, 1996, 2, 129 - 35 Suc1: cdc2 affinity reagent or essential cdk adaptor protein? Vogel L, Baratte B. CKS proteins, for which the original member, p13suc1, was identified as a suppressor of cdc2 alleles in S . Pombe, have long served as a reagent for the purification of p34cdc2, whereas their biological function has remained elusive . Apparently conflicting data derived from different model systems may indicate a diversity of function for these proteins . Several new observations in yeast and Xenopus egg extracts together with new structural information tends to enhance the hypothesis that CKS proteins function to alter the activity of cdc2 at several important points in the cell cycle . Here we review previous observations and recent data that suggest CKS proteins serve as adaptor proteins that modify the functions of cdc2 throughout the cell cycle. Prog Cell Cycle Res, 1996, 2, 91 - 7 Tyrosine kinases wee1 and mik1 as effectors of DNA replication checkpoint control; Tourret J et al.; Cell cycle studies have revealed mechanisms that prevent cell division if DNA fails to be completely replicated or sustains damage . Here we focus on the evidence from yeast genetics that the wee1 and mik1 tyrosine kinases cooperate in the inhibitory phosphorylation of cdc2p, and the possibility that these kinases function in pathways that ensure the integrity of the genome prior to cell division . We also review the progress in cloning and analysing wee1-like tyrosine kinases from higher eukaryotes, and the evidence for and against their functioning in ensuring DNA replication prior to mitosis . Finally, we discuss the genes involved in these feedback controls and suggest that wee1p and mik1p might be the ultimate effectors that prevent mitosis when a checkpoint is triggered. Prog Cell Cycle Res, 1995, 1, 287 - 97 The MAP kinase cascade: its role in Xenopus oocytes, eggs and embryos; Gotoh Y et al.; Mitogen-activated protein kinase (MAPK) was originally identified as a serine/threonine kinase that is activated by mitogens . Now MAPK and its activator, MAPK kinase (MAPKK), are thought to function in a wide variety of intracellular signalling pathways from yeast to vertebrate . We describe here a brief summary of the dissection of the MAPK cascade and its possible functions, especially in Xenopus oocytes and embryos. J Immunol, 1998 Jan 1, 160(1), 197 - 208 Characterization and mapping to human chromosome 8q24.3 of Ly-6-related gene 9804 encoding an apparent homologue of mouse TSA-1; Shan X et al.; The 9804 gene, which encodes a human Ly-6 protein most similar to mouse differentiation Ag TSA-1/Sca-2, has also been called RIG-E . Like mouse TSA-1, it has a broad tissue distribution with varied expression levels in normal human tissues and tumor cell lines . Like some members of the murine Ly-6 family, the 9804 gene is responsive to IFNs, particularly IFN-alpha . Overlapping genomic fragments spanning the 9804 gene (5543 bp) have been isolated and characterized . The gene organization is analogous to that of known mouse Ly-6 genes . The first exon, 2296 bp upstream from exon II, is entirely untranslated . The three coding exons (II, III, and IV) are separated by short introns of 321 and 131 bp, respectively . Primers were developed for specific amplification of 9804 gene fragments . Screening of human-hamster somatic cell hybrids and yeast artificial chromosomes (YACs) indicated that the gene is distal to c-Myc, located in the q arm of human chromosome 8 . No positives were detected from the Centre d'Etude du Polymorphisme Humain mega-YAC A or B panels, nor from bacterial artificial chromosome libraries; two positive cosmids (c101F1 and c157F6) were isolated from a human chromosome 8 cosmid library (LA08NC01) . Fluorescence in situ hybridization of metaphase spreads of chromosome 8, containing hybrid cell line 706-B6 clone 17 (CL-17) with cosmid c101F1, placed the 9804 gene close to the telomere at 8q24.3 . This mapping is significant, since the region shares a homology with a portion of mouse chromosome 15, which extends into band E where Ly-6 genes reside . Moreover, the gene encoding E48, the homologue of mouse Ly-6 molecule ThB, has also been mapped to 8q24. Structure, 1998 Mar 15, 6(3), 293 - 307 Copper amine oxidase from Hansenula polymorpha: the crystal structure determined at 2.4 A resolution reveals the active conformation; Li R et al.; BACKGROUND: Copper-containing amine oxidases (CAOs) are widespread in nature . These enzymes oxidize primary amine substrates to the aldehyde product, reducing molecular oxygen to hydrogen peroxide in the process . CAOs contain one type 2 copper atom and topaquinone (TPQ), a modified tyrosine sidechain utilized as a redox cofactor . The methylamine oxidase from the yeast Hansenula polymorpha (HPAO) is an isoform of CAO with a preference for small aliphatic amine or phenethylamine substrates . The enzyme is dimeric with a subunit molecular weight of 78 kDa . Structural studies are directed at understanding the basis for cofactor biogenesis and catalytic efficiency . RESULTS: The X-ray crystal structure of HPAO has been solved at 2.4 A resolution by a combination of molecular replacement and single isomorphous replacement followed by refinement using sixfold symmetry averaging . The electron density at the catalytic site shows that the TPQ conformation corresponds to that of the active form of the enzyme . Two channels, one on either side of TPQ, are observed in the structure that provide access between the active site and the bulk solvent . CONCLUSIONS: The structure shows TPQ in a position poised for catalysis . This is the first active CAO structure to reveal this conformation and may help further our understanding of the catalytic mechanism . On the substrate side of TPQ a water-containing channel leading to the protein surface can serve as an entrance or exit for substrate and product . On the opposite side of TPQ there is direct access from the bulk solvent of the dimer interface by which molecular oxygen may enter and hydrogen peroxide depart . In addition, a network of conserved water molecules has been identified which may function in the catalytic mechanism. J Clin Pharmacol, 1998 Feb, 38(2 Suppl), 25S - 35S Preclinical enantioselective pharmacology of (R)- and (S)- ketorolac; Handley DA et al.; Many of the nonsteroidal anti-inflammatory drugs (NSAIDs) are marketed as racemic mixtures, composed of (R)- and (S)- enantiomers . Racemic NSAIDs are potent cyclooxygenase (COX) inhibitors only through the action of the (S)- enantiomers, as the (R)- enantiomers do not exhibit COX inhibition . However, the (R)- enantiomer of ketoprofen exhibits potent analgesic activity and minimal ulcerogenic potential . To extend these observations, we examined the (R)- and (S)- enantiomers of RS- ketorolac, (S)- ketorolac exhibited potent COX1 and COX2 enzyme inhibition, whereas (R)- ketorolac was > 100-fold less active on both COX subtypes . Both enantiomers did not affect norepinephrine or serotonin uptake sites, and nitric oxidase or lipoxygenase activities, nor did they demonstrate any affinity for opioid receptors (mu, delta, or kappa) . In experimental models, (S)- ketorolac exhibited about 10-fold greater activity than (R)- ketorolac in the murine phenylquinone writhing model . In this model, morphine sulfate was effective at much lower doses, however, and neither (R)- nor (S)- ketorolac showed any morphine-sparing effect . In the rat gait test for analgesia in the foot paw after injection of brewers yeast suspension, neither (R)- nor (S)- ketorolac affected paw volume . However, both provoked changes in gait scores, the (S)- enantiomer being 30-fold more potent than the (R)- enantiomer . A similar reduction was observed with respect to ulcerogenic potential, measured by direct microscopic changes after test conclusion . These findings suggest that (R)- ketorolac may possess analgesic activity that is independent of COX inhibition and may be associated with reduced ulcerogenic potential compared to effects exhibited by (S)- ketorolac. Mycopathologia, 1997, 139(2), 79 - 85 Humoral immune response to Malassezia furfur in patients with pityriasis versicolor and seborrheic dermatitis; Silva V et al.; Humoral immune responses against exoantigen components of oval, elliptic and round yeast forms of Malassezia furfur were analysed by ELISA and Western blotting assays, using sera from patients with pityriasis versicolor (PV), seborrheic dermatitis (SD) and healthy adults (HA), as control . Sera from patients with SD showed IgG anti-oval M . furfur titers ranging from 1/400 to 1/6400 showing geometric mean (GM) of 1/1472, higher than those obtained with sera from patients with PV (1/200 to 1/6400, GM = 1/1239) . Both patient groups showed mean titres statistically superior (P < 0.05) than those obtained form HA (GM = 1/229) . Similar data were also obtained with the elliptic and round antigens . However, the anti-oval IgG mean titers from patients' sera were much higher than those obtained with elliptic or round antigenic components (p < 0.05) Anti-M furfur IgM titers obtained from patient's sera with PV against all three exoantigens were statistically superior (p < 0.05) than HA group . Patients with SD showed IgM titers statistically superior (p < 0.05) only to oval yeasts of M . furfur . The IgA mean titers from patients' groups against the different morphological antigens were shown be slightly higher than those HA group . By Western blot, using rabbit anti-sera, the different antigenic components of M.furfur showed a close relationship mainly between oval and elliptic yeast cells antigens . The 70 kDa component of the M . furfur exoantigen of oval morphology was recognized by 84% of the PV patients' sera . On the other hand, SD patients' sera recognized 3 principal components of 70 kDa (100%), 65 kDa (67%) and 84 kDa (53%) . These components may be considered immunological markers for PV and SD . Twenty-five percent of HA sera recognized the components of 65, 70 and 94 kDa . This investigation shows that M . furfur antigens can sensitize the host, mainly the oval yeast form of M . furfur with a very important specific IgG response in patients with SD and PV. Genome, 1998 Feb, 41(1), 62 - 9 Collinearity between a 30-centimorgan segment of Arabidopsis thaliana chromosome 4 and duplicated regions within the Brassica napus genome; Cavell AC et al.; Arabidopsis thaliana (the model dicotyledonous plant) is closely related to Brassica crop species . Genome collinearity, or conservation of marker order, between Brassica napus (oilseed rape) and A . thaliana was assessed over a 7.5-Mbp region of the long arm of A . thaliana chromosome 4, equivalent to 30 cM . Estimates of copy number indicated that sequences present in a single copy in the haploid genome of A . thaliana (n = 5) were present in 2-8 copies in the haploid genome of B . napus (n = 19), while sequences present in multiple copies in A . thaliana were present in over 10 copies in B . napus . Genetic mapping in B . napus of DNA markers derived from a segment of A . thaliana chromosome 4 revealed duplicated homologous segments in the B . napus genome . Physical mapping in A . thaliana of homologues of Brassica clones derived from these regions confirmed the identity of six duplicated segments with substantial homology to the 7.5-Mbp region of chromosome 4 in A . thaliana . These six duplicated Brassica regions (on average 22 cM in length) are collinear, except that two of the six copies contain the same large internal inversion . These results have encouraging implications for the feasibility of shuttling between the physical map of A . thaliana and genetic maps of Brassica species, for identifying candidate genes and for map based gene cloning in Brassica crops. J Biol Chem, 1998 Mar 27, 273(13), 7358 - 66 The role of DnaJ-like proteins in glucocorticoid receptor.hsp90 heterocomplex assembly by the reconstituted hsp90.p60.hsp70 foldosome complex; Dittmar KD et al.; The glucocorticoid receptor (GR) is recovered from hormone-free cells in a heterocomplex with the molecular chaperone hsp90, which is required to produce the proper folding state for steroid binding . GR.hsp90 heterocomplexes are formed by a multiprotein system that appears to exist in all eukaryotic cells . Recently, we have reconstituted a receptor.hsp90 heterocomplex assembly system with purified rabbit hsp90 and hsp70 and bacterially expressed human p23 and p60 . We have shown that hsp90, p60, and hsp70 form an hsp90.p60 . hsp70 complex that converts the GR from a non-steroid binding to a steroid binding form (Dittmar, K . D., and Pratt, W . B . (1997) J . Biol . Chem . 272, 13047-13054) . The resulting GR.hsp90 heterocomplex rapidly disassembles unless p23 is present to bind to the ATP-dependent conformation of hsp90 and stabilize its association with the receptor (Dittmar, K . D., Demady, D . R., Stancato, L . F., Krishna, P., and Pratt, W . B . (1997) J . Biol . Chem . 272, 21213-21220) . In the current work, we show that the purified rabbit hsp70 utilized in prior studies is contaminated with a small amount of the rabbit DnaJ homolog hsp40 . Elimination of the hsp40 from the purified GR.hsp90 assembly system reduces assembly activity, and the activity is restored by addition of the purified yeast DnaJ homolog YDJ-1 . hsp40 is a component of the hsp90.p60.hsp70 foldosome complex isolated from reticulocyte lysate with antibody against p60 . Under conditions that promote binding of p23 to hsp90 (elevated temperature, ATP, Nonidet P-40, molybdate), a five-membered (p23 . hsp90.p60.hsp70.hsp40) complex of chaperone proteins is formed in reticulocyte lysate or from purified proteins . The hsp40-free, purified assembly system has a modest level of assembly activity that is maximally potentiated by YDJ-1 when it is present at about one-twentieth the concentration of hsp70 . Although hsp40 is not in the final GR.hsp90 heterocomplex isolated from L cell cytosol, it is in the GR.hsp90 heterocomplex assembled in reticulocyte lysate . We conclude that hsp40 is a component of the multiprotein hsp90-based chaperone system where it potentiates GR.hsp90 heterocomplex assembly. Genes Dev, 1998 Mar 15, 12(6), 858 - 67 A cooperative interaction between U2AF65 and mBBP/SF1 facilitates branchpoint region recognition; Berglund JA et al.; During the early events of pre-mRNA splicing, intronic cis-acting sequences are recognized and interact through a network of RNA-RNA, RNA-protein, and protein-protein contacts . Recently, we identified a branchpoint sequence binding protein in yeast (BBP) . The mammalian ortholog (mBBP/SF1) also binds specifically to branchpoint sequences and interacts with the well studied mammalian splicing factor U2AF65, which binds to the adjacent polypyrimidine (PY) tract . In this paper we demonstrate that the mBBP/SF1-U2AF65 interaction promotes cooperative binding to a branchpoint sequence-polypyrimidine tract-containing RNA, and we suggest that this cooperative RNA binding contributes to initial recognition of the branchpoint sequence (BPS) during pre-mRNA splicing . We also demonstrate the essential nature of the third RBD of U2AF65 for the interaction between the two proteins, both in the presence and absence of RNA. Bioconjug Chem, 1998 Mar-Apr, 9(2), 152 - 9 Cell targeting by glycosidic telomers . Specific recognition of the Kb CWL1 lectin by galactosylated telomers; Coulon J et al.; This work deals with the synthesis and lectinic recognition ability of galactosylated telomers . To investigate if telomeric carriers could exhibit cellular recognition properties, we have synthesized mono- and polygalactosylated tris(hydroxymethyl)acrylamidomethane (THAM) telomers . The affinity of such macromolecular drug carriers toward a receptor, the yeast Kb CWL1 lectin, was defined, and the influence of mono- or polygalactosylation of THAM units on the recognition phenomenon was assessed . The lectinic affinity of the compounds was estimated by measuring the inhibition of yeast aggregation . The average degree of polymerization as well as the hydrophilic-lipophilic balance of such galactosylated telomers affects their recognition ability for the lectin. Eur J Cell Biol, 1998 Feb, 75(2), 128 - 39 Adenosine inhibits actin dynamics in human neutrophils: evidence for the involvement of cAMP; Zalavary S et al.; The mechanisms by which adenosine regulates the inflammatory reaction are poorly characterized . In this study, we investigated the effects of adenosine on neutrophil actin polymerization elicited by the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP) or IgG-opsonized yeast particles . We used bodipy-phallacidin staining in combination with flow cytometry and found that adenosine markedly reduced actin polymerization triggered by IgG-yeast, whereas the effect on the fMLP-response was less pronounced . Similar or even more pronounced effects were obtained with the adenosine A2 receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA), suggesting an A2 receptor-mediated mechanism . The following observations indicate that the A2 receptor-induced effects involve the cAMP-protein kinase A (PKA) signaling pathway: (1) a combination of NECA and the cAMP-specific phosphodiesterase (PDE) inhibitor Ro 20-1724 raised the cAMP content in both unstimulated and stimulated neutrophils and also further inhibited the actin dynamics; (2) the PKA inhibitor H89 reversed the inhibitory effects of NECA on the actin dynamics; (3) Ro 20-1724, isoproterenol and dibutyryl cAMP (DBcAMP) reduced actin polymerization in almost the same way as NECA did . NECA together with Ro 20-1724 impaired the fMLP-induced shape changes and cortical accumulation of actin filaments . In contrast, H89 potentiated the fMLP-induced formation of a submembranous ring of actin filaments . Neutrophils phagocytosing yeast particles in the presence of NECA and Ro 20-1724 were predominantly round in shape, and their ability to extend actin-rich pseudopods around the prey was reduced . These effects were partly antagonized by H89 . In correlation with the effects on actin polymerization, NECA more effectively diminished IgG-induced upregulation of the beta2 integrin CD11b/CD18 than such upregulation induced by fMLP . The inhibitory effects of A2-receptor activation on actin dynamics and beta2 integrin expression in neutrophils exposed to IgG-yeast were also associated with a cAMP-dependent reduction of the phagocytic capacity . In conclusion, we show that adenosine inhibits actin dynamics and shape changes in neutrophils via a cAMP-dependent pathway . This finding further characterizes the mechanisms by which adenosine functions as an important modulator of the inflammatory response. No To Hattatsu, 1998 Mar, 30(2), 128 - 33 {Molecular analysis of peroxisomal disorders}; Shimozawa N; Peroxisome biogenesis disorders (PBD) include Zellweger syndrome (ZS), neonatal adrenoleukodystrophy (NALD) and infantile Refsum disease (IRD) . They are classified into ten complementation groups . Five pathogenic genes have been identified using different model systems of peroxisome deficient mutants . PAF-1 and 2 were identified from CHO mutants and were responsible genes for PBD group F and C . Human PEX 5, 12 and 1, responsible genes for group 2, 3 and 1, respectively, were cloned by homology search between yeast PEX genes and human genes on the cDNA data base . Adrenoleukodystrophy (ALD), the most frequent peroxisomal disorder, shows phenotypic heterogeneity . Its responsible gene was cloned by positional cloning . It encodes a 75 kDa peroxisomal membrane protein (ALDP) that is a member of the ATP-binding cassette transporter family . There are about 120 different mutations including missense, nonsense and splice mutations, as well as insertions and deletions of a few base pairs . There is no correlation between the clinical phenotype and the ALDP gene mutation . Recently, animal models have been produced by targeted mutation of the PBD and ALD genes . The mouse model should facilitate researches on PBD and ALD, especially those on regulatory factors of their phenotypic heterogeneity and on new therapeutic approaches. Genomics, 1998 Mar 15, 48(3), 384 - 8 Chromosomal locations of three human nuclear genes (RPSM12, TUFM, and AFG3L1) specifying putative components of the mitochondrial gene expression apparatus; Shah ZH et al.; We have mapped the chromosomal locations of three human nuclear genes for putative components of the apparatus of mitochondrial gene expression, using a combination of in situ hybridization and interspecies hybrid mapping . The genes RPMS12 (mitoribosomal protein S12, a conserved protein component of the mitoribosomal accuracy center), TUFM (mitochondrial elongation factor EF-Tu), and AFG3L1 (similar to the yeast genes Afg3 and Rca1 involved in the turnover of mistranslated or misfolded mtDNA-encoded polypeptides) were initially characterized by a combination of database sequence analysis, PCR, cloning, and DNA sequencing . RPMS12 maps to chromosome 19q13.1, close to the previously mapped gene for autosomal dominant hearing loss DFNA4 . The TUFM gene is located on chromosome 16p11.2, with a putative pseudogene or variant (TUFML) located very close to the centromere of chromosome 17 . AFG3L1 is located on chromosome 16q24, very close to the telomere . By virtue of their inferred functions in mitochondria, these genes should be regarded as candidates of disorders sharing features with mitochondrial disease syndromes, such as sensorineural deafness, diabetes, and retinopathy. Genomics, 1998 Mar 15, 48(3), 377 - 80 Genomic organization of the human SPOCK gene and its chromosomal localization to 5q31; Charbonnier F et al.; SPOCK, previously identified as testican, is a modular proteoglycan that carries both chondroitin and heparan sulfate glycosaminoglycan side chains . The overall genomic organization has been established . The SPOCK gene spans at least 70 kb and is composed of 11 exons: the first half of the gene is dramatically expanded, but the second half is more compact . In situ hybridization and YAC mapping independently linked the SPOCK gene to 5q31, a region containing an impressive number of genes encoding growth factors, cytokines, and neurotransmitter and hormone receptors . The gene is located between the IL9 and the EGR1 genes, bordering the smallest commonly deleted region of chromosome 5. Genomics, 1998 Mar 15, 48(3), 277 - 88 Structure and methylation-based silencing of a gene (DBCCR1) within a candidate bladder cancer tumor suppressor region at 9q32-q33; Habuchi T et al.; Loss of heterozygosity (LOH) on chromosome 9q is the most frequent genetic alteration in transitional cell carcinoma (TCC) of the bladder, indicating the presence of one or more relevant tumor suppressor genes . We previously mapped one of these putative tumor suppressor loci to 9q32-q33 and localized the candidate region within a single YAC 840 kb in size . This locus has been designated DBC1 (for deleted in bladder cancer gene 1) . We have identified a novel gene, DBCCR1, in this candidate region by searching for expressed sequence tags (ESTs) that map to YACs spanning the region . Database searching using the entire DBCCR1 cDNA sequence identified several human ESTs and a few homologous mouse . ESTs . However, the predicted 761-amino-acid sequence had no significant homology to known protein sequences . Mutation analysis of the coding region and Southern blot analysis detected neither somatic mutations nor gross genetic alterations in primary TCCs . Although DBCCR1 was expressed in multiple normal human tissues including urothelium, mRNA expression was absent in 5 of 10 (50%) bladder cancer cell lines . Methylation analysis of the CpG island at the 5' region of the gene and the induction of de novo expression by a demethylating agent indicated that this island might be a frequent target for hypermethylation and that hypermethylation-based silencing of the gene occurs in TCC . These findings make DBCCR1 a good candidate for DBC1. Proc Natl Acad Sci U S A, 1998 Mar 17, 95(6), 3275 - 80 Alpha2-macroglobulin associates with beta-amyloid peptide and prevents fibril formation; Hughes SR et al.; We have used the yeast two-hybrid system to isolate cDNAs encoding proteins that specifically interact with the 42-aa beta-amyloid peptide (Abeta), a major constituent of senile plaques in Alzheimer's disease . The carboxy terminus of alpha2-macroglobulin (alpha2M), a proteinase inhibitor released in response to inflammatory stimuli, was identified as a strong and specific interactor of Abeta, utilizing this system . Direct evidence for this interaction was obtained by co-immunoprecipitation of alpha2M with Abeta from the yeast cell, and by formation of SDS-resistant Abeta complexes in polyacrylamide gels by using synthetic Abeta and purified alpha2M . The association of Abeta with alpha2M and various purified amyloid binding proteins was assessed by employing a method measuring protein-protein interactions in liquid phase . The dissociation constant by this technique for the alpha2M-Abeta association using labeled purified proteins was measured (Kd = 350 nM) . Electron microscopy showed that a 1:8 ratio of alpha2M to Abeta prevented fibril formation in solution; the same ratio to Abeta of another acute phase protein, alpha1-antichymotrypsin, was not active in preventing fibril formation in vitro . These results were corroborated by data obtained from an in vitro aggregation assay employing Thioflavine T . The interaction of alpha2M with Abeta suggests new pathway(s) for the clearance of the soluble amyloid peptide. Proc Natl Acad Sci U S A, 1998 Mar 17, 95(6), 2844 - 9 Caenorhabditis elegans orthologs of the aryl hydrocarbon receptor and its heterodimerization partner the aryl hydrocarbon receptor nuclear translocator; Powell-Coffman JA et al.; The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor, until now described only in vertebrates, that mediates many of the carcinogenic and teratogenic effects of certain environmental pollutants . Here, we describe orthologs of AHR and its dimerization partner AHR nuclear translocator (ARNT) in the nematode Caenorhabditis elegans, encoded by the genes ahr-1 and aha-1, respectively . The corresponding proteins, AHR-1 and AHA-1, share biochemical properties with their mammalian cognates . Specifically, AHR-1 forms a tight association with HSP90, and AHR-1 and AHA-1 interact to bind DNA fragments containing the mammalian xenobiotic response element with sequence specificity . Yeast expression studies indicate that C . elegans AHR-1, like vertebrate AHR, requires some form of post-translational activation . Moreover, this requirement depends on the presence of the domains predicted to mediate binding of HSP90 and ligand . Preliminary experiments suggest that if AHR-1 is ligand-activated, its spectrum of ligands is different from that of the mammalian receptor: C . elegans AHR-1 is not photoaffinity labeled by a dioxin analog, and it is not activated by beta-naphthoflavone in the yeast system . The discovery of these genes in a simple, genetically tractable invertebrate should allow elucidation of AHR-1 function and identification of its endogenous regulators. Proc Natl Acad Sci U S A, 1998 Mar 17, 95(6), 2795 - 800 Characterization of a human RPD3 ortholog, HDAC3; Emiliani S et al.; Histone acetylation levels in cells result from a dynamic equilibrium between competing histone acetylases and deacetylases . Changes in histone acetylation levels occur during both transcriptional activation and silencing . Cloning of the cDNA for a human histone deacetylase (HDAC1) has shown that it represents a human ortholog of the yeast transcriptional regulator RPD3 . We have screened the expressed sequence tag database (National Center for Biotechnology Information) with the yeast RPD3 sequence and identified a human ortholog of RPD3, HDAC3 . This cDNA encodes a protein of 428 amino acids with 58% sequence identity with HDAC1p . By using a specific polyclonal antiserum recognizing the C-terminal domain of HDAC3p and Western blotting, we detected a single approximately 49-kDa band in several tumor cell lines . HDAC3p is expressed predominantly in the nuclear compartment . Immunoprecipitation experiments with either an antiserum against HDAC3p or an anti-FLAG antiserum and a flagged HDAC3 cDNA showed that HDAc3p exhibits deacetylase activity both on free histones and on purified nucleosomes . This deacetylase activity is inhibited by trichostatin, trapoxin, and butyrate in vitro to the same degree as the deacetylase activity associated to HDAC1p . These observations identify another member of a growing family of human HDAC genes. J Neurosci, 1998 Mar 15, 18(6), 2017 - 27 Yotiao, a novel protein of neuromuscular junction and brain that interacts with specific splice variants of NMDA receptor subunit NR1; Lin JW et al.; The molecular machinery underlying neurotransmitter receptor immobilization at postsynaptic sites is poorly understood . The NMDA receptor subunit NR1 can form clusters in heterologous cells via a mechanism dependent on the alternatively spliced C1 exon cassette in its intracellular C-terminal tail, suggesting a functional interaction between NR1 and the cytoskeleton . The yeast two-hybrid screen was used here to identify yotiao, a novel coiled coil protein that interacts with NR1 in a C1 exon-dependent manner . Yotiao mRNA (11 kb) is present modestly in brain and abundantly in skeletal muscle and pancreas . On Western blots, yotiao appears as an approximately 230 kDa band that is present in cerebral cortex, hippocampus, and cerebellum . Biochemical studies reveal that yotiao fractionates with cytoskeleton-associated proteins and with the postsynaptic density . With regard to immunohistochemistry, two anti-yotiao antibodies display a somatodendritic staining pattern similar to each other and to the staining pattern of NR1 . Yotiao was colocalized by double-label immunocytochemistry with NR1 in rat brain and could be coimmunoprecipitated with NR1 from heterologous cells . Thus yotiao is an NR1-binding protein potentially involved in cytoskeletal attachment of NMDA receptors . Consistent with a general involvement in postsynaptic structure, yotiao was also found to be specifically concentrated at the neuromuscular junction in skeletal muscle. EMBO J, 1998 Mar 2, 17(5), 1395 - 404 A new rac target POSH is an SH3-containing scaffold protein involved in the JNK and NF-kappaB signalling pathways; Tapon N et al.; The Rho, Rac and Cdc42 GTPases coordinately regulate the organization of the actin cytoskeleton and the JNK MAP kinase pathway . Mutational analysis of Rac has previously shown that these two activities are mediated by distinct cellular targets, though their identity is not known . Two Rac targets, p65(PAK) and MLK, are ser/thr kinases that have been reported to be capable of activating the JNK pathway . We present evidence that neither is the Rac target mediating JNK activation in Cos-1 cells . We have used yeast two-hybrid selection and identified a new target of Rac, POSH . This protein consists of four SH3 domains and ectopic expression leads to the activation of the JNK pathway and to nuclear translocation of NF-kappaB . When overexpressed in fibroblasts, POSH is a strong inducer of apoptosis . We propose that POSH acts as a scaffold protein and contributes to Rac-induced signal transduction pathways leading to diverse gene transcriptional changes. EMBO J, 1998 Mar 2, 17(5), 1371 - 84 Axin, a negative regulator of the Wnt signaling pathway, forms a complex with GSK-3beta and beta-catenin and promotes GSK-3beta-dependent phosphorylation of beta-catenin; Ikeda S et al.; Glycogen synthase kinase-3 (GSK-3) mediates epidermal growth factor, insulin and Wnt signals to various downstream events such as glycogen metabolism, gene expression, proliferation and differentiation . We have isolated here a GSK-3beta-interacting protein from a rat brain cDNA library using a yeast two-hybrid method . This protein consists of 832 amino acids and possesses Regulators of G protein Signaling (RGS) and dishevelled (Dsh) homologous domains in its N- and C-terminal regions, respectively . The predicted amino acid sequence of this GSK-3beta-interacting protein shows 94% identity with mouse Axin, which recently has been identified as a negative regulator of the Wnt signaling pathway; therefore, we termed this protein rAxin (rat Axin) . rAxin interacted directly with, and was phosphorylated by, GSK-3beta . rAxin also interacted directly with the armadillo repeats of beta-catenin . The binding site of rAxin for GSK-3beta was distinct from the beta-catenin-binding site, and these three proteins formed a ternary complex . Furthermore, rAxin promoted GSK-3beta-dependent phosphorylation of beta-catenin . These results suggest that rAxin negatively regulates the Wnt signaling pathway by interacting with GSK-3beta and beta-catenin and mediating the signal from GSK-3beta to beta-catenin. EMBO J, 1998 Mar 2, 17(5), 1328 - 35 The polo-like kinase Plx1 is required for M phase exit and destruction of mitotic regulators in Xenopus egg extracts; Descombes P et al.; Polo-like kinases (Plks), named after the Drosophila gene product polo, have been implicated in the regulation of multiple aspects of mitotic progression, including the activation of the Cdc25 phosphatase, bipolar spindle formation and cytokinesis . Genetic analyses performed in yeast and Drosophila suggest a function for Plks at late stages of mitosis, but biochemical data to support such a function in vertebrate organisms are lacking . Here we have taken advantage of Xenopus egg extracts for exploring the function of Plx1, a Xenopus Plk, during the cell cycle transition from M phase to interphase (I phase) . We found that the addition of a catalytically inactive Plx1 mutant to M phase-arrested egg extracts blocked their Ca2+-induced release into interphase . Concomitantly, the proteolytic destruction of several targets of the anaphase-promoting complex and the inactivation of the Cdc2 protein kinase (Cdk1) were prevented . Moreover, the M to I phase transition could be abolished by immunodepletion of Plx1, but was restored upon the addition of recombinant Plx1 . These results demonstrate that the exit of egg extracts from M phase arrest requires active Plx1, and they strongly suggest an important role for Plx1 in the activation of the proteolytic machinery that controls the exit from mitosis. EMBO J, 1998 Aug 10, 17(5), 1315 - 27 The mitotic peptidyl-prolyl isomerase, Pin1, interacts with Cdc25 and Plx1; Crenshaw DG et al.; The cis/trans peptidyl-prolyl isomerase, Pin1, is a regulator of mitosis that is well conserved from yeast to man . Here we demonstrate that depletion of Pin1-binding proteins from Xenopus egg extracts results in hyperphosphorylation and inactivation of the key mitotic regulator, Cdc2/cyclin B . We show biochemically that this phenotype is a consequence of Pin1 interaction with critical upstream regulators of Cdc2/cyclin B, including the Cdc2-directed phosphatase, Cdc25, and its known regulator, Plx1 . Although Pin1 could interact with Plx1 during interphase and mitosis, only the phosphorylated, mitotically active form of Cdc25 was able to bind Pin1, an event we have recapitulated using in vitro phosphorylated Cdc25 . Taken together, these data suggest that Pin1 may modulate cell cycle control through interaction with Cdc25 and its activator, Plx1. Plant Cell, 1998 Jan, 10(1), 63 - 73 AtKuP1: a dual-affinity K+ transporter from Arabidopsis; Fu HH et al.; Plant roots contain both high- and low-affinity transport systems for uptake of K+ from the soil . In this study, we characterize a K+ transporter that functions in both high- and low-affinity uptake . Using yeast complementation analysis, we isolated a cDNA for a functional K+ transporter from Arabidopsis (referred to as AtKUP1 for Arabidopsis thaliana K+ uptake) . When expressed in a yeast mutant, AtKUP1 dramatically increased K+ uptake capacity at both a low and high {K+} range . Kinetic analyses showed that AtKUP1-mediated K+ uptake displays a "biphasic" pattern similar to that observed in plant roots . The transition from the high-affinity phase (K(m) of 44 microM) to the low-affinity phase (K(m) of 11 mM) occurred at 100 to 200 microM external K+ . Both low- and high-affinity K+ uptake via AtKUP1 were inhibited by 5 mM or higher concentrations of NaCl . In addition, AtKUP1-mediated K+ uptake was inhibited by K+ channel blockers, including tetraethylammonium, Cs+, and Ba2+ . Consistent with a possible function in K+ uptake from the soil, the AtKUP1 gene is primarily expressed in roots . We conclude that the AtKUP1 gene product may function as a K+ transporter in Arabidopsis roots over a broad range of {K+} in the soil. Hum Mol Genet, 1998 Mar, 7(3), 557 - 62 Rodent Y chromosome TSPY gene is functional in rat and non-functional in mouse; Mazeyrat S et al.; Recombination is believed to prevent genetic deterioration in sexual populations because it allows conservation of functional genotypes by removing deleterious mutations . Moreover, evidence that non-recombining segments of a genome deteriorate is provided by genetic experiments in Drosophila and yeast . Y chromosomes generally do not recombine along most of their length, and thus Y chromosome genes, despite having been selectively maintained for their function, could be lost from the genome . Here we present definitive evidence that functional Y genes can be lost from the mammalian genome . TSPY genes must have been selectively maintained on the mammalian Y chromosome since before the radiation of eutheria, 80 million years ago, as they are found conserved on the Y chromosome in two mammalian orders: primate and artiodactyl . We have now identified TSPY on the rodent Y chromosome, in mouse and rat . The gene structure and expression of rat TSPY suggest that it is a functional, testis-specific gene, but the closely related mouse gene, Tspy, has clearly become non-functional, producing only low levels of aberrantly spliced transcripts . Thus TSPY lost its function in the mouse lineage after its divergence from the rat lineage . So, in the case of Tspy at least, the absence of recombination does appear to have led to the loss of a functional gene. Hum Mol Genet, 1998 Mar, 7(3), 501 - 5 Detection of a homozygous four base pair deletion in the protein X gene in a case of pyruvate dehydrogenase complex deficiency; Ling M et al.; While the presence of a lipoyl-containing protein (protein X) separate from lipoyl transacetylase in the pyruvate dehydrogenase complex (PDC) has been known for some time, until recently only the cDNA for the yeast enzyme has been cloned . We have cloned, sequenced and characterized the cDNA encoding the human protein X and localized the protein X gene to chromosome 11p13 . We also report here a new case of protein X deficiency identified immunologically, with decreased activity of PDC and without mutations in the E1alpha subunit or E1beta subunit . We report that the cDNA and gene of this patient for protein X has a homozygous 4 bp deletion, specifically in the putative mitochondrial targeting signal sequence which results in a premature stop codon . This is the first documented case of a molecular defect in pyruvate dehydrogenase protein X. Hum Genet, 1998 Mar, 102(3), 282 - 8 Isolation of the human BACH1 transcription regulator gene, which maps to chromosome 21q22.1; Blouin JL et al.; In order to contribute to the development of the transcriptional map of chromosome 21, we performed exon trapping using cosmid clones mapped in the region 21q22.1-22.2 and identified a number of potential exons . One of the trapped exons (Genbank No . AF026200) showed a strong homology with the mouse Bach1 gene (Genbank No . D86603), a transcription factor regulating gene expression . We then isolated the full-length coding region of the human BACH1 gene using expressed sequence tags, reverse transcription-polymerase chain reaction and rapid amplification of cDNA ends . The predicted BACH1 protein contains 736 amino acids and is 88% identical to its mouse homolog . It contains basic leucine zipper and BTB-zinc finger domains (which are directly involved in DNA binding for transcription regulation) . The BACH1 gene maps in a relatively gene-poor region on 21q22.1 in yeast artificial chromosome 814c1 of the collection of Chumakov et al . Northern blot analysis revealed that it is expressed as an mRNA species of approximately 5.8 kb in all 16 adult and 4 fetal tissues examined; an additional mRNA species of 2.8 kb was observed in adult testis . The contribution of the BACH1 gene to the pathophysiology of trisomy or monosomy 21 is unknown . In addition, no monogenic disorders associated with mutations in the BACH1 gene have yet been identified. Can J Gastroenterol, 1998 Jan-Feb, 12(1), 57 - 60 Antibody levels in Ethiopian children five years after vaccination with two different doses of hepatitis B vaccine: is there a need for booster vaccine? Tsega E, Horton J, Nordenfelt E, Hansson BJ, Tafesse B, Wolde-Hawariat G, Lindberg J. It was hypothesized that, following effective initial vaccination, a booster dose of hepatitis B vaccine will not be necessary in areas of hyperendemicity for hepatitis B virus (HBV) infection . A total of 314 Ethiopian children, ranging from two to 14 years old, were alternatively vaccinated with 10 and 20 micrograms hepatitis B vaccine doses, using the initial, one- and six-month schedule . Five years later, 210 of the vaccinees were retested for anti-HBV surface antibody titres . Both 10 and 20 micrograms doses of hepatitis B rDNA yeast vaccine were equally immunogenic and protective against HBV infection for at least five years despite marked reduction of mean antibody levels and geometric mean titres, with 11% of the vaccinees showing antibodies below the protective level . For firm further recommendations a longer follow-up period of vaccinees is suggested. FEBS Lett, 1998 Mar 20, 425(1), 7 - 13 Identification and characterization of a novel member of the dystrobrevin gene family; Puca AA et al.; A new member of the dystrobrevin gene family was identified using a bioinformatics approach . Sequence analysis indicates that this gene, named DTN-B, is highly homologous to the rabbit A0, the previously described dystrobrevin (DTN), Torpedo 87 kDa and to the C-terminus of dystrophin . The coiled-coil domain, shown to be the site of interaction between dystrobrevins and dystrophin, is highly conserved . Immunostaining studies indicate that DTN-B and DTN expression is absent in affected muscle fibers from DMD patients and carriers. Biochim Biophys Acta, 1998 Mar 9, 1396(2), 158 - 62 Structure and expression of the human ubiquitin fusion-degradation gene (UFD1L); Novelli G et al.; We report the genomic organization, RNA and protein expression patterns of the gene encoding for the human homolog of the yeast ubiquitin fusion-degradation protein-1 (UFD1L) . This enzyme is involved in a ubiquitin-dependent proteolytic pathway (UFD), firstly described in yeast . The human UFD1L gene is organized into 12 exons ranging in size from 33 to 161 bp . Sequence analysis of the 5'-flanking region of the gene revealed a high GC content, multiple CCAAT-binding motifs, CREB, CFT, and AP-2 sites . RNA transcripts were detected in all tissues and cell lines examined, including thymus, thymocytes, T- and B-cells, fibroblasts, chorionic villi, and amniocytes . In Western blot, the UFD1L antibody demonstrated the presence of multiple protein isoforms in all the tested tissues . Expression profile and promoter characteristics suggest UFD1L is a housekeeping gene with implications in the pathogenesis of DiGeorge/velo-cardio-facial syndrome, due to 22q11.2 deletions. Biochim Biophys Acta, 1998 Mar 9, 1396(2), 153 - 7 Cloning and characterization of the human RNA polymerase I subunit hRPA40; Dammann R et al.; The cloning of the human RNA polymerase I 40 kDa subunit, and the comparison of its amino acid sequence to other related RNA polymerase subunits are described . The amino acid sequence of hRPA40 has high homology to the mouse RNA polymerase I 40 kDa subunit (93%), to two Arabidopsis thaliana subunits (47%), the yeast RPC40 subunit (46%) and the human RNA polymerase II hRPB33 subunit (40%) . Southern blot analysis shows that this gene is single copy and Northern blot analysis indicates that the mRNA of 1.3 kb is expressed in different cell types. Mutat Res, 1998 Feb, 407(1), 67 - 84 Search for DNA repair pathways in Drosophila melanogaster; de Buendia PG; The knowledge about the existence of different pathways for the repairing of DNA lesions has made possible a better understanding of mutation processes . The double mutant method has been shown to be useful for grouping rad mutants in yeast . Through this method, three different groups of repair mechanisms were found: (a) RAD3 group corresponding to the excision repair of UV lesions, (b) RAD6 group corresponding to the translesion type of post-replication repair and (c) RAD52 group corresponding to the recombination type of post-replication repair . In this work, a search for a classification of Drosophila mus mutants in groups analogous to yeast RAD groups is done . Information obtained by double mutant studies was integrated with that obtained by biochemical, recombination, DNA damaging agent sensitivity and mutation studies . The following groups were found: (a) group of mei9 and mus201, analogous to RAD3, (b) group of mei41 and mus302 analogous to RAD52 and, (c) group of mus104 and mus101 analogous to RAD6 . In addition, there are mutants that belong to a group corresponding to pre-replication repair of MMS lesions such as mus103, mus306 and mus207 . As a peculiarity of Drosophila, it was found that interaction between pre- and post-replication repair mechanisms is indifferent and not synergistic as was found in yeast . A possible explanation could be a weaker control of post-replication repair mechanisms in Drosophila than in yeast . It is expected that this research could help for a better understanding of repair mechanisms in complex organisms. Neuron, 1998 Mar, 20(3), 565 - 73 Slob, a novel protein that interacts with the Slowpoke calcium-dependent potassium channel; Schopperle WM et al.; Slob, a novel protein that binds to the carboxy-terminal domain of the Drosophila Slowpoke (dSlo) calcium-dependent potassium channel, was identified with a yeast two-hybrid screen . Slob and dSlo coimmunoprecipitate from Drosophila heads and heterologous host cells, suggesting that they interact in vivo . Slob also coimmunoprecipitates with the Drosophila EAG potassium channel but not with Drosophila Shaker, mouse Slowpoke, or rat Kv1.3 . Confocal fluorescence microscopy demonstrates that Slob and dSlo redistribute in cotransfected cells and are colocalized in large intracellular structures . Direct application of Slob to the cytoplasmic face of detached membrane patches containing dSlo channels leads to an increase in channel activity . Slob may represent a new class of multi-functional channel-binding proteins. DNA Cell Biol, 1998 Mar, 17(3), 249 - 63 Expression and processing of G0/G1 switch gene 24 (G0S24/TIS11/TTP/NUP475) RNA in cultured human blood mononuclear cells; Heximer SP et al.; The human G0/G1 switch (G0S) gene, G0S24, and its rodent immediate-early homolog (TIS11, TTP, NUP475) are part of a mammalian gene family whose members encode CCCH zinc finger domains and domains similar to part of the large subunit of RNA polymerase II and to the Mei2 regulator of G1 arrest in fission yeast . We compared the RNA expression of G0S24 with that of other G0S genes in cultured blood mononuclear cells and examined the levels of various RNA processing intermediates . Freshly isolated cells contained high levels of several G0S RNAs, which declined by 24 h, suggesting transient spontaneous stimulation during cell purification (Heximer et al., 1996) . However, in cells preincubated for 24 h, G0S24 RNA levels remained much higher than those of other G0S genes (107+/-42 x 10(6) molecules/microg of RNA); stimulation with lectin (Con-A) further increased G0S24 RNA, much of which remained nuclear . Like those of FOS/G0S7, EGR1/G0S30 and of the gene encoding the regulator of G protein signalling 1 (RGS1), G0S24 RNA levels increased more in response to a protein kinase C activator than to a calcium ionophore, whereas the opposite held for FOSB/G0S3 and RGS2/G0S8 . With appropriate PCR primer pairs, we showed a G0S24 RNA processing intermediate, which crossed the exon-1/intron boundary, and nonpolyadenylated nuclear RNA extending into the 3' flank, where there is a second CpG island . The concentration of the latter intermediate (1.2+/-0.2 x 10(6) molecules/microg of RNA), which increased transiently on cell stimulation, did not account for all G0S24 nuclear RNA . The levels of G0S24 RNA and both intermediates were increased by the protein synthesis inhibitor cycloheximide, consistent with regulation by a labile repressor. DNA Cell Biol, 1998 Mar, 17(3), 221 - 30 Human leukotriene B4 omega-hydroxylase (CYP4F3) gene: molecular cloning and chromosomal localization; Kikuta Y et al.; Leukotriene B4 (LTB4) omega-hydroxylase catalyzes the conversion of LTB4 into a biologically less active product, 20-hydroxy-LTB4 . In a preceding paper (Kikuta et al., 1993), we showed human polymorphonuclear leukocyte (PMN) LTB4 omega-hydroxylase to be a novel form of cytochrome P450, designated CYP4F3, on the basis of its cDNA cloning and expression in yeast cells . Here, we have isolated the gene encoding CYP4F3 and determined its genomic organization and chromosomal localization . The CYP4F3 gene contained 13 exons and spanned approximately 22.2 kb . The cDNA of CYP4F3 contained 5050 nucleotides excluding the poly(A) tail . The translation initiation codon (ATG) was present in exon II . Primer extension and S1 mapping analyses indicated that the transcription initiation site is 49 nucleotides upstream from the 3' end of exon I, and no other initiation sites were detected . A TATA-box-like sequence (TACAT) and 120-b GC-rich sequence were observed just before transcription initiation site . Several putative regulating elements recognized by the GATA family, MZF-1, CACCC binding protein, and C/EBP, were identified in its 5' flanking region . Genomic DNA screening for CYP4F3 and Southern blot analysis suggested the existence of other CYP4F genes in addition to CYP4F3 and CYP4F2 in the human genome . Fluorescence in situ hybridization demonstrated that the CYP4F3 gene is located at 19p13.2. FEBS Lett, 1998 Mar 6, 424(1-2), 63 - 8 Cloning and characterization of MUPP1, a novel PDZ domain protein; Ullmer C et al.; Using the yeast two-hybrid system we isolated a cDNA clone encoding a novel protein interacting with the C-terminal domain of the 5-HT2C receptor . The protein, named MUPP1 (multi-PDZ-domain protein), contains thirteen PDZ domains and no obvious catalytic domain; it is related to hINADL and a putative C . elegans polypeptide referred to as C52A11.4 containing six or ten PDZ domains, respectively . Domains highly similar to those of MUPP1 are arrayed in the same order in all three proteins . The MUPP1 gene is localized on human chromosome 9p24-p22 . Transcripts encoding MUPP1 are abundant in the brain as well as in several peripheral organs. Nat Genet, 1998 Apr, 18(4), 354 - 9 Neurofibromatosis 2 tumour suppressor schwannomin interacts with betaII-spectrin; Scoles DR et al.; NF2 is the most commonly mutated gene in benign tumours of the human nervous system . The NF2 protein, called schwannomin or merlin, is absent in virtually all schwannomas, and many meningiomas and ependymomas . Using the yeast two-hybrid system, we identified betaII-spectrin (also known as fodrin) as a schwannomin-binding protein . Interaction occurred between the carboxy-terminal domain of schwannomin isoform 2 and the ankyrin-binding region of betaII-spectrin . Isoform 1 of schwannomin, in contrast, interacted weakly with betaII-spectrin, presumably because of its strong self-interaction . Thus, alternative splicing of NF2 may regulate betaII-spectrin binding . Schwannomin co-immunoprecipitated with betaII-spectrin at physiological concentrations . The two proteins interacted in vitro and co-localized in several target tissues and in STS26T cells . Three naturally occurring NF2 missense mutations showed reduced, but not absent, betaII-spectrin binding, suggesting an explanation for the milder phenotypes seen in patients with missense mutations . STS26T cells treated with NF2 antisense oligonucleotides showed alterations of the actin cytoskeleton . Schwannomin itself lacks the actin binding sites found in ezrin, radixin and moesin, suggesting that signalling to the actin cytoskeleton occurs via actin-binding sites on betaII-spectrin . Thus, schwannomin is a tumour suppressor directly involved in actin-cytoskeleton organization, which suggests that alterations in the cytoskeleton are an early event in the pathogenesis of some tumour types. Appl Microbiol Biotechnol, 1998 Feb, 49(2), 182 - 8 Beta-glycosidase (amygdalase and linamarase) from Endomyces fibuliger (LU677): formation and crude enzyme properties; Brimer L et al.; In our previous studies, the yeast Endomyces fibuliger LU677 was found to degrade amygdalin in bitter apricot seeds . The present investigation shows that E . fibuliger LU677 produces extracellular beta-glycosidase activity when grown in malt extract broth (MEB) . Growth was very good at 25 degrees C and 30 degrees C and slightly less at 35 degrees C . When grown in MEB of pH 5 and pH 6 with addition of 0, 10 or 100 ppm amygdalin, E . fibuliger produced only slightly more biomass at pH 5, and was only slightly inhibited in the presence of amygdalin . Approximately, 60% of the added amygdalin was degraded (fastest at 35 degrees C) during an incubation period of 5 days . Supernatants of cultures grown at 25 degrees C and pH 6 for 5 days were tested for the effects of pH and temperature on activity (using amygdalin, linamarin and prunasin as substrates) . Prunase activity had two pH optima (pH 4 and pH 6), amygdalase and linamarase only one each at pH 6 and pH 4-5 respectively . The linamarase activity evolved earlier than amygdalase (2 days and 4 days respectively) . The data thus indicate the presence of at least two different glycosidases having different pH optima and kinetics of excretion . In the presence of amygdalin, lower glycosidase activities were generally produced . However, the amygdalin was degraded from the start of the growth, strongly indicating an uptake of amygdalin by the cells . The temperature optimum for all activities was at 40 degrees C . Activities of amygdalase (assayed at pH 4) and linamarase (at pH 6) evolving during the growth of E . fibuliger were generally higher in cultures grown at 25 degrees C and 30 degrees C . TLC analysis of amygdalin degradation products show a two-stage sequential mechanism as follows: (1) amygdalin to prunasin and (2) prunasin to cyanohydrin. Curr Biol, 1998 Feb 26, 8(5), 279 - 82 Reconstitution of telomerase activity in normal human cells leads to elongation of telomeres and extended replicative life span; Vaziri H et al.; Normal somatic cells have a finite life span {1} and lose telomeric DNA, present at the ends of chromosomes, each time they divide as a function of age in vivo or in culture {2-4} . In contrast, many cancer cells and cell lines established from tumours maintain their telomere length by activation of an RNA-protein complex called telomerase, an enzyme originally discovered in Tetrahymena {5}, that synthesizes telomeric repeats {6-8} . These findings have led to the formation of the 'telomere hypothesis', which proposes that critical shortening of telomeric DNA due to the end-replication problem {9} is the signal for the initiation of cellular senescence {10,11} . In yeast, the EST2 gene product, the catalytic subunit of telomerase, is essential for telomere maintenance in vivo {12-14} . The recent cloning of the cDNA encoding the catalytic subunit of human telomerase (hTERT) {15,16} makes it possible to test the telomere hypothesis . In this study, we expressed hTERT in normal human diploid fibroblasts, which lack telomerase activity, to determine whether telomerase activity could be reconstituted leading to extension of replicative life span . Our results show that retroviral-mediated expression of hTERT resulted in functional telomerase activity in normal aging human cells . Moreover, reconstitution of telomerase activity in vivo led to an increase in the length of telomeric DNA and to extension of cellular life span . These findings provide direct evidence in support of the telomere hypothesis, indicating that telomere length is one factor that can determine the replicative life span of human cells. Curr Biol, 1998 Feb 26, 8(5), R161 - 4 DNA dynamics: different means to a common end? Lustig AJ. A ribosomal frameshift is required for the synthesis of an essential component of the yeast telomerase pathway; this and other findings on telomerases from many species raise interesting questions regarding the evolutionary relationship between telomerases and retrotransposons lacking long terminal repeats. J Biol Chem, 1998 Mar 13, 273(11), 6218 - 22 Identification of PLC210, a Caenorhabditis elegans phospholipase C, as a putative effector of Ras; Shibatohge M et al.; Mammalian Ras proteins regulate multiple effectors including Raf, Ral guanine nucleotide dissociation stimulator (RalGDS), and phosphoinositide 3-kinase . In the nematode Caenorhabditis elegans, LIN-45 Raf has been identified by genetic analyses as an effector of LET-60 Ras . To search for other effectors in C . elegans, we performed a yeast two-hybrid screening for LET-60-binding proteins . The screening identified two cDNA clones encoding a phosphoinositide-specific phospholipase C (PI-PLC) with a predicted molecular mass of 210 kDa, designated PLC210 . PLC210 possesses two additional functional domains unseen in any known PI-PLCs . One is the C-terminal Ras-associating domain bearing a structural homology with those of RalGDS and AF-6 . This domain, which could be narrowed down to 100 amino acid residues, associated in vitro with human Ha-Ras in a GTP-dependent manner and competed with yeast adenylyl cyclase for binding Ha-Ras . The binding was abolished by specific mutations within the effector region of Ha-Ras . The other functional domain is the N-terminal CDC25-like domain, which possesses a structural homology to guanine nucleotide exchange proteins for Ras . These results strongly suggest that PLC210 belongs to a novel class of PI-PLC, which is a putative effector of Ras. J Biol Chem, 1998 Mar 6, 273(10), 5970 - 8 The conserved carboxyl terminus and zinc finger-like domain of the co-chaperone Ydj1 assist Hsp70 in protein folding; Lu Z et al.; Ydj1 is a member of the Hsp40 (DnaJ-related) chaperone family that facilitates cellular protein folding by regulating Hsp70 ATPase activity and binding unfolded polypeptides . Ydj1 contains four conserved subdomains that appear to represent functional units . To define the action of these regions, protease-resistant Ydj1 fragments and Ydj1 mutants were analyzed for activities exhibited by the unmodified protein . The Ydj1 mutant proteins analyzed were unable to support growth of yeast at elevated temperatures and were found to have alterations in the J-domain (Ydj1 H34Q), zinc finger-like region (Ydj1 C159T), and conserved carboxyl terminus (Ydj1 G315D) . Fragment Ydj1 (1-90) contains the J-domain and a small portion of the G/F-rich region and could regulate Hsp70 ATPase activity but could not suppress the aggregation of the model protein rhodanese . Ydj1 H34Q could not regulate the ATPase activity of Hsp70 but could bind unfolded polypeptides . The J-domain functions independently and was sufficient to regulate Hsp70 ATPase activity . Fragment Ydj1 (179-384) could suppress rhodanese aggregation but was unable to regulate Hsp70 . Ydj1 (179-384) contains the conserved carboxyl terminus of DnaJ but is missing the J-domain, G/F-rich region, and a major portion of the zinc finger-like region . Ydj1 G315D exhibited severe defects in its ability to suppress rhodanese aggregation and form complexes with unfolded luciferase . The conserved carboxyl terminus of Ydj1 appeared to participate in the binding of unfolded polypeptides . Ydj1 C159T could form stable complexes with unfolded proteins and suppress protein aggregation but was inefficient at refolding denatured luciferase . The zinc finger-like region of Ydj1 appeared to function in conjunction with the conserved carboxyl terminus to fold proteins . However, Ydj1 does not require an intact zinc finger-like region to bind unfolded polypeptides . These data suggest that the combined functions of the J-domain, zinc finger-like region, and the conserved carboxyl terminus are required for Ydj1 to cooperate with Hsp70 and facilitate protein folding in the cell. J Biol Chem, 1998 Mar 6, 273(10), 5892 - 902 Association of activating transcription factor 2 (ATF2) with the ubiquitin-conjugating enzyme hUBC9 . Implication of the ubiquitin/proteasome pathway in regulation of ATF2 in T cells; Firestein R et al.; Activating transcription factor 2 (ATF2) is regulated by phosphorylation via the Jun N-terminal kinase, and its binding activity is markedly induced at late stages of T and B lymphocyte activation (Feuerstein, N., Firestein, R., Aiyer, N., Xiao, H., Murasko, D., and Cristofalo, V . (1996) J . Immunol . 156, 4582-4593) . To identify proteins that interact specifically with ATF2 in lymphocytes, the yeast two-hybrid interaction system was employed using ATF2 cDNA as a "bait." In two separate screenings, a clone was identified that revealed a novel sequence with homology to several members of the ubiquitin-conjugating enzyme family . An identical sequence was recently reported as the human homolog of the yeast UBC9, hUBC9 . Northern blot analysis revealed a 1.3-kilobase RNA transcript, which showed differential levels of expression in various human tissues and a moderate induction after a 48-h stimulation of peripheral blood T lymphocytes . An antibody that was generated against the bacterially expressed glutathione S-transferase-hUBC9 detected a approximately 19-kDa protein, which localizes predominantly in the nuclei of T cells . Further quantitative assays using the yeast two-hybrid system confirmed a high and specific level of interaction of hUBC9 with ATF2 and lack of interaction with lamin or control vectors . Two other cyclic AMP-responsive element-binding transcription factors, CREB and ATF1, also showed significant levels of interaction with hUBC9 . However, this interaction was severalfold lower as compared with ATF2 . Far Western blot analysis confirmed the specific binding of ATF2 and hUBC9 also in vitro . Evidence is presented that indicates a physiological significance for the interaction of hUBC9 with ATF2 . (a) We show that ATF2 is ubiquitinated in vivo and in vitro, and (b) ATF2 ubiquitination in vitro is facilitated by addition of purified hUBC9 . (c) ATF2 is shown to undergo a proteolytic process, which is rapidly regulated upon T cell activation concomitant with induction of ATF2 phosphorylation . (d) A proteasome inhibitor delays the down-regulation of ATF2 phophorylation after T cell activation . Taken collectively, these results implicate a role for hUBC9 and the ubiquitin/proteasome pathway in regulation of ATF2 in T cells. J Biol Chem, 1998 Mar 6, 273(10), 5468 - 77 Cloning of a phosphatidic acid-preferring phospholipase A1 from bovine testis; Higgs HN et al.; We report the molecular cloning and expression of a phosphatidic acid-preferring phospholipase A1 from bovine testis . The open reading frame encoded an 875-amino acid protein with a calculated molecular mass of 97,576 daltons and a pI of 5.61 . The sequence included a region similar to a lipase consensus sequence containing the putative active site serine and also included a potential, coiled-coil-forming region . Expression of the open reading frame in COS1 cells resulted in a 20-44-fold increase in phosphatidic acid phospholipase A1 activity over that of control cells . Mutation of the putative active site serine (amino acid 540) demonstrated that it was essential for this increase in enzyme activity . Northern blot analysis revealed at least five different messages with the highest overall message levels in mature testis, but detectable message in all tissues examined . Two possible alternately spliced regions in the open reading frame also were identified . Finally, a search of the data base identified six related proteins: a potential counterpart of the phospholipase A1 in Caenorhabditis elegans, two putative lipases in yeast, and three proteins separately encoded by the Drosophila retinal degeneration B gene and its mouse and human homologues. Cytogenet Cell Genet, 1997, 79(1-2), 125 - 31 Detailed map of a region commonly amplified at 11q13-->q14 in human breast carcinoma; Bekri S et al.; Amplification of loci present on band q13 of human chromosome 11 is a feature of a subset of estrogen receptor positive breast carcinomas prone to metastasis . As many as five distinct amplification units have been described on 11q13 . They include particularly a genomic area encompassing the GARP gene at 11q13.5-->q14.1 . We have reassessed our current knowledge of this region, located telomeric to CCND1 and EMS1, which is amplified in 7-10% of mammary tumors . The loose definition of the driving forces of these amplification events led us to map accurately the boundaries of the amplifiable region, and thus to contribute a physical and transcriptional map of a 3-Mb region of chromosome 11 . Four new genes were placed on the regional map, namely CBP2, CLNS1A, UVRAG, and PAK1 . We have narrowed the core of the 11q13-->q14 amplicon to a 350-kb area encompassing D11S533, mostly on its telomeric side . The map reported here represents an indispensable step toward sequencing the entire region, and thus toward uncovering gene(s) which play(s) a critical role in breast cancer progression. Cytogenet Cell Genet, 1997, 79(1-2), 109 - 13 Molecular cloning and chromosome mapping of rat phospholipase D genes, Pld1a, Pld1b and Pld2; Nakashima S et al.; We have previously obtained three partial rat phospholipase D (PLD) cDNA fragments by a reverse transcriptase-polymerase chain reaction (RT-PCR) method using degenerate primers based on two conserved amino acid sequences in PLDs of human and yeast . The entire coding regions of these genes were isolated and sequenced . The longest clone, Pld1a encodes a 1075 amino acid (aa) protein that was highly similar (89% identity) to human PLD1a, especially in four conserved regions present in other PLDs . The nucleotide sequence of the second clone was identical to that of Pld1a except that the clone lacked 114 nucleotides corresponding to 38 aa in the middle . A shorter alternatively spliced form of human PLD1 (PLD1b) lacking the corresponding 38 aa was also identified . Therefore, the second clone (Pld1b) was considered to correspond to the rat counterpart of human PLD1b . The third clone, Pld2 encoding 933 aa was smaller than that of Pld1 and its aa identity to rat Pld1 was 56% . However, it contains four conserved regions and aa sequences of these regions are homologous to those of rat Pld1 and human PLD1 . Its entire aa sequence was very similar (96% identity) to the recently cloned mouse PLD, Pld2 . Chromosome locations of the Pld1a, Pld1b and Pld2 genes were determined in the rat and mouse by fluorescent in situ hybridization . As expected, both Pld1a and Pld1b clones were hybridized to the same chromosome regions . The Pld1 and Pld2 genes were localized to rat chromosome 2q23.3-->q24 proximal end and the proximal region of mouse Chromosome 3B, and rat chromosome 10q23.3-->q24 proximal end and mouse Chromosome 11B3, respectively . They were mapped in regions where conserved linkage homology has been identified between the two species. Cytogenet Cell Genet, 1997, 79(1-2), 97 - 100 Mapping of 22 new ESTs around a tumor suppressor gene and a senescence gene at 6q16-->q21; Morelli C et al.; Twenty two expressed sequence tags (ESTs) have been mapped at the border of 6q16-->q21 and at the proximal end of 6q21, a candidate for two tumor suppressor genes and a senescence gene . Use of a translocation and deletion hybrid panel together with a 4-Mb YAC contig allowed us to precisely define the position of the ESTs . Thirteen ESTs were placed within the 4-Mb interval at the proximal portion of 6q21 using a restriction map of the YAC contig, seven ESTs span a 2-Mb region on the 6q16-->q21 border, and two are distal to the contig . Refinement of the localization of these ESTs will provide substantial assistance in identifying new genes within the region 6q16-->q21. Cytogenet Cell Genet, 1997, 79(1-2), 88 - 91 Mapping FRA11A, a folate-sensitive fragile site in human chromosome band 11q13.3; Perucca-Lostanlen D et al.; FRA11A, a rare folate-sensitive fragile site assigned to 11q13.3, lies in an area of genomic instability associated with several diseases and amplification events . To map FRA11A, we used fluorescence in situ hybridization with yeast artificial chromosome and cosmid probes on metaphase chromosomes of patients expressing the fragile site . FRA11A was found situated centromeric to ACTN3 and telomeric to D11S913, these markers being within an interval of approximately 1 Mb in the 11q13.3 region. Cytogenet Cell Genet, 1997, 79(1-2), 64 - 70 Construction of two near-kilobase resolution restriction maps of the 5' regulatory region of the human apolipoprotein B gene by quantitative DNA fiber mapping (QDFM); Duell T et al.; Quantitative DNA fiber mapping (QDFM) is a high-resolution technique for physical mapping of DNA . The method is based on hybridization of fluorescently labeled DNA probes to individual DNA molecules stretched on a chemically modified glass surface . We now demonstrate and validate a rapid QDFM-based approach for the mapping of multiple restriction sites and precise localization of restriction fragments in large genomic clones . Restriction fragments of a 70-kb P1 clone (P1-70) containing the 5' region of the human apolipo-protein B gene (APOB) were subcloned and mapped along straightened P1-70 DNA molecules . Multicolor fluorescence in situ hybridization (FISH) and digital image analysis allowed us to rapidly position 29 restriction fragments, ranging in size from 0.5 kb to 8 kb, and to map 43 restriction sites . The restriction map obtained by QDFM was in excellent agreement with information obtained by RecA-assisted restriction endonuclease (RARE) cleavage, long-range PCR, and DNA sequence analyses of the P1-70 clone . These data demonstrate that QDFM is a rapid, reliable method for detailed restriction site-mapping of large DNA clones. Planta, 1998 Mar, 204(3), 345 - 51 Arabidopsis SKP1, a homologue of a cell cycle regulator gene, is predominantly expressed in meristematic cells; Porat R et al.; The yeast SKP1 gene and its human homolog p19skp1 encode a kinetochore protein required for cell cycle progression at both the DNA synthesis and mitosis phases of the cell cycle . In orchids we identified a cDNA (O 108) that is expressed in early stages of ovule development and is homologous to the yeast SKP1 . Based on the orchid O 108 cDNA clone, we identified and characterized an Arabidopsis thaliana (L.) Heynh . cDNA designated ATskp1 that also has high sequence similarity to yeast SKP1 . The Arabidopsis ATskp1 is a single-copy gene that mapped to chromosome 1 . The expression of the ATskp1 gene was highly correlated with meristem activity in that its mRNA accumulated in all of the plant meristems including the vegetative shoot meristem, inflorescence and floral meristems, root meristem, and in the leaf and floral organ primordia . In addition, ATskp1 was also highly expressed in the dividing cells of the developing embryo, and in other cells that become multinucleate or undergo endoreplication events such as the endosperm free nuclei, the tapetum and the endothelium . Based on its spatial pattern of expression, ATskp1 is a marker for cells undergoing division and may be required for meristem activity. Am J Physiol, 1998 Mar, 274(3 Pt 1), G578 - 83 The primary and final effector mechanisms required for kinin-induced epithelial chloride secretion; Cuthbert AW et al.; The short-circuit current technique was used to examine the effects of N2-L-lysylbradykinin (LBK) on chloride secretion in the mucosae of the mouse intestine . It was found to be a potent chloride secretagogue in the mucosa lining the colon, jejunum, and cecum, as it is in most mammals, with 2 nM being sufficient to cause half-maximal secretion . The extent of the responses was in the order cecum > colon > jejunum . In cystic fibrosis (CF) null mice, with no CF transmembrane conductance regulator (CFTR) chloride channels, LBK caused no chloride secretion, but transporting activities for other ions were revealed . Introduction of the human CF gene into the genome of CF null mice at the zygote stage restored the chloride secretory activity of LBK, with only minor differences in potency . In mice in which the kinin B2 receptor gene had been disrupted, LBK had no effect, whereas the responses to forskolin were unchanged . Thus the acute effects of kinins on chloride secretion depend uniquely on kinin B2 receptors and CFTR chloride channels, which form the primary and final effector mechanisms of the secretory process. Cell, 1998 Mar 20, 92(6), 747 - 58 BiP maintains the permeability barrier of the ER membrane by sealing the lumenal end of the translocon pore before and early in translocation; Hamman BD et al.; Secretory proteins are cotranslationally translocated across the mammalian ER membrane through an aqueous pore in the translocon while the permeability barrier is maintained by a tight ribosome-membrane junction . The lumenal end of the pore is also blocked early in translocation . Extraction of soluble lumenal proteins from microsomes and reconstitution with purified proteins demonstrate, by fluorescence collisional quenching, that BiP seals the lumenal end of this pore . BiP also seals translocons that are assembled but are not engaged in translocation . These ribosome-free translocons have smaller pores (9-15 A diameter versus 40-60 A in functioning translocons) and are generated when ribosomes dissociate from functioning translocons with large pores . BiP therefore maintains the permeability barrier by sealing both nontranslocating and newly targeted translocons. Endocrinology, 1998 Apr, 139(4), 1588 - 93 Association of gonadotropin receptor precursors with the protein folding chaperone calnexin; Rozell TG et al.; The lutropin/choriogonadotropin receptor (LHR) and follitropin receptor (FSHR) are members of the superfamily of G protein-coupled receptors . The carboxyl half of each receptor is composed of the classical seven membrane spanning regions connected by intracellular and extracellular loops . In addition, each receptor contains a large extracellular domain . Despite the complexity of the structure of G protein-coupled receptors, little is known about how these receptors assume their correct conformations during biosynthesis . Although the role of chaperone proteins in the folding of other proteins has been well documented, their role in the folding of G protein-coupled receptors has been an enigma . To better understand the folding of the LH and FSH receptors, we examined their association with the general chaperone proteins calnexin, binding protein (BiP), and the 94-kDa glucose-regulated protein (GRP94) . Clonal 293 cell lines expressing comparably high levels of each receptor were solubilized, and the extracts were incubated with the appropriate antibody bound to Protein A-sepharose beads . Experiments were performed using two approaches: 1) coimmunoprecipitation of receptor/chaperone complexes with one of the antireceptor antibodies, then SDS-PAGE and Western blotting using either anticalnexin or anti-KDEL (which recognizes BiP and GRP94) antibodies; or 2) coimmunoprecipitation of receptor/chaperone complexes with anticalnexin or anti-KDEL, then Western blotting with one of the antireceptor antibodies . Using these protocols, we found that the immature forms of both the rLHR and rFSHR are associated with calnexin, but little or no association was observed for either receptor with BiP or GRP94 . These experiments show that the precursor forms of the wild-type LHR and FSHR can associate with calnexin, raising the possibility that this chaperone protein may facilitate in the folding of the gonadotropin receptors. Oncogene, 1998 Mar 5, 16(9), 1149 - 59 Mmip1: a novel leucine zipper protein that reverses the suppressive effects of Mad family members on c-myc; Gupta K et al.; C-myc, a member of the basic helix-loop-helix-leucine zipper (bHLH-ZIP) protein family activates target genes in heterodimeric association with another bHLH-ZIP protein, Max . Max readily homodimerizes, competes with C-myc-Max heterodimers, and represses transcription . Four additional bHLH-ZIP proteins, Mad1, Mxi1, Mad3 and Mad4, heterodimerize with Max and also repress transcription of c-myc-responsive genes . We employed a yeast two-hybid approach to identify proteins which interact with Mxi . We identified a novel ZIP-containing protein, Mmip1 (Mad member-interacting protein 1) that strongly dimerizes with all four Mad members, but not with c-myc, Max, or with unrelated HLH proteins . The Mmip1-Mxi association is mediated by the ZIP domain of each polypeptide and is as strong or stronger than the associations between c-myc and Max or Max and Mxi1 . In vitro, Mmip1 can inhibit DNA binding by Max-Mad heterodimers and, in vivo, can reverse the suppressive effects of Mad proteins on c-myc functions . Mmipl is found in a variety of cells types, is induced by serum stimulation, and can be co-immunoprecipitated from fibroblasts in association with Mxi1 . By interfering with the dimerization between Max and Mad family member proteins, Mmip1 can indirectly up-regulate the transcriptional activity of c-myc and suppress the antiproliferative actions of Mad proteins. Oncogene, 1998 Mar 5, 16(9), 1125 - 30 Increased transversions in a novel mutator colon cancer cell line; Eshleman JR et al.; We describe a novel mutator phenotype in the Vaco411 colon cancer cell line which increases the spontaneous mutation rate 10-100-fold over background . This mutator results primarily in transversion base substitutions which are found infrequently in repair competent cells . Of the four possible types of transversions, only three were principally recovered . Spontaneous mutations recovered also included transitions and large deletions, but very few frameshifts were recovered . When compared to known mismatch repair defective colon cancer mutators, the distribution of mutations in Vaco411 is significantly different . Consistent with this difference, Vaco411 extracts are proficient in assays of mismatch repair . The Vaco411 mutator appears to be novel, and is not an obvious human homologue of any of the previously characterized bacterial or yeast transversion phenotypes . Several hypotheses by which this mutator may produce transversions are presented. Mol Cell Biol, 1998 Apr, 18(4), 2023 - 8 The carboxy-terminal domain of Hsc70 provides binding sites for a distinct set of chaperone cofactors; Demand J et al.; The modulation of the chaperone activity of the heat shock cognate Hsc70 protein in mammalian cells involves cooperation with chaperone cofactors, such as Hsp40; BAG-1; the Hsc70-interacting protein, Hip; and the Hsc70-Hsp90-organizing protein, Hop . By employing the yeast two-hybrid system and in vitro interaction assays, we have provided insight into the structural basis that underlies Hsc70's cooperation with different cofactors . The carboxy-terminal domain of Hsc70, previously shown to form a lid over the peptide binding pocket of the chaperone protein, mediates the interaction of Hsc70 with Hsp40 and Hop . Remarkably, the two cofactors bind to the carboxy terminus of Hsc70 in a noncompetitive manner, revealing the existence of distinct binding sites for Hsp40 and Hop within this domain . In contrast, Hip interacts exclusively with the amino-terminal ATPase domain of Hsc70 . Hence, Hsc70 possesses separate nonoverlapping binding sites for Hsp40, Hip, and Hop . This appears to enable the chaperone protein to cooperate simultaneously with multiple cofactors . On the other hand, BAG-1 and Hip have recently been shown to compete in binding to the ATPase domain . Our data thus establish the existence of a network of cooperating and competing cofactors regulating the chaperone activity of Hsc70 in the mammalian cell. Mol Cell Biol, 1998 Apr, 18(4), 1927 - 34 Estrogen response elements function as allosteric modulators of estrogen receptor conformation; Wood JR et al.; The estrogen receptor (ER) is a ligand-dependent transcription factor that regulates the expression of estrogen-responsive genes . ER-mediated transcriptional changes are brought about by interaction of the ER with the estrogen response element (ERE) . In this study, we examined the interaction of the Xenopus laevis ER DNA binding domain (DBD) and the intact ER with the X . laevis vitellogenin A2 ERE and the human pS2 ERE . Using gel mobility shift, DNase I footprinting, and methylation interference assays, we demonstrated that the DBD bound only as a dimer to the A2 ERE . However, the DBD bound as a monomer to the consensus pS2 ERE half site at lower DBD concentrations and then as a homodimer to the consensus and imperfect pS2 ERE half site at higher DBD concentrations . Antibody supershift experiments carried out with partially purified, yeast-expressed full-length ER demonstrated that three ER-specific antibodies interacted differentially with A2 and pS2 ERE-bound ER, indicating that receptor epitopes were differentially exposed . Furthermore, partial digestion of the A2 and pS2 ERE-bound ER with chymotrypsin or trypsin produced distinct protease cleavage patterns . Taken together, these data provide evidence that differential interaction of the DBD with the A2 and pS2 EREs brings about global changes in ER conformation . The conformational changes in ER induced by individual ERE sequences could lead to association of the receptor with different transcription factors and assist in the differential modulation of estrogen-responsive genes in target cells. Mol Cell Biol, 1998 Apr, 18(4), 1903 - 10 The absence of enhancer competition between Igf2 and H19 following transfer into differentiated cells; Webber AL et al.; H19 and Igf2 are reciprocally imprinted genes that lie 90 kb apart on mouse chromosome 7 . The two genes are coexpressed during development, with the H19 gene expressed exclusively from the maternal chromosome and Igf2 from the paternal chromosome . It has been proposed that their reciprocal imprinting is governed by a competition between the genes for a common set of enhancers . The competition on the paternal chromosome is influenced by extensive allele-specific methylation of the H19 gene and its 5' flank, which acts to inhibit H19 transcription and thus indirectly leads to the activation of the Igf2 gene . In contrast, no allele-specific methylation has been detected on the maternal chromosome, and the basis for the preference for H19 transcription on that chromosome is unresolved . In this investigation, the mechanism controlling the silencing of the Igf2 gene on the maternal chromosome was explored by studying the transcriptional activity of a yeast artificial chromosome (YAC) containing Igf2 and H19 following transfer into differentiated tissue culture cells . Contrary to expectations, both H19 and Igf2 were expressed from a single integrated copy of the YAC . Furthermore, Igf2 expression appeared to be independent of the H19 locus, based on deletions of the H19 gene promoter and its enhancers . These results suggest that an active process is responsible for the transcriptional bias toward H19 on the maternal chromosome and that the hypomethylated state of this chromosome cannot be viewed as a "default" state . Moreover, the active process is not reproduced in a differentiated cell and may require passage through the female germ line. Z Naturforsch {C}, 1998 Jan-Feb, 53(1-2), 101 - 6 Hemolytic activity of aminoethyl-dodecanoates; Kleszczynska H et al.; The effect of new lysosomotropic compounds on red blood cell hemolysis and erythrocyte membrane fluidity has been investigated . In earlier studies it was shown that the compounds inhibit the growth of yeast and plasma membrane H(+)-ATPase activity . The study was performed with eight aminoethyl esters of lauric acid variously substituted at nitrogen atom . Esters of dodecanoic acid were chosen for study because at that chain length dimethylaminoethyl esters showed maximum activity . The hemolytic activity of the substances studied exhibits diversified activity in their interaction with the erythrocyte membrane: they differ in hemolytic activity and affect membrane fluidity differently . Erythrocyte membrane fluidity changes under the effect of those compounds which possess highest hemolytic activity . The hemolytic activity of the aminoesters investigated was found to follow a sequence that depended on basicity (i.e . ability of the protonated form formation) of the compound and its polar head group size . The most active are the compounds that possess not more than four carbon atoms substituted at nitrogen and highest pKa value. Mol Pharmacol, 1998 Mar, 53(3), 408 - 14 Topology inversion of CYP2D6 in the endoplasmic reticulum is not required for plasma membrane transport; Loeper J et al.; The presence of CYP2D6 at the surface of isolated rat and human hepatocytes and its recognition by autoantibodies were reported recently . We wondered whether the unexpected outside orientation at the plasma membrane could be related to topological inversion (luminal-oriented form) of cytochrome P450 in the endoplasmic reticulum . To examine the potential role of cDNA polymorphism, a CYP2D6 variant carrying three positive charges at the amino terminus (2D6ext) was constructed and expressed in yeast . Immunoblotting, flow cytometry, and electron microscopy showed that wild-type CYP2D6 expressed in yeast was present on the outer face of the cell plasma membrane in addition to the regular microsomal location . This location reproduces the hepatocyte situation . 2D6ext expressed in yeast and COS7 cells seemed to be partially N-glycosylated and was located at the plasma membrane surface . Nevertheless, the glycosylated form was not enriched in the plasma membranes compared with microsomes . The relationship between CYP2D6 and 2D6ext topologies and catalytic competence was tested . Cumene hydroperoxide-dependent dextromethorphan demethylation was performed on microsomal vesicles after combined proteolysis and immunoinhibition experiments . CYP2D6 activity was completely abolished, whereas the glycosylated and luminal-oriented fraction of 2D6ext remained active . This suggests that a luminal-oriented glycosylated form is not involved in cytochrome P450 transport to the plasma membrane . Yeast thus reproduces the unusual CYP2D6 plasma membrane location and orientation, which do not require sequence alteration, glycosylation, or even an inverted endoluminal orientation. J Virol, 1998 Apr, 72(4), 3037 - 44 Human immunodeficiency virus type 1 (HIV-1) viral protein R (Vpr) interacts with Lys-tRNA synthetase: implications for priming of HIV-1 reverse transcription; Stark LA et al.; The vpr gene of human immunodeficiency virus type 1 (HIV-1) encodes a 96-amino-acid 14-kDa protein (viral protein R {Vpr}), which is produced late in the viral life cycle and is incorporated into the virion . Although Vpr is not required for viral replication in transformed cell lines and primary T lymphocytes, it is essential for productive infection of macrophages and monocytes and appears to be important for pathogenesis in vivo . To establish the role of Vpr in HIV-1 replication and pathogenesis, we have isolated cellular proteins with which Vpr interacts . By using the yeast two-hybrid system, Lys-tRNA synthetase (LysRS) was identified as a Vpr-interacting protein . The interaction between Vpr and LysRS was characterized both in vitro and in vivo, and the domains of Vpr required for the interaction were defined . In the presence of Vpr, LysRS-mediated amino-acylation of tRNA(Lys) is inhibited . Since tRNA(Lys) is the primer for reverse transcription of the HIV-1 genome, this suggests that the interaction between Vpr and LysRS may influence the initiation of HIV-1 reverse transcription. Ciba Found Symp, 1997, 211, 53 - 67; discussion 67-70 Drosophila telomere elongation; Biessmann H et al.; Drosophila melanogaster has an unusual telomere elongation mechanism . Instead of short repeats that are synthesized by telomerase, long retrotransposons, HeT-A and TART, transpose to the ends of chromosomes . This mechanism generates tandem arrays of these elements at the chromosome ends, in which all elements are oriented with their oligo(A) tails towards the centromere . Structural features of HeT-A and TART elements may provide clues as to their transposition mechanism . Drosophila telomere length polymorphism is mainly due to terminal retrotransposon arrays that differ between chromosome tips and that change with time . In addition, stable terminal chromosome deletions can be generated that do not contain terminal HeT-A and TART arrays, suggesting that, unlike the equivalent terminal repeats in yeast and humans, the presence and length of terminal arrays in Drosophila may not be critical for cell cycle progression. Ciba Found Symp, 1997, 211, 20 - 8; discussion 28-34 Telomerase and the chromosome end replication problem; Cech TR et al.; Telomerase, the enzyme that extends chromosomal DNA ends in most eukaryotes, contains essential RNA and protein subunits . We have been studying telomere replication in hypotrichous ciliates such as Euplotes aediculatus, which have numerous short macronuclear DNA molecules and therefore are highly enriched in telomeres and in telomerase . Cloning and sequencing genes for the RNA subunits from several ciliates revealed that telomerase RNAs with insignificant nucleotide sequence homology nevertheless form a common secondary structure . Affinity chromatography based on the sequence of the RNA subunit was used to purify the Euplotes telomerase as an active ribonucleoprotein enzyme . Two protein subunits, 123 kDa and 43 kDa, were identified . The finding of a yeast homologue to the 123 kDa subunit suggests that telomerase protein components may be much more highly conserved in evolution than the RNA subunits . The purified Euplotes telomerase has no activity with blunt-ended DNA primers, but instead requires a four to six nucleotide single-stranded 3' tail . This result supports a model for telomere replication in which other activities such as helicases or nucleases activate replicated DNA for extension by telomerase, a model that may be applicable to telomere replication in diverse eukaryotes. APMIS, 1998 Jan, 106(1), 245 - 51 Detection of oestrogenic chemicals by assaying the expression level of oestrogen regulated genes; Jorgensen M et al.; The oestrogen receptor belongs to the superfamily of nuclear receptors . Classically, nuclear receptors are thought to reside either in the nucleus or in the cytoplasm where they interact with their ligand which induces a conformational change that exposes the DNA binding domain . This is followed by dimerisation and binding of their corresponding response elements . By interacting with the transcriptional apparatus they then either activate or repress the transcription of target genes . However, this is a highly simplified view, since the activated oestrogen receptor interacts with other signal transduction pathways and its intrinsic transcriptional activity is highly influenced by phosphorylation and by its interaction with other proteins . This is clearly observed when the oestrogenicity of antioestrogens is tested since some compounds activate the receptor in yeast, but not in mammalian cells . However, when specific kinases are activated antioestrogens can also function as oestrogens in mammalian cells . Moreover, components of the MAP kinase and perhaps the cAMP and other pathways are activated before the receptor even enters the nucleus . Thus, when analysing the effects of oestrogenic compounds, it is important to assay both their potency as activators of transcription as the effects caused by interactions with other signal transduction pathways . This may be possible by combining assay methods, such as direct in vitro measurement of interaction between a potential oestrogenic chemical and the receptor or the yeast E-screen, with methods that are based on mammalian cells or whole animals . An alternative is to assay gene expression directly by methods such as differential display, where the expression of both genes known to be regulated directly by the receptor and genes regulated by other pathways can be monitored . Thereby it may be possible to assign different responses to the activation of distinct pathways. Matrix Biol, 1998 Feb, 16(7), 357 - 68 Prolyl 4-hydroxylases and their protein disulfide isomerase subunit; Kivirikko KI et al.; Prolyl 4-hydroxylases (EC 1.14,11.2) catalyze the formation of 4-hydroxyproline in collagens and other proteins with collagen-like sequences . The vertebrate type I and type II enzymes are {alpha (I)}2 beta 2 and {alpha (II)}2 beta 2 tetramers, respectively, whereas the enzyme from the nematode Caenorhabditis elegans is an alpha beta dimer . The type I enzyme is the major form in most but not all vertebrate tissues . The catalytic properties of the various enzyme forms are highly similar, but there are distinct, although small, differences in K(m) values for various peptide substrates between the enzyme forms and major differences in Ki values for the competitive inhibitor, poly(L-proline) . Prolyl 4-hydroxylase requires Fe2+, 2-oxoglutarate, O2 and ascorbate . Kinetic studies and theoretical considerations have led to elucidation of the reaction mechanism, and recent extensive site-directed mutagenesis studies have identified five critical residues at the cosubstrate binding sites . A number of compounds have been characterized that inhibit it competitively with respect to some of the cosubstrates, and three groups of suicide inactivators have also been identified . The beta subunit in all forms of prolyl 4-hydroxylase is identical to protein disulfide isomerase (PDI), a multifunctional polypeptide that also serves as a subunit in the microsomal triglyceride transfer protein, as a chaperone-like polypeptide that probably assists folding of a number of newly synthesized proteins, and in several other functions . The main role of the PDI polypeptide as a protein subunit is probably related to its chaperone function . Recent expression studies of recombinant human prolyl 4-hydroxylase subunits in a yeast have indicated that the formation of a stable enzyme tetramer in vivo requires coexpression of collagen polypeptide chains. Gene, 1998 Feb 27, 208(2), 307 - 14 Identification of N-terminal minimal transactivation domain of CBP, p300 and caenorhabditis elegans homologues; Yoshida E et al.; CBP/p300 is a multidomain transcriptional cofactor that acts in junction with other factors to regulate transcription . To elucidate the domain function of CBP, we fused its dissected fragments to Ga14 DNA-binding domain and transfected the deletion mutants into several cell lines . First, we found that the minimal transactivation domain (MTD) at the N-terminal portion maps to between 344 and 451 aa, and shows activity in a cell-type dependent manner . Second, we cloned C . elegans homologues corresponding to the MTD by RT-PCR and identified the three related products, two of which exhibited weak transcriptional activity . Finally, by means of the yeast two hybrid screening using MTD as a bait, we cloned hypoxia-inducible factor (HIF) 1 alpha and Stat2 cDNAs . These results suggested a functional role of MTD located at the N-terminal region of CBP/p300 in connecting to transcriptional factors. Gene, 1998 Feb 27, 208(2), 253 - 8 The number of kringle IV repeats 3-10 is invariable in the human apo(a) gene; Haibach C et al.; The human apolipoprotein(a) (apo(a)) gene is a member of a family of related genes including plasminogen, apo(a)rg-B and apo(a)rg-C, which are clustered on chromosome 6q 2,7 . Apo(a) contains ten different types of plasminogen-like kringle IV repeats (K-IV 1-10) one of which (K-IV 2) varies in number resulting in a remarkable size polymorphism of the protein . Sequence analysis of human apo(a) alleles and indirect evidence have suggested that K-IV 1 and K-IV 3-10 are each present once in individual alleles and that the 3' apo(a) region encompassing kringles IV 3-10, kringle V and the protease domain is invariable . To directly test this, we have constructed a restriction map of the apo(a) gene region from genomic DNA and from a yeast artificial chromosome (YAC) (K-IV 13) which contains the entire apo(a) gene . The presence of a 63 kb ClaI fragment encompassing kringles IV 3-10, kringle V and the protease domain and a 46 kb SwaI fragment, spanning kringles IV 5-10, kringle V and the protease domain was demonstrated by PFGE/Southern blotting in 30 unrelated subjects, who represented a range of apo(a) size alleles containing from 11 to 49 kringles . Our analysis demonstrates that the number of kringles IV 3-10 is invariable in the human apo(a) gene, suggesting that the 3'domain of Apo(a) is functionally important. Gene, 1998 Feb 27, 208(2), 191 - 9 Isolation and characterisation of smallminded, a Drosophila gene encoding a new member of the Cdc48p/VCP subfamily of AAA proteins; Long AR et al.; Smallminded (smid) encodes a new member of the cdc48p/VCP subfamily of AAA proteins in Drosophila . The gene was isolated by plasmid rescue from a GAL4 enhancer trap line which shows reporter gene expression in neuroblasts, imaginal disks and a subset of sensory neurons . Larvae homozygous for the insert arrest development as second instar larvae and die without pupating . The most obvious defect in these larvae is a significantly reduced CNS, hence the naming of the gene as smallminded . The deduced amino acid sequence of smid contains a tandem duplication of the AAA nucleotide binding domain characteristic of the cdc48p/VCP subfamily . Overall, smid shares 33% identical residues with its closest r