Yeasts

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 relative, yeast L0919-chrXII and 26-29% with other members of the cdc48p/VCP subfamily . The most highly conserved regions of the predicted protein structure are found in and around the nucleotide binding domains . The gene is expressed at all developmental stages.

Gene, 1998 Feb 27, 208(2), 147 - 56
22-Mb integrated physical and genetic map based on YAC/STS content spanning the interval DXS1125-DXS95 in human Xq12-q21.31; Mumm S et al.; A YAC/STS map has been assembled spanning 22 Mb across Xq12-q21.31, between markers DXS1125 and DXS95 . In addition to the landmark loci for the X-inactivation center XIST and the ATRX, ATP7A, phosphoglycerate kinase, POU3F4, and choroideremia genes, the candidate disease gene regions for torsion dystonia 3 and two X-linked mental retardation syndromes are included . Also, the human voltage-dependent anion channel gene (HVDAC1) has been placed near DXS986 . The current map incorporates 211 YACs from five different libraries, formatted with 185 STSs that comprise 26 genetic linkage markers, 60 newly-developed YAC-end STSs, and eight ESTs . The multiple clone coverage and average resolution of one STS per 120 kb provide resources for disease gene searches and are facilitating complete sequencing of the region.

Biol Trace Elem Res, 1997 Winter, 59(1-3), 195 - 206
Effects of dietary selenium and vitamin E concentrations on phospholipid hydroperoxide glutathione peroxidase expression in reproductive tissues of pubertal maturing male rats; Lei XG et al.; Phospholipid hydroperoxide glutathione peroxidase (PHGPX) is the second intracellular selenium (Se)-dependent glutathione peroxidase (GSH-Px) identified in mammals . Our objectives were to determine the effect of dietary vitamin E and Se levels on PHGPX activity expression in testis, epididymis, and seminal vesicles of pubertal maturing rats, and the relationship of PHGPX expression with testicular development and sperm quality . Forty Sprague-Dawley male weanling rats (21-d old), were initially fed for 3 wk a torula yeast basal diet (containing 0.05 mg Se/kg) supplemented with marginal levels of Se (0.1 mg/kg as Na2SeO3) and vitamin E (25 IU/kg as all-rac-alpha-tocopheryl acetate) . Then, rats were fed the basal diets supplemented with 0 or 0.2 mg Se/kg and 0 or 100 IU vitamin E/kg diet during the 3-wk period of pubertal maturing . Compared with the Se-supplemented rats, those fed the Se-deficient diets retained 31, 88, 67, and 50% of Se-dependent GSH-Px activities in liver, testis, epididymis, and seminal vesicles, respectively . Testes and seminal vesicles had substantially higher (5- to 20-fold) PHGPX activity than liver . Dietary Se deficiency did not affect PHGPX activities in the reproductive tissues, but reduced PHGPX activity in liver by 28% (P < 0.0001) . Dietary vitamin E supplementation did not affect PHGPX activity in liver, whereas it raised PHGPX activity in seminal vesicles by 43% (P < 0.005) . Neither dietary vitamin E nor Se levels affected body weight gains, reproductive organ weights, or sperm counts and morphology . In conclusion, expression of PHGPX activity in testis and seminal vesicles was high and regulated by dietary Se and vitamin E differently from that in liver.

Eur J Biochem, 1998 Mar 1, 252(2), 290 - 8
Identification of a specific effector of the small GTP-binding protein Rap2; Janoueix-Lerosey I et al.; Rap2 is a small GTP-binding protein that belongs to the Ras superfamily and whose function is still unknown . To elucidate Rap2 function, we searched for potential effectors by screening a mouse brain cDNA library in a yeast two-hybrid system using as a bait a Rap2A protein bearing a mutation of Gly to Val at position 12 . This strategy lead to the identification of a protein that interacts specifically with Rap2A complexed with GTP, and requires an intact effector domain of Rap2A for interaction; we designated this protein Rap2-interacting protein 8 (RPIP8) . Biochemical data obtained from in vitro studies with purified proteins confirmed the genetic results . Mouse RPIP8 consists of 446 amino acids, bears a coiled-coil domain between residues 265 and 313, and is expressed principally in brain . Its human counterpart, of 400 amino acids, exhibits 93.7% identity in their common region . A search for similar sequences in expressed-sequence-tags databanks revealed the presence in human and rodents of mRNAs encoding the 400-residue and 446-residue forms of RPIP8 . Furthermore a doublet of 45-50 kDa, corresponding to the 400-residue and 446-residue forms of the protein, was detected by western blotting of mouse brain extracts and lysates from pheochromocytoma PC12 cells and the pancreatic beta-cell lines HIT-T15 and RIN-m5F . Using transient transfections of HIT-T15 cells it was possible to demonstrate that {Val12}Rap2 and wild-type Rap2 could be immunoprecipitated with RPIP8 . These data therefore argue for RPIP8 being a specific effector of the Rap2 protein in cells exhibiting neuronal properties.

Eur J Biochem, 1998 Mar 1, 252(2), 237 - 44
Purification, properties and in situ localization of the amphibolic enzymes D-ribulose 5-phosphate 3-epimerase and transketolase from spinach chloroplasts; Teige M et al.; The amphibolic enzymes D-ribulose 5-phosphate 3-epimerase and transketolase have been purified from stroma extracts of spinach chloroplasts using ammonium sulfate fractionation and FPLC . For the native enzymes, a molecular mass of 180 kDa for epimerase and 160 kDa for transketolase was found and the molecular masses of the subunits was determined to be 23 kDa for epimerase and 74 kDa for transketolase . Protein sequencing of the purified chloroplast enzymes revealed the NH2-terminal amino acid sequences of mature epimerase (NH2-TSRVDKFSKSDIIVSP) and transketolase (NH2-AAVEALESTDTDQLVEG) . The enzymic properties of both enzymes such as Km values or pH optima, were found to be very similar to those for epimerases and transketolases from other sources, including yeast and animal cells . In contrast to the light-activated enzymes of the Calvin cycle, the activity of these amphibolic enzymes was not redox-dependent . Immunogold electron microscopy on spinach leaf thin sections revealed that about 90% of the total epimerase and transketolase, and 96% of the total chloroplast H+-ATP synthase portion CF1 are associated with thylakoid membranes in situ . Ribulose-1,5-bisphosphate carboxylase/oxygenase, in contrast, was evenly distributed throughout chloroplasts . These and other results indicate that minor chloroplast enzymes are arranged in a thin layer on thylakoid membrane surfaces in vivo.

Plant Cell Physiol, 1997 Dec, 38(12), 1389 - 96
cDNA cloning and tissue specific expression of a gene for sucrose transporter from rice (Oryza sativa L.); Hirose T et al.; We describe the cloning and expression analysis of a sucrose transporter cDNA from a monocot (the rice plant, Oryza sativa L.) . The cDNA clone (OsSUT1) encoded an open reading frame of 1,611 bp (537 amino acids) and showed 76.8 to 79.7% similarity at the amino acid level to other sucrose transporters of dicot species . The predicted membrane topology of OsSUT1 protein is made up of 12 transmembrane helices which is consistent with most of the mono- and disaccharide transporters previously identified . When OsSUT1 cDNA was introduced into yeast and expressed, the cells rapidly accumulated sucrose demonstrating that OsSUT1 does, in fact, encode a sucrose transporter . From genomic Southern hybridization OsSUT1 appeared to be a single copy gene . OsSUT1 was expressed in source organs such as leaf blade, leaf sheath and germinating seed whereas little or no expression was observed in some sink organs such as the panicles before heading and the roots . Transcript was observed at high levels in panicles after heading, particularly in the portion containing endosperm and embryo . In addition, expression of OsSUT1 was high in etiolated seedlings and decreased during light-induced greening.

DNA Seq, 1997, 8(1-2), 105 - 8
Primary structure of Drosophila ribosomal protein L14 and identification of conserved protein motifs; Haynes SR; Determination of the primary structure of individual ribosomal proteins is important for understanding their functions and organization within the ribosome . I have sequenced a cDNA that encodes a Drosophila homolog of the rat ribosomal protein L14 . The cDNA sequence was 601 nucleotides long, with an open reading frame encoding a protein of 166 amino acids . Homology searches revealed 34-38% sequence identity to the rat and yeast L14 ribosomal proteins . There were also extensive homologies to sequences in the EST database, which are likely to encode portions of L14 . Analysis of sequence comparisons revealed several highly conserved regions, one of which is related to a portion of ribosomal protein L27 . The sizes of the L14 proteins vary between different species, with most of the variability confined to the C-terminal region.

Genomics, 1998 Mar 1, 48(2), 242 - 7
cDNA cloning, mRNA expression, and chromosomal mapping of human and mouse periplakin genes; Aho S et al.; A portion of the intracellular domain of Type XVII collagen, used as a bait in a yeast two-hybrid screen of an epidermal keratinocyte cDNA library, identified overlapping cDNA clones that showed a high degree of homology to envoplakin and other members of the plakin family of intermediate filament connector molecules . Subsequent cloning allowed identification of contiguous cDNA sequences with an open reading frame of 5268 bp encoding a putative polypeptide of 1756 amino acids with a computed molecular mass of 204.7 kDa . Northern analysis using these cDNA clones revealed a prominent band of approximately 6.5 kb in keratinocytes, which was barely detectable in fibroblasts . Multiple tissue RNA analysis showed that this protein is highly expressed in tissues with a prominent component of epithelial cells . This novel member of the plakin family was designated periplakin . The human gene (PPL) was mapped to the interval between D16S510 and D16S509 by radiation hybrid mapping, corresponding to chromosomal band 16p13 . Murine ESTs having 97.2% amino acid identity to the human sequence were identified . Interspecific backcross mapping was used to place the murine periplakin gene (Ppl) 0.53 cM distal to marker D16mit32 on the proximal part of murine chromosome 16, close to the locus of mahoganoid (md), a mouse hair mutant . Mapping of this gene in human and mouse will allow evaluation of periplakin as a candidate locus for disorders of epithelial fragility, with or without other phenotypes.

Genomics, 1998 Mar 1, 48(2), 203 - 8
Genomic organization of the 70-kDa peroxisomal membrane protein gene (PXMP1); Gartner J et al.; The 70-kDa peroxisomal membrane protein (PMP70) is a member of a family of half-ATP-binding cassette (ABC) transporter proteins located in the human peroxisomal membrane . Other members include the PMP70-related peroxisomal membrane protein, the adrenoleukodystrophy protein (ALDP), and the adrenoleukodystrophy-related protein . The functions of ABC transporters in the peroxisomal membrane are poorly understood . Evidence from yeast and human mutants suggests that they are involved in the peroxisomal import of fatty acids and/or fatty acyl-CoAs into the organelle . We report the cloning and characterization of the human PMP70 structural gene (gene symbol: PXMP1) localized on human chromosome 1p21-p22 . PXMP1 is approximately 65 kb in length, contains 23 exons, and is quite different in structure from the gene (ALD) that encodes the related protein, ALDP . We also analyzed the 5' flanking region of the human PXMP1 gene and the corresponding region of murine Pxmp-1 . Both promoters have features of housekeeping genes, including a high GC content and multiple consensus Sp1 binding sequences . In more than 3 kb of Pxmp-1 5' flanking sequence we did not identify a consensus peroxisomal proliferator responsive element.

Genomics, 1998 Mar 1, 48(2), 171 - 7
Homozygosity and physical mapping of the autosomal recessive retinitis pigmentosa locus (RP14) on chromosome 6p21.3; Banerjee P et al.; Retinitis pigmentosa (RP) is a heterogeneous genetic disorder with autosomal dominant, autosomal recessive, and X-linked forms . We previously mapped an additional arRP locus to chromosome 6p21 (RP14) in a single extended kinship from the Dominican Republic . Aided by a second linked RP pedigree from the same region of the Dominican Republic, we have refined the disease locus to a 2-cM region that is homozygous-by-descent in both pedigrees . A complete YAC, and a partial BAC, contig of the RP14 locus was constructed between the markers D6S1560 and D6S291, encompassing approximately 2.1 Mb . The contig contains 12 YACs and 31 BACs and is characterized by 45 markers including 8 microsatellite markers, 6 gene-derived sequences/ESTs obtained from the databases, and 28 new STSs and 4 new ESTs obtained by BLAST search using DNA sequence from the ends of the BAC and YAC inserts . With a STS density of approximately 1 every 20 kilobases, this contig significantly enhances available maps of the region.

Nature, 1998 Mar 19, 392(6673), 300 - 3
Mutations of mitotic checkpoint genes in human cancers; Cahill DP et al.; Genetic instability was one of the first characteristics to be postulated to underlie neoplasia . Such genetic instability occurs in two different forms . In a small fraction of colorectal and some other cancers, defective repair of mismatched bases results in an increased mutation rate at the nucleotide level and consequent widespread microsatellite instability . In most colorectal cancers, and probably in many other cancer types, a chromosomal instability (CIN) leading to an abnormal chromosome number (aneuploidy) is observed . The physiological and molecular bases of this pervasive abnormality are unknown . Here we show that CIN is consistently associated with the loss of function of a mitotic checkpoint . Moreover, in some cancers displaying CIN the loss of this checkpoint was associated with the mutational inactivation of a human homologue of the yeast BUB1 gene; BUB1 controls mitotic checkpoints and chromosome segregation in yeast . The normal mitotic checkpoints of cells displaying microsatellite instability become defective upon transfer of mutant hBUB1 alleles from either of two CIN cancers.

Mol Gen Genet, 1998 Feb, 257(3), 283 - 91
Isolation and characterisation of the RAD51 and DMC1 homologs from Arabidopsis thaliana; Doutriaux MP et al.; By using RT-PCR and degenerate oligonucleotides based on the sequence homology between the yeast RAD51 and DMC1 genes, two genes belonging to the RAD51 and DMC1 families were isolated from Arabidopsis thaliana ecotype Columbia . A RAD51 genomic DNA was also sequenced which is almost identical to its Landsberg erecta counterpart, except for a few translationally silent substitutions and for the presence of a 527-bp element downstream of the polyadenylation site . This element is repeated in the genome of Arabidopsis . Northern analyses were conducted to characterize the expression pattern of both these genes . AtRAD51 and AtDMC1 are expressed in flower buds, but also in the mitotically active cells from a suspension culture . AtRAD51, but not AtDMC1, transcript level increases after gamma irradiation of the cells . Finally, a synchronisation experiment conducted with the suspension culture indicated that not only AtRAD51 but also AtDMC1 are regulated during the cell cycle, with S-phase-specific induction . Since DMC1 genes have always been regarded as being specifically meiotic, we discuss the significance of this mitotic transcriptional regulation in Arabidopsis.

Plant Physiol, 1998 Mar, 116(3), 1023 - 8
Expression of an arabidopsis aspartate Kinase/Homoserine dehydrogenase gene is metabolically regulated by photosynthesis-related signals but not by nitrogenous compounds
Zhu-Shimoni JX, Galili G.
Although the control of carbon fixation and nitrogen assimilation has been studied in detail, relatively little is known about the regulation of carbon and nitrogen flow into amino acids . In this paper we report our study of the metabolic regulation of expression of an Arabidopsis aspartate kinase/homoserine dehydrogenase (AK/HSD) gene, which encodes two linked key enzymes in the biosynthetic pathway of aspartate family amino acids . Northern blot analyses, as well as expression of chimeric AK/HSD-beta-glucuronidase constructs, have shown that the expression of this gene is regulated by the photosynthesis-related metabolites sucrose and phosphate but not by nitrogenous compounds . In addition, analysis of AK/HSD promoter deletions suggested that a CTTGACTCTA sequence, resembling the binding site for the yeast GCN4 transcription factor, is likely to play a functional role in the expression of this gene . Nevertheless, longer promoter fragments, lacking the GCN4-like element, were still able to confer sugar inducibility, implying that the metabolic regulation of this gene is apparently obtained by multiple and redundant promoter sequences . The present and previous studies suggest that the conversion of aspartate into either the storage amino acid asparagine or aspartate family amino acids is subject to a coordinated, reciprocal metabolic control, and this biochemical branch point is a part of a larger, coordinated regulatory mechanism of nitrogen and carbon storage and utilization.

Jpn J Pharmacol, 1998 Jan, 76(1), 75 - 86
Anti-inflammatory, analgesic and anti-pyretic effects of d-2-{4-(3-methyl-2-thienyl)phenyl}propionic acid (M-5011), a new non-steroidal anti-inflammatory drug, in rats and guinea pigs; Kido H et al.; Anti-inflammatory, analgesic and anti-pyretic effects of d-2-{4-(3-methyl-2-thienyl)phenyl}propionic acid (M-5011), a new non-steroidal anti-inflammatory drug (NSAID), were compared with those of indomethacin, diclofenac sodium and ketoprofen in rats and guinea pigs . Anti-inflammatory effect of M-5011 on ultraviolet-induced erythema in guinea pigs was 11.7 and 1.8 times more potent than that of indomethacin and ketoprofen, respectively . Inhibitory effect of M-5011 on carrageenin-induced paw edema was 2 and 1.5 times more potent than that of indomethacin and diclofenac sodium, respectively . Analgesic effect of M-5011 on dry yeast-induced hyperalgesia or adjuvant-induced arthritic pain was equipotent to that of indomethacin, diclofenac sodium or ketoprofen . Anti-pyretic effect of M-5011 on yeast-induced pyrexia in rats was 4.2 and 4.6 times more potent than that of indomethacin and ketoprofen, respectively . Inhibitory effect of M-5011 on prostaglandin E2 production in the exudate of air-pouch inflammation induced by carrageenin was 1.75 times more potent than that in the non-inflamed site (stomach) . As a result, gastric ulcerogenic activity of M-5011 was half that of indomethacin in rat . These results suggest that M-5011 shows more potent anti-inflammatory and anti-pyretic effects and equipotent analgesic effect with low gastro-ulcerogenic activity compared with classical NSAIDs.

Plant Cell Physiol, 1998 Jan, 39(1), 115 - 23
The influences of two plant nuclear matrix attachment regions (MARs) on gene expression in transgenic plants; Liu JW et al.; Nuclear matrix attachment regions (MARs) are thought to influence gene expression by anchoring active chromatin to the proteinaceous nuclear matrix . In this study, two plant DNA fragments with strong MAR activity were selected and tested for their effects on expression of a linked reporter gene in transgenic tobacco . One MAR was isolated from the 5' flanking region of a pea vicilin gene previously reported to be expressed in a copy number-dependent manner in transgenic tobacco . A second MAR was isolated from the genome of Arabidopsis thaliana by preselection for autonomously replicating sequence (ARS) activity in yeast . Flanking copies of the A . thaliana MAR stimulated median reporter gene expression in transgenic plants by five to ten fold . Neither MAR significantly reduced the variation in transgene expression between individual transformants, or conferred copy number-dependence in gene expression.

Gene, 1998 Feb 16, 208(1), 67 - 72
Characterization of the sequence coding for the clathrin coat assembly protein AP17 (sigma2) associated with the plasma membrane from Zea mays and constitutive expression of its gene; Roca R et al.; The cDNA and genomic sequences coding for the clathrin coat assembly protein AP17 (sigma2) from maize and its corresponding mRNA accumulation have been analyzed . This protein in yeast and mammals has been shown to be part of the associated protein (AP) complex of clathrin in the plasma membrane . The availability of this sequence as well as a previous AP19 in a plant allows one to propose that specific AP complexes exist in plants in the Golgi complex and in the plasma membrane . The AP17 protein is encoded in maize by a single gene, and its mRNA accumulates in all the organs studied . In the immature embryo, it displays a pattern of expression typical of constitutively expressed genes.

J Biol Chem, 1998 Feb 27, 273(9), 5101 - 8
Neurofilament (NF) assembly; divergent characteristics of human and rodent NF-L subunits; Carter J et al.; Previous studies have shown that rodent neurofilaments (NF) are obligate heteropolymers requiring NF-L plus either NF-M or NF-H for filament formation . We have assessed the competence of human NF-L and NF-M to assemble and find that unlike rat NF-L, human NF-L is capable of self-assembly . However, human NF-M cannot form homopolymers and requires the presence of NF-L for incorporation into filaments . To investigate the stage at which filament formation is blocked, the rod domains or the full-length subunits of human NF-L, human NF-M, and rodent NF-L were analyzed in the yeast "interaction trap" system . These studies demonstrated that the fundamental block to filament formation in those neurofilaments that do not form homopolymers is at the level of dimer formation . Based on theoretical biophysical considerations of the requirements for the formation of coiled-coil structures, we predicted which amino acid differences were likely to be responsible for the differing dimerization potentials of the rat and human NF-L rod domains . We tested these predictions using site-specific mutagenesis . Interestingly, single amino acid changes in the rod domains designed to restore or eliminate the coiled-coil propensity were found respectively to convert rat NF-L into a subunit capable of homopolymerization and human NF-L into a protein that is no longer able to self-assemble . Our results additionally suggest that the functional properties of the L12 linker region of human NF-L, generally thought to assume an extended beta-sheet conformation, are consonant with an alpha-helix that positions the heptad repeats before and after it in an orientation that allows coiled-coil dimerization . These studies reveal an important difference between the assembly properties of the human and rodent NF-L subunits possibly suggesting that the initiating events in neurofilament assembly may differ in the two species.

J Biol Chem, 1998 Feb 27, 273(9), 4976 - 81
Enzymological characterization of recombinant xenopus DG42, a vertebrate hyaluronan synthase; Pummill PE et al.; We have characterized the hyaluronan (HA) synthase activity of the Xenopus DG42 gene product in vitro . The recombinant enzyme produced in yeast does not possess a nascent HA chain and, therefore, is an ideal model system for kinetic studies of the synthase's glycosyltransferase activity . The enzymatic rate was optimal from pH 7.6 to 8.1 . Only the authentic sugar nucleotide precursors, UDP-glucuronic acid (UDP-GlcA) and UDP-N-acetylglucosamine (UDP-GlcNAc), were utilized to produce a large molecular weight polymer . UDP-glucose or the galactose epimers of the normal substrates did not substitute . The Michaelis constant, Km, of recombinant DG42 in membranes was 60 +/- 20 and 235 +/- 40 microM for UDP-GlcA and UDP-GlcNAc, respectively, which is comparable to values obtained previously from membranes derived from vertebrate cells . The apparent energy of activation for HA elongation is about 15 kilocalories/mol . DG42 polymerizes HA at average rates of about 80 to 110 monosaccharides/s in vitro . The resulting HA polysaccharide possessed molecular weights spanning 2 x 10(6)-10(7) Da, corresponding to about 10(4) sugar residues . This is the first report characterizing a defined eukaryotic enzyme that can produce a glycosaminoglycan.

J Biol Chem, 1998 Feb 13, 273(7), 4096 - 105
Characterization of a human lysophosphatidic acid acyltransferase that is encoded by a gene located in the class III region of the human major histocompatibility complex; Aguado B et al.; Sequence analysis of cDNA clones corresponding to a number of genes located in the class III region of the human major histocompatibility complex (MHC), in the chromosome band 6p21.3, has shown that the G15 gene encodes a 283-amino acid polypeptide with significant homology over the entire polypeptide with the enzyme lysophosphatidic acid acyltransferase (LPAAT) from different yeast, plant, and bacterial species . The amino acid sequence of the MHC-encoded human LPAAT (hLPAATalpha) is 48% identical to the recently described hLPAAT (Eberhardt, C., Gray, P . W., and Tjoelker, L . W . (1997) J . Biol . Chem . 272, 20299-20305), which is encoded by a gene located on chromosome 9p34.3 . LPAAT is the enzyme that in lipid metabolism converts lysophosphatidic acid (LPA) into phosphatidic acid (PA) . The expression of the hLPAATalpha polypeptide in the baculovirus system and in mammalian cells has shown that it is an intracellular protein that contains LPAAT activity . Cell extracts from insect cells overexpressing hLPAATalpha were analyzed in different LPAAT enzymatic assays using, as substrates, different acyl acceptors and acyl donors . These cell extracts were found to contain up to 5-fold more LPAAT activity compared with control cell extracts, indicating that the hLPAATalpha specifically converts LPA into PA, incorporating different acyl-CoAs with different affinities . The hLPAATalpha polypeptide expressed in the mammalian Chinese hamster ovary cell line was found, by confocal immunofluorescence, to be localized in the endoplasmic reticulum . Due to the known role of LPA and PA in intracellular signaling and inflammation, the hLPAATalpha gene represents a candidate gene for some MHC-associated diseases.

J Biol Chem, 1998 Feb 13, 273(7), 4089 - 95
Isolation and characterization of PNUTS, a putative protein phosphatase 1 nuclear targeting subunit; Allen PB et al.; Protein phosphatase 1 (PP1) is found in the cell nucleus and has been implicated in several aspects of nuclear function . We report here the cloning and initial characterization of a novel protein approximately named phosphatase 1 nuclear targeting subunit (PNUTS) . This protein interacts with PP1 in a yeast two-hybrid assay, is found in a stable complex with PP1 in mammalian cell lysates, and exhibits a potent modulation of PP1 catalytic activity toward exogenous substrate in vitro . PNUTS is a ubiquitously expressed protein that exhibits a discreet nuclear compartmentalization and is colocalized with chromatin at distinct phases during mitosis . The subcellular localization of PP1 and the activity toward substrates involved in many aspects of cell physiology have previously been shown to be regulated by association with noncatalytic targeting subunits . The properties of PNUTS are consistent with its role as a targeting subunit for the regulation of nuclear PP1 function.

J Biol Chem, 1998 Feb 13, 273(7), 3948 - 53
Isozyme function of n-alkane-inducible cytochromes P450 in Candida maltosa revealed by sequential gene disruption; Ohkuma M et al.; An n-alkane-assimilating yeast Candida maltosa contains multiple n-alkane-inducible forms of cytochromes P450 (P450alk), which can be assumed to catalyze terminal hydroxylation of n-alkanes in the assimilation pathway . Eight structurally related P450alk genes have been identified . In the present study, the function of four major isoforms of P450alk (encoded by ALK1, ALK2, ALK3, and ALK5 genes) was investigated by sequential gene disruption . Auxotrophic markers used for the selection of disrupted strains were regenerated repeatedly through either mitotic recombination between heterozygous alleles of the diploid genome or directed deletion of the marker gene, to allow sequential gene disruptions within a single strain . The strain depleted of all four isoforms could not utilize n-alkanes for growth, providing direct evidence that P450alk is essential for n-alkane assimilation . Growth properties of a series of intermediate disrupted strains, plasmid-based complementation, and enzyme assays after heterologous expression of single isoforms revealed (i) that each of the four individual isoforms is alone sufficient to allow growth on long chain n-alkane; (ii) that the ALK1-encoding isoform is the most versatile and efficient P450alk form, considering both its enzymatic activity and its ability to confer growth on n-alkanes of different chain length; and (iii) that the ALK5-encoding isoform exhibits a rather narrow substrate specificity and thus cannot support the utilization of short chain n-alkanes.

Curr Biol, 1998 Jan 1, 8(1), 56 - 64
Regulation of the MAP kinase pathway by mammalian Ksr through direct interaction with MEK and ERK; Yu W et al.; BACKGROUND: Genetic screens in Drosophila melanogaster and Caenorhabditis elegans identified the kinase suppressor of Ras, Ksr, as a new component in the Ras intracellular signaling pathway . In these organisms, mutations in Ksr resulted in attenuation of Ras-mediated signaling . Homologs of Ksr have also been isolated from mice and humans; their precise role in Ras signaling is not well defined . Here, we present data showing interactions between the murine form of Ksr (mKsr-1) and other components of the Ras pathway . RESULTS: To gain insight into the biological function of Ksr, we used a yeast two-hybrid screen and found an interaction between the carboxy-terminal region of mKsr-1 and mitogen-activated protein (MAP) kinase kinase 1 (MAPKK-1 or MEK-1) . An interaction was also detected between MAP kinase (also called extracellular signal-regulated kinase; ERK), and the amino-terminal region of mKsr-1 . These interactions were recapitulated in COS-7 cells . Further, when COS-7 cells were transfected with either full-length mKsr-1 or only its carboxy-terminal region, an inhibition of serum-stimulated MAP kinase activation was observed . Microinjection of full-length mKsr-1 or its carboxy-terminal, but not its amino-terminal region, blocked serum-induced DNA synthesis in rat embryo fibroblasts . Co-injection of mKsr-1 with MEK-1 reversed the blockade . CONCLUSIONS: Together with the data from genetic analyses, our findings lead us to propose that mKsr-1 may control MAP kinase signaling by serving as a scaffold protein that links MEK and its substrate ERK.

Curr Biol, 1998 Jan 1, 8(1), 46 - 55
Murine Ksr interacts with MEK and inhibits Ras-induced transformation; Denouel-Galy A et al.; BACKGROUND: Ksr (kinase supressor of Ras) was identified as a regulator of the Ras-MAP kinase (mitogen-activated protein kinase) pathway by genetic screens in Drosophila and Caenorhabditis elegans . Ksr is a kinase with similarities to the three conserved regions of Raf kinases, especially within the kinase domain . To investigate whether these structural similarities correlated with common functional properties, we examined the ability of mKsr-1, the murine homolog of Ksr, to interact with components of the vertebrate MAP kinase pathway . RESULTS: In the yeast two-hybrid interaction assay, mKsr-1 did not bind to either Ras, B-Raf or Raf-1, but interacted strongly with both MEK-1 and MEK-2, activators of MAP kinase . The Ksr-MEK interaction was confirmed by co-immunoprecipitation experiments . Ectopically expressed mKsr-1 co-precipitated with endogenous MEK-1 in COS-1 cells, and endogenous Ksr and MEK co-precipitated from PC12 cells . Phosphorylation of MEK by mKsr-1 was not detected, however . In contrast, the MEK subpopulation complexed with mKsr-1 in COS-1 cells or PC12 cells did not display kinase activity . This ability of Ksr to block MEK in an inactive form correlated with a biological response: mKsr-1 did not transform NIH3T3 cells, and, furthermore, mKsr-1 reduced Ras-induced transformation . Similarly, mKsr-1 inhibited the proliferation of embryonic neuroretina cells induced by Ras and B-Raf but not that induced by MEK . CONCLUSIONS: Our results suggest a novel mechanism for Ksr in regulating the MAP kinase pathway, at least in part through an ability to interact with MEK.

N Engl J Med, 1998 Mar 26, 338(13), 879 - 87
CDKN2A mutations in multiple primary melanomas; Monzon J et al.; BACKGROUND: Germ-line mutations in the CDKN2A tumor-suppressor gene (also known as p16, p16INK4a, and MTS1) have been linked to the development of melanoma in some families with inherited melanoma . Whether mutations in CDKN2A confer a predisposition to sporadic (nonfamilial) melanoma is not known . In some patients with sporadic melanoma, one or more additional primary lesions develop, suggesting that some of these patients have an underlying genetic susceptibility to the cancer . We hypothesized that this predisposition might be due to germ-line CDKN2A mutations . METHODS: We used the polymerase chain reaction, single-strand conformation polymorphism analysis, and direct DNA sequencing to identify germ-line mutations in the CDKN2A gene in patients with multiple primary melanomas who did not have family histories of the disease . A quantitative yeast two-hybrid assay was used to evaluate the functional importance of the CDKN2A variants . RESULTS: Of 33 patients with multiple primary melanomas, 5 (15 percent; 95 percent confidence interval, 4 percent to 27 percent) had germ-line CDKN2A mutations . These included a 24-bp insertion at the 5' end of the coding sequence, three missense mutations (Arg24Pro, Met53Ile, and Ser56Ile), and a 2-bp deletion at position 307 to 308 (resulting in a truncated CDKN2A protein) . In three families, CDKN2A mutations identical to those in the probands were found in other family members . In two families with mutations, we uncovered previously unknown evidence of family histories of melanoma . CONCLUSIONS: Some patients with multiple primary melanomas but without family histories of the disease have germ-line mutations of the CDKN2A gene . The presence of multiple primary melanomas may signal a genetic susceptibility to melanoma not only in the index patient but also in family members, who may benefit from melanoma-surveillance programs.

Clin Exp Allergy, 1998 Feb, 28(2), 169 - 74
Immune-reactivity of recombinant isoforms of the major house dust mite allergen Der p 2; Hakkaart GA et al.; BACKGROUND: Recombinant Der p 2, expressed in yeast, lacked reactivity with 5 monoclonal antibodies against natural Der p 2 . OBJECTIVE: The aim of this study was to investigate whether the lack of reactivity with recombinant Der p 2 can be explained by the existence of isoforms . METHODS: By site-directed mutagenesis three recombinant isoforms of Der p 2 were produced . Reactivity with monoclonal antibodies and human IgE was analysed by means of RAST and RAST-inhibition . RESULTS: All five monoclonals that lacked reactivity with the originally selected isoform, showed reactivity upon replacement of aspartic acid by asparagine at position 114 . The other two substitutions (at position 26 and 47) had no effect . Binding of human IgE (n = 10) was not significantly influenced by the isogenetic variation at position 114 . CONCLUSIONS: Monoclonal antibodies raised against natural Der p 2 can sometimes discriminate between different isoforms, allowing the study of the natural occurrence of isoforms . For application in allergen-measurement assays, non-discriminating monoclonal antibodies should be selected.

Am J Med Genet, 1998 Feb 7, 81(1), 81 - 91
Bipolar affective disorder partially cosegregates with a balanced t(9;11)(p24;q23.1) chromosomal translocation in a small pedigree; Baysal BE et al.; Analysis of an extended pedigree in which a balanced t(9;11)(p24;q23.1) translocation was found to cosegregate with bipolar affective disorder revealed that five of 11 translocation carriers had bipolar affective disorder and one carrier had unipolar depression . There were no affected individuals in the pedigree without the balanced translocation . We hypothesized that gene(s) or gene regulatory regions disrupted by the translocation might be contributing to the bipolar affective disorder in a dominant fashion . To test this hypothesis, we isolated the derivative chromosome 9 and derivative chromosome 11 in somatic cell hybrids and identified the nearest flanking markers on chromosome 9 (D9S230 and D9S2011E/HRFX3) and chromosome 11 (EST00652 and CRYA2) . YAC contigs were constructed in the region of flanking markers for both chromosomes 9 and 11 . Chromosome 11 breakpoint was localized within an 8-kb region in a small insert (100 kb) YAC . Chromosome 9 breakpoint was localized within approximately 2 Mb region . Several genes and ESTs including EST00652, CRYA2, DRD2, 5HTR3 on chromosome 11 and VLDLR and SLC1A1 on chromosome 9 were mapped within the vicinity of the breakpoint but were shown not to be disrupted by the translocation breakpoint . Although several possibilities exist regarding the role of the balanced translocation in developing bipolar affective disorder in this pedigree, including a chance cosegregation, identification of a disrupted gene or gene regulatory region with the help of physical mapping resources described in this study may help to identify the presence of a susceptibility gene for this disorder.

J Comp Neurol, 1998 Mar 23, 392(4), 428 - 38
Twofold overexpression of human beta-amyloid precursor proteins in transgenic mice does not affect the neuromotor, cognitive, or neurodegenerative sequelae following experimental brain injury; Murai H et al.; By using transgenic mice that overexpress human beta-amyloid precursor proteins (APPs) at levels twofold higher than endogenous APPs, following introduction of the human APP gene in a yeast artificial chromosome (YAC), we examined the effects of controlled cortical impact (CCI) brain injury on neuromotor/cognitive dysfunction and the development of Alzheimer's disease (AD)-like neuropathology . Neuropathological analyses included Nissl-staining and immunohistochemistry to detect APPs, beta-amyloid (Abeta), neurofilament proteins, and glial fibrillary acidic protein, whereas Abeta levels were measured in brain homogenates from mice subjected to CCI and control mice by using a sensitive sandwich enzyme-linked immunosorbent assay . Twenty APP-YAC transgenic mice and 17 wild type (WT) littermate controls were anesthetized and subjected to CCI (velocity, 5 m/second; deformation depth, 1 mm) . Sham (anesthetized but uninjured) controls (n = 10 APP-YAC; n = 8 WT) also were studied . Motor function was evaluated by using rotarod, inclined-plane, and forelimb/hindlimb flexion tests . The Morris water maze was used to assess memory . Although CCI induced significant motor dysfunction and cognitive deficits, no differences were observed between brain-injured APP-YAC mice and WT mice at 24 hours and 1 week postinjury . By 1 week postinjury, both cortical and hippocampal CA3 neuron loss as well as extensive astrogliosis were observed in all injured animals, suggesting that overexpression of human APPs exhibited no neuroprotective effects . Although AD-like pathology (including amyloid plaques) was not observed in either sham or brain-inj ured animals, a significant decrease in brain concentrations of only Abeta terminating at amino acid 40 (Abeta x-40) was observed following brain injury in APP-YAC mice (P < 0.05 compared with sham control levels) . Our data show that the APP-YAC mice do not develop AD-like neuropathology following traumatic brain injury . This may be because this injury does not induce elevated levels of the more amyloidogenic forms of human Abeta (i.e., Abeta x-42/43) in these mice.

J Mol Biol, 1998 Feb 13, 276(1), 85 - 104
Prediction of splice sites in plant pre-mRNA from sequence properties; Brendel V et al.; Heterologous introns are often inaccurately or inefficiently processed in higher plants . The precise features that distinguish the process of pre-mRNA splicing in plants from splicing in yeast and mammals are unclear . One contributing factor is the prominent base compositional contrast between U-rich plant introns and flanking G + C-rich exons . Inclusion of this contrast factor in recently developed statistical methods for splice site prediction from sequence inspection significantly improved prediction accuracy . We applied the prediction tools to re-analyze experimental data on splice site selection and splicing efficiency for native and more than 170 mutated plant introns . In almost all cases, the experimentally determined preferred sites correspond to the highest scoring sites predicted by the model . In native genes, about 90% of splice sites are the locally highest scoring sites within the bounds of the flanking exon and intron . We propose that, in most cases, local context (about 50 bases upstream and downstream from a potential intron end) is sufficient to account for intrinsic splice site strength, and that competition for transacting factors determines splice site selection in vivo . We suggest that computer-aided splice site prediction can be a powerful tool for experimental design and interpretation.

Biochem Soc Symp, 1998, 63, 141 - 7
Production of therapeutic proteins in the milk of transgenic livestock; Colman A; With the advent of the Human Genome Project and associated developments in 'functional genomics', there are going to be increasing numbers of proteins identified and developed for clinical use . There are a number of production methods available, although only three, bacterial, yeast and mammalian cell culture, have produced recombinant proteins that have been approved for clinical use . Nevertheless other production systems are under development, and one, the production of human proteins in the milk of transgenic livestock, is showing great promise, with two proteins now in clinical trials . This chapter will compare and contrast the various competing technologies and will then concentrate on factors influencing the choice and use of the transgenic system.

Immunogenetics, 1998 Apr, 47(5), 371 - 80
BAC/YAC contigs from the H2-M region of mouse Chr 17 define gene order as Znf173-Tctex5-mog-D17Tu42-M3-M2; Yoshino M et al.; A yeast artificial chromosome (YAC) contig from the C57BL/6 (H2(b)) mouse was created from the major histocompatibility complex (Mhc, H2 in mouse) class Ib subregion, H2-M . It spans approximately 1.2 megabase (Mb) pairs and unites the previous >1.5-Mb YAC contigs (Jones et al . 1995) into a single contig, which includes 21 Mhc class I genes distal to H2-T1 . A bacterial artificial chromosome (BAC) contig from the 129 (H2(bc)) mouse, spanning approximately 600 kilobases, was also built from Znf173 (Afp, a gene for acid finger protein), through Tctex5 (t-complex testis expressed-5) and Mog (myelin oligodendrocyte glycoprotein), to H2-M2 . Twenty-four sequence-tagged site (STS) markers were newly developed, and 35 markers were mapped in the YAC/BAC contigs, which define the marker order as Cen - Znf173 - Tctex5 - Mog - D17Tu42 - D17Mit232 - H2-M3 - D17Leh525 - H2-M2 - Tel . The gene order of Znf173 - Tctex5 - Mog - D17Tu42 is conserved between mouse and human, showing that the middle H2-M region corresponds to the subregion of the human Mhc surrounding HLA-A.

Curr Biol, 1998 Mar 12, 8(6), 347 - 50
Developmental regulation of MCM replication factors in Xenopus laevis; Sible JC et al.; At the midblastula transition (MBT) during Xenopus laevis development, zygotic transcription begins {1}, and the rapid, early cleavage cycles are replaced by cell-division cycles that lengthen and acquire G (gap) phases {2} and checkpoints {3-5} . This cell-cycle remodeling may result from either a loss of maternal products, the transcription of zygotic genes, or the replacement of maternal proteins by zygotic gene products . We have identified an example of the third possibility: distinct maternal and zygotic genes encoding a member of the minichromosome maintenance (MCM) protein family . The mcm genes were identified in yeast by mutations that blocked replication of artificial chromosomes or perturbed the G1/S transition in the cell cycle {6,7} . In Xenopus eggs, the MCM2-MCM7 proteins assemble as multimeric complexes at chromosomal origins of replication {8-14} . The sequential, cell-cycle-dependent assembly of the origin replication complex (ORC), CDC6 protein and the MCM complex at origins of replication ensures that DNA replicates only once per cell cycle {15,16} . The periodic association of the MCM complex with chromatin may be regulated via phosphorylation by cyclin-dependent kinases (Cdks) {11} . We have cloned the first example of a developmentally regulated mcm gene, zygotic mcm6 (zmcm6), expressed only after gastrulation when the cell cycle is remodeled . The zMCM6 protein assembles into MCM complexes and differs from maternal MCM6 (mMCM6) in having a carboxy-terminal extension and a consensus cyclin-Cdk phosphorylation site . There may also be maternal-zygotic pairs of other MCMs . These data suggest that MCMs are critical for cell-cycle remodeling during early Xenopus development.

Curr Biol, 1998 Mar 12, 8(6), 305 - 14
Identification of a nuclear export receptor for tRNA; Arts GJ et al.; BACKGROUND: Transport of macromolecules between the nucleus and cytoplasm of eukaryotic cells is mediated by nuclear import and export receptors . The receptors identified to date are members of a family of Ran GTPase-binding proteins whose founding member is importin-beta . Interaction between these receptors and their cargo is regulated by the GTP-bound form of Ran . Export complexes form and import complexes disassemble on binding of RanGTP to the receptor . Yeast Los 1 p is a member of the importin-beta family with a poorly defined role in tRNA production . RESULTS: A human member of the importin-beta family that is distantly related to Los 1 p (21% identity) has been characterized . The protein shuttled between the nucleus and cytoplasm and interacts with tRNA in a RanGTP-dependent manner . Injection of the protein into the nuclei of Xenopus oocytes resulted in a specific stimulation of the export of tRNA from the nucleus and in relief of the competitive inhibition of tRNA export caused by the introduction of saturating amounts of nuclear tRNA . CONCLUSIONS: The human protein has the functional properties expected of a transport receptor that mediates export of tRNA from the nucleus . We therefore name the protein Exportin(tRNA).

Mycopathologia, 1997, 139(1), 9 - 14
Isolation of ergosterol peroxide and its reversion to ergosterol in the pathogenic fungus Sporothrix schenckii; Sgarbi DB et al.; Ergosterol peroxide, a presumed product of the H2O2-dependent enzymatic oxidation of ergosterol, has been isolated from yeast forms of the pathogenic fungus Sporothrix schenckii . The substance, which may have a role in fungal virulence, has been characterized mainly using spectroscopic methods (1H and 13C nuclear magnetic resonance and high resolution mass spectra) . The purified compound showed a molecular formula of C28H44O3, displaying characteristic features of epidioxy sterols and was reverted to ergosterol when submitted to S . schenckii enzymatic extract.

Nature, 1998 Mar 5, 392(6671), 97 - 101
The 1.7 A crystal structure of the regulator of chromosome condensation (RCC1) reveals a seven-bladed propeller; Renault L et al.; The gene encoding the regulator of chromosome condensation (RCC1) was cloned by virtue of its ability to complement the temperature-sensitive phenotype of the hamster cell line tsBN2, which undergoes premature chromosome condensation or arrest in the G1 phase of the cell cycle at non-permissive temperatures . RCC1 homologues have been identified in many eukaryotes, including budding and fission yeast . Mutations in the gene affect pre-messenger RNA processing and transport, mating, initiation of mitosis and chromatin decondensation, suggesting that RCC1 is important in the control of nucleo-cytoplasmic transport and the cell cycle . Biochemically, RCC1 is a guanine-nucleotide-exchange factor for the nuclear Ras homologue Ran; it increases the dissociation of Ran-bound GDP by 10(5)-fold . It may also bind to DNAvia a protein-protein complex . Here we show that the structure of human RCC1, solved to 1.7-A resolution by X-ray crystallography, consists of a seven-bladed propeller formed from internal repeats of 51-68 residues per blade . The sequence and structure of the repeats differ from those of WD40-domain proteins, which also form seven-bladed propellers and include the beta-subunits of G proteins . The nature of the structure explains the consequences of a wide range of known mutations . The region of the protein that is involved in guanine-nucleotide exchange is located opposite the region that is thought to be involved in chromosome binding.

Tissue Antigens, 1998 Feb, 51(2), 183 - 94
A first map of the porcine major histocompatibility complex class I region; Velten F et al.; A map of the SLA complex, or swine major histocompatibility complex (MHC), class I region was constructed by alignment of yeast artificial chromosomes (YACs) harboring MHC class I genes as well as anchor genes already mapped within the human MHC complex (HLA) . Five YACs containing 9 anchor genes built a contig of about 1.0-1.2 Mb between the SLA class III BAT1 locus and the olfactory receptor-like genes OLF42 . Ten different SLA class I sequences, including putative allelic forms of published classical and non-classical SLA class I genes, were assigned to the 400-kb enclosing centromeric part of the contig . Three additional YACs comprising the OLF89 genes and two YACs containing the butyrophilin gene were located telomeric to the contig . Comparison between the human and porcine MHC complexes showed a perfect conserved order of anchor genes, whereas no orthologous relationships were found for the class I loci.

Mech Dev, 1998 Jan, 70(1-2), 193 - 5
Localization of mRNAs to the oocyte is common in Drosophila ovaries; Dubowy J et al.; Patterns of mRNA accumulation are sometimes dictated post-transcriptionally . Striking examples of mRNA localized to subcellular sites within cells have been described in animal oocytes, a variety of somatic cells in both animals and plants, and most recently in yeast (St Johnston, 1995; Bouget et al., 1996; Long et al., 1997; Takizawa et al., 1997) . Some of the best characterized of the localized mRNAs come from the Drosophila ovary, where localization of three mRNAs--bicoid, oskar and gurken--within the oocyte defines the anteroposterior and dorsoventral body axes (reviewed in St Johnston, 1995) . About 20 other mRNAs display a related pattern of localization, in which they become concentrated in the oocyte after transcription in the adjacent and interconnected nurse cells . Despite considerable work on the roles and mechanisms of mRNA localization, there have been no clear indications of the prevalence of this phenomenon, even in the much studied Drosophila system . Here we address this issue . We have examined the distributions of a random collection of individual Drosophila ovarian mRNAs, and find that a significant fraction, almost a tenth, are localized to the oocyte.

Bioessays, 1998 Jan, 20(1), 20 - 9
Cell morphogenesis in Arabidopsis; Hulskamp M et al.; Cell morphogenesis encompasses all processes required to establish a three-dimensional cell shape . Cells acquire the architecture specific to their developmental context by using the spatial information provided by internal or external cues . As a response to these signals, cells become reorganized and establish functionally distinct subcellular domains that ultimately lead to morphological changes . In its simplest form, cell morphogenesis results in the establishment of asymmetry along one axis, a cell polarity . Although cell polarity has been studied intensively in budding yeast and epithelial cells, little is known about more complex modes of cell morphogenesis involving multiple axes . In this review we compare the regulation of cell morphogenesis of different genetically well-characterized cell types in Arabidopsis thaliana.

Plant Cell, 1998 Feb, 10(2), 135 - 54
The AmMYB308 and AmMYB330 transcription factors from antirrhinum regulate phenylpropanoid and lignin biosynthesis in transgenic tobacco
Tamagnone L, Merida A, Parr A, Mackay S, Culianez-Macia FA, Roberts K, Martin C.
MYB-related transcription factors are known to regulate different branches of flavonoid metabolism in plants and are believed to play wider roles in the regulation of phenylpropanoid metabolism in general . Here, we demonstrate that overexpression of two MYB genes from Antirrhinum represses phenolic acid metabolism and lignin biosynthesis in transgenic tobacco plants . The inhibition of this branch of phenylpropanoid metabolism appears to be specific to AmMYB308 and AmMYB330, suggesting that they recognize their normal target genes in these transgenic plants . Experiments with yeast indicate that AmMYB308 can act as a very weak transcriptional activator so that overexpression may competitively inhibit the activity of stronger activators recognizing the same target motifs . The effects of the transcription factors on inhibition of phenolic acid metabolism resulted in complex modifications of the growth and development of the transgenic plants . The inhibition of monolignol production resulted in plants with at least 17% less lignin in their vascular tissue . This reduction is of importance when designing strategies for the genetic modification of woody crops.

Proc Natl Acad Sci U S A, 1998 Mar 3, 95(5), 2027 - 8
The rice genome project in japan
Sasaki T.
Since 1991, the Rice Genome Research Program in Japan has carried out rice genomics, such as large-scale cDNA analysis, construction of a fine-scale restriction fragment length polymorphism map, and physical mapping of the rice genome with yeast artificial chromosome clones . These studies have made a great impact on research into grass genomes and made rice a model plant for other cereal crop research . Starting in 1998, the Rice Genome Research Program will step into a new stage of genomics-that of genome sequencing . This project eventually should reveal all of the genomic sequence information in the rice plant and be an indispensable aid in understanding the genomics of other grass species.

Mutat Res, 1998 Jan 13, 412(1), 69 - 81
Analysis of the in vivo nitrosation capacity of the larvae used in the wing somatic mutation and recombination test of Drosophila melanogaster; Guzman Rincon J et al.; The in vivo nitrosation capacity of third-instar larvae of Drosophila melanogaster was assessed using the wing somatic mutation and recombination test (SMART) . Larvate derived from two different crosses, the standard cross (ST) and the high bioactivation cross (HB) both involving the recessive wing cell markers multiple wing hairs (mwh) and flare (flr3), were used . The HB cross is characterised by an increased cytochrome P450-dependent bioactivation capacity for promutagens and procarcinogens . The larvae were treated either with methyl urea, sodium nitrite or its combination . N-Nitrosomethylurea was used as a positive control . The wings of the resulting flies were analysed for the occurrence of mutant spots produced by various types of mutational events or by mitotic recombination . Methyl urea is negative in the ST and the HB cross, whereas sodium nitrite is weakly genotoxic in both crosses . However, the combination of both compounds produces highly increased frequencies of mutations and recombinations predominantly in the HB cross . The genotoxic effects produced by the combined treatments were considerably increased when mashed potatoes or an agar-yeast medium were used for the treatment instead of the standard instant medium . Treatment of larvae with the mixture resulting from the in vitro reaction of nitrosation precursors also resulted in high frequencies of induced spots comparable to those recorded with the potent genotoxin N-nitrosomethylurea . Further experiments showed that the genotoxic effect resulting from the in vivo exposure to nitrosation precursors can be reduced by co-treatment with catechin, a known nitrosation inhibitor . The present study demonstrates that the wing spot test is well suited for the determination of genotoxicity produced by in vivo nitrosation processes and for the study of their modulation by individual compounds or dietary complex mixtures.

J Med Genet, 1998 Feb, 35(2), 146 - 50
Characterisation of an inverted X chromosome (p11.2q21.3) associated with mental retardation using FISH; Sloan-Bena F et al.; We report on a patient with a pericentric inversion of the X chromosome, 46,Y,inv(X) (p11.2q21.3), who was referred for cytogenetic analysis because of mild mental retardation, short stature, prepubescent macro-orchidism, and submucous cleft palate . The same chromosomal abnormality was found in the proband's mother . The inverted X chromosome was late replicating in all the mother's lymphocytes studied, indicative of a likely unbalanced inversion . We show, by fluorescence in situ hybridisation (FISH) using a panel of ordered yeast artificial chromosome (YAC) clones, that the Xp breakpoint is localised in Xp11.23 between DXS146 and DXS255 and that the Xq breakpoint is assigned to the X-Y homologous region in Xq21.3 . YACs crossing the Xp and Xq breakpoints have been identified . One of these two breakpoints could be linked to the mental retardation in this patient as many non-specific mental retardation (MRX) loci have previously been located in the pericentromeric region of the X chromosome . Morever, the elucidation at the molecular level of this rearrangement will also indicate if cleft palate or prepubescent macro-orchidism, or both, in this boy are related to one of the two X breakpoints.

J Biochem Biophys Methods, 1997 Dec 17, 36(1), 51 - 61
Approaches to determine the specific role of the delta isoform of protein kinase C; Garrone B et al.; Two dimensional gel electrophoresis of proteins from HL-60 human leukaemia cells treated with bistratene A, a specific activator of protein kinase C (PKC) delta, was performed in conjunction with sequencing in order to identify components of the signal transduction pathway of this isoform of PKC . Stathmin (oncoprotein 18) was identified in this way and the phosphorylation of this protein after treatment with bistratene A, was confirmed by Western blotting of 2D gels . Since stathmin has phosphorylation sites for mitogen activated protein (MAP) kinases, cyclin dependent kinases and calcium/calmodulin dependent protein kinases, it is assumed that one of these enzymes, acting downstream from PKC delta, is responsible for the phosphorylation . Another approach to determining the role of PKC delta involves the identification of interacting proteins using the yeast two hybrid screen . The sequence of nine out of ten independently isolated clones from a two hybrid screen showed perfect homology to human ribosomal protein L8 . This protein has previously been shown to exist in complexes with ribosomal RNA, aminoacyl-tRNA and elongation factor-1 alpha, a known substrate of PKC delta, suggesting a role for PKC delta in protein synthesis regulation.

Br J Haematol, 1998 Mar, 100(3), 521 - 33
Correlation of cytogenetic, molecular cytogenetic, and clinical findings in 59 patients with ANLL or MDS and abnormalities of the short arm of chromosome 12; Streubel B et al.; Abnormalities of the short arm of chromosome 12 (12p) are found in about 5% of acute nonlymphocytic leukaemias (ANLL) and myelodysplastic syndromes (MDS) . They are described to be characteristic of secondary leukaemias, especially after prior mutagenic exposure, and to be associated with a poor prognosis . In our series of 59 patients with 12p abnormalities and ANLL or MDS, exposure to genotoxic agents was proven only in five patients, but in 13/44 patients ANLL evolved from an MDS . Patients with a small deletion del(12)(p11.2p13) having a mild clinical course were distinguished from those with a large del(12)(p11.2), additional chromosomal anomalies, and a poor clinical course . Among the 31 patients with translocations or dicentric chromosomes involving 12p, a group of eight with t/dic(12;13) was the most frequent and was associated with a poor prognosis . The clinical outcome was adverse in the majority of patients with complex karyotype abnormalities, but in some patients a milder clinical course seems likely . A new, hitherto undescribed, abnormality in an MDS case with a duplication dup(12)(p11.2p13) was the amplification of the signal of the yeast artificial chromosome (YAC) clone 964c10 (D12S736) . In 38 cases with deletions or unbalanced translocations/dicentrics one YAC signal was lost . Five patients with balanced translocations demonstrated breakpoints within the YAC, containing the ETV6 (TEL) gene . The breakpoints were telomeric to the YAC 964c10 in seven cases and centromeric in one patient.

Genomics, 1998 Feb 15, 48(1), 71 - 8
Genomic organization and promoter identification of the human protein kinase CK2 catalytic subunit alpha (CSNK2A1); Wirkner U et al.; The isolation and characterization of the complete gene coding for human protein kinase CK2 catalytic subunit alpha is described . The gene spans 70 kb and consists of 13 exons, and the exon/intron boundaries conform to the gt/ag rule . Exons range in size from 51 to 2960 bp, introns from 527 to around 34000 bp . The translation start site is located in Exon 2, the stop codon in Exon 13 . Two transcription start sites were identified by primer extension analysis: The further 5'-located site defines position 1 of the gene, the second site is located at position 50 . The 5' region of the CK2 alpha gene shows features of a housekeeping promoter, such as lack of a TATA box and presence of a CpG island and GC boxes . The region was analyzed by reporter gene assay, and promoter activity was detected within the region ranging from position -256 to 144 . Six potential polyadenylation signals were identified in the 3' noncoding region of the CK2 alpha gene . As indicated by comparison with expressed sequence tags from the EMBL databank and by Northern-blot analysis, the most 3' located, active polyadenylation signal seems to be the fourth signal, defining the end of the gene.

Genomics, 1998 Feb 15, 48(1), 12 - 23
Transcriptional map of the 2.5-Mb CBR-ERG region of chromosome 21 involved in Down syndrome; Dahmane N et al.; The region of chromosome 21 between genes CBR and ERG (CBR-ERG region), which spans 2.5 Mb on 21q22.2, has been defined by analysis of patients with partial trisomy 21 . It contributes significantly to the pathogenesis of many characteristics of Down syndrome, including morphological features, hypotonia, and mental retardation . Cosmid contigs covering 80% of the region were constructed and EcoRI maps produced . These cosmids were used for exon trapping and cDNA selection from three cDNA libraries (fetal brain, fetal liver, and adult skeletal muscle) . Isolated exons and cDNAs were mapped on the EcoRI map, organized into contigs, sequenced, and used as probes for Northern blot analysis of RNA from fetal and adult tissues . We identified 27 genuine or highly probable transcriptional units evenly distributed along the CBR-ERG region . Eight of the transcriptional units are known genes.

J Immunol Methods, 1997 Dec 15, 210(1), 93 - 101
Development of a two-site enzyme-linked immunosorbent assay for alpha-amylase from Aspergillus oryzae based on monoclonal antibodies; Sander I et al.; A two-site monoclonal antibody ELISA was developed to quantify the allergen Asp o 2 (alpha-amylase from Aspergillus oryzae) . Two mAbs recognizing distinct epitopes were selected, enriched by in vitro production in a modular minifermenter and affinity-purified . The first antibody was bound to microtiter plates which were then incubated with samples containing the allergen . Bound allergen was detected using a biotinylated second antibody and peroxidase-polymer-labelled streptavidin . The assay had a sensitivity of 0.6 ng/ml and did not react to high concentrations of wheat and rye flour or yeast proteins . The mAb ELISA will be useful in individual or epidemiological studies of baker's asthma to assess workplace allergen concentrations and the efficacy of allergen exposure prevention . It can be used as a standard assay for the quantification of alpha-amylase and the establishment and control of threshold limits in European bakeries.

Chem Pharm Bull (Tokyo), 1998 Feb, 46(2), 279 - 86
Studies on anti-inflammatory agents . VI . Synthesis and pharmacological properties of 2,3-diarylthiophenes; Tsuji K et al.; A series of novel 5-substituted-2,3-diarylthiophenes has been synthesized and found to be active in the rat adjuvant arthritis (AA) model and/or in the yeast-induced hyperalgesia (Randall-Selitto) assay . Among the compounds synthesized herein, 2-(4-fluorophenyl)-3-{4-(methylsulfonyl)phenyl}-5-(trifluoromethyl)thiop hene (6a) exhibited the most potent activities on AA, collagen-induced arthritis (CIA) and the delayed-type hypersensitivity response to type II collagen . 5-Bromo-2-{4-(methylamino)phenyl}-3-{4-(methylsulfinyl)phenyl}thiophene (38) is also a potent inhibitor of AA, CIA, hyperalgesia and in vitro tumor necrosis factor-alpha production.

Nat Genet, 1998 Mar, 18(3), 257 - 61
The meiotic checkpoint monitoring synapsis eliminates spermatocytes via p53-independent apoptosis; Odorisio T et al.; Evidence is accumulating that meiosis is subject to 'checkpoints' that monitor the quality of this complex process . In yeast, unresolved double strand breaks (DSBs) in DNA are thought to trigger a 'recombination checkpoint' that leads to pachytene arrest . In higher eukaryotes, there is evidence for a checkpoint that monitors chromosome synapsis and in mammals the most compelling evidence relates to the sex chromosomes . In normal male mice, there is synapsis between the X and Y pseudoautosomal regions; in XSxr(a)O mice, with a single asynaptic sex chromosome, there is arrest at the first meiotic metaphase, the arrested cells being eliminated by apoptosis (our unpublished data) . Satisfying the requirement for pseudoautosomal synapsis by providing a pairing partner for the XSxr(a) chromosome avoids this arrest . We have considered that this 'synapsis checkpoint' may be a modification of the yeast 'recombination checkpoint' with unresolved DSBs (a corollary of asynapsis) providing the trigger for apoptosis . DSBs induced by irradiation are known to trigger apoptosis in a number of cell types via a p53-dependent pathway, and we now show that irradiation-induced spermatogonial apoptosis is also p53-dependent . In contrast, the apoptotic elimination of spermatocytes with synaptic errors proved to be p53-independent.

Am J Obstet Gynecol, 1998 Feb, 178(2), 300 - 4
The effect of fluconazole on circulating ethinyl estradiol levels in women taking oral contraceptives; Sinofsky FE et al.; OBJECTIVE: This open-label, two-period, crossover study was conducted to evaluate the effect of a single 150 mg dose of fluconazole on the pharmacokinetics of ethinyl estradiol in healthy female subjects . STUDY DESIGN: Ten subjects regularly taking Ortho-Novum 7/7/7 (Ortho Pharmaceutical, Raritan, N.J.) and 10 subjects regularly taking Triphasil (Wyeth-Ayerst Laboratories, Philadelphia), which contain ethinyl estradiol 35 microg and 30 microg during days 1 to 6, respectively, were randomly assigned to receive a single 150 mg dose of fluconazole 2 hours before the oral contraceptive, on pill day 6 of one of two menstrual cycles . Ethinyl estradiol serum concentrations were measured at baseline and up to 24 hours after oral contraceptive intake . No fluconazole was administered during the other menstrual cycle, which served as the control . RESULTS: Mean serum concentrations of ethinyl estradiol were increased after fluconazole administration in both oral contraceptive groups . Maximum observed serum concentration and area under the concentration-time curve values were significantly (p < 0.05) greater during the fluconazole regimens (vs regimens without fluconazole) for both oral contraceptive groups and for combined values of the two oral contraceptive groups . The mean time to reach the maximum concentration was not altered by concomitant fluconazole administration . CONCLUSIONS: These findings suggest that there is a potential for a clinically significant interaction between coadministration of fluconazole and ethinyl estradiol in oral contraceptivesPIP: The effect of a single dose of the imidazole antifungal agent fluconazole on circulating ethinyl estradiol levels in oral contraceptive (OC) users was investigated in an open-label, two-period, crossover study . 10 regular users of Ortho-Novum 7/7/7 and 10 women regularly taking Triphasil were randomly assigned to receive 150 mg of fluconazole 2 hours before OC ingestion on pill day 6 of one of two menstrual cycles . Mean serum concentrations of ethinyl estradiol were increased after fluconazole administration in both OC groups . Maximum observed serum concentration and area under the concentration time curve values were significantly greater during the fluconazole cycle for both OC groups and for the combined values of the two groups (p 0.05) . The mean time to reach the maximum concentration was not altered by concomitant fluconazole administration . Mean serum ethinyl estradiol concentrations were greater at all time points in Ortho-Novum 7/7/7 users than Triphasil users . These findings suggest there is potential for a clinically significant drug interaction between co-administration of fluconazole and ethinyl estradiol . The most likely site of this drug interaction is the hepatic cytochrome P450 enzyme system . Since a single dose of 150 mg of fluconazole is standard treatment for common vaginal yeast infections, such concomitant therapy is likely in OC users at some point and its implications should be considered by prescribing clinicians .

J Mol Biol, 1998 Feb 6, 275(5), 719 - 24
Cytochrome c folding traps are not due solely to histidine-heme ligation: direct demonstration of a role for N-terminal amino group-heme ligation; Hammack B et al.; In previous work, heme ligation effects on the folding of cytochrome c have been attributed to histidine side-chains . A variant of yeast iso-1-cytochrome c designated TM, which lacks all histidine residues except His18, still shows evidence of denatured state heme ligation in the pH range between 5 and 6 where normally only histidine ligation is expected . Conversion of the N-terminal amino group of TM to a carbonyl group through a transamination reaction with glyoxylate produced a protein (ModTM) with no terminal amino group . The midpoint pH (pH1/2) for loss of heme ligation in 3 M guanidine-HCl shifts from 5.9 to 7.4 as a result of this modification, providing direct evidence for N-terminal amino group-heme ligation under these conditions . The N-terminal amino group thus competes with histidine for misligation of iso-1-cytochrome c under denaturing conditions . To assess the effect of denatured state N-terminal amino group-heme ligation on the folding of iso-1-cytochrome c, stopped-flow kinetics experiments were conducted . At pH 6.2, the major refolding lifetimes (3 M-->0.27 M guanidine-HCl) for ModTM, TM and the wild-type protein are 11.6 ms, 30 ms and 1.3 seconds, respectively . Denatured state ligation of the N-terminal amino group thus slows folding 2.6-fold .

Genome Res, 1998 Feb, 8(2), 146 - 57
Integrated YAC contig map of the Prader-Willi/Angelman region on chromosome 15q11-q13 with average STS spacing of 35 kb; Christian SL et al.; Prader-Willi syndrome and Angelman syndrome are associated with parent-of-origin-specific abnormalities of chromosome 15q11-q13, most frequently a deletion of an approximately 4-Mb region . Because of genomic imprinting, paternal deficiency of this region leads to PWS and maternal deficiency to AS . Additionally, this region is frequently involved in other chromosomal rearrangements including duplications, triplications, or supernumerary marker formation . A detailed physical map of this region is important for elucidating the genes and mechanisms involved in genomic imprinting, as well as for understanding the mechanism of recurrent chromosomal rearrangments . An initial YAC contig extended from D15S18 to D15S12 and was comprised of 23 YACs and 21 STSs providing an average resolution of about one STS per 200 kb . To close two gaps in this contig, YAC screening was performed using two STSs that flank the gap between D15S18 and 254B5R and three STSs located distal to the GABRA5-149A9L gap . Additionally, we developed 11 new STSs, including seven polymorphic markers . Although several groups have developed whole-genome genetic and radiation hybrid maps, the depth of coverage for 15q11-q13 has been somewhat limited and discrepancies in marker order exist between the maps . To resolve the inconsistencies and to provide a more detailed map order of STSs in this region, we have constructed an integrated YAC STS-based physical map of chromosome 15q11-q13 containing 118 YACs and 118 STSs, including 38 STRs and 49 genes/ESTs . Using an estimate of 4 Mb for the size of this region, the map provides an average STS spacing of 35 kb . This map provides a valuable resource for identification of disease genes localized to this region as well as a framework for complete DNA sequencing.

Genome Res, 1998 Feb, 8(2), 100 - 10
Physical and genetic mapping of the human X chromosome centromere: repression of recombination; Mahtani MM et al.; Classical genetic studies in Drosophila and yeast have shown that chromosome centromeres have a cis-acting ability to repress meiotic exchange in adjacent DNA . To determine whether a similar phenomenon exists at human centromeres, we measured the rate of meiotic recombination across the centromere of the human X chromosome . We have constructed a long-range physical map of centromeric alpha-satellite DNA (DXZ1) by pulsed-field gel analysis, as well as detailed meiotic maps of the pericentromeric region of the X chromosome in the CEPH family panel . By comparing these two maps, we determined that, in the proximal region of the X chromosome, a genetic distance of 0.57 cM exists between markers that span the centromere and are separated by at least the average 3600 kb physical distance mapped across the DXZ1 array . Therefore, the rate of meiotic exchange across the X chromosome centromere is <1 cM/6300 kb (and perhaps as low as 1 cM/17,000 kb on the basis of other physical mapping data), at least eightfold lower than the average rate of female recombination on the X chromosome and one of the lowest rates of exchange yet observed in the human genome.

J Biol Chem, 1998 Feb 20, 273(8), 4740 - 6
A rho-associated protein kinase, ROKalpha, binds insulin receptor substrate-1 and modulates insulin signaling; Farah S et al.; Insulin receptor substrate-1 (IRS-1) is phosphorylated on multiple tyrosine residues by ligand-activated insulin receptors . These tyrosine phosphorylation sites serve to dock several Src homology 2-containing signaling proteins . In addition, IRS-1 contains a pleckstrin homology domain and a phosphotyrosine binding domain (PTB) implicated in protein-protein and protein-lipid interactions . In a yeast two-hybrid screening using Xenopus IRS-1 (xIRS-1) pleckstrin homology-PTB domains as bait, we identified a Xenopus homolog of Rho-associated kinase alpha (xROKalpha) as a potential xIRS-1-binding protein . The original clone contained the carboxyl terminus of xROKalpha (xROK-C) including the putative Rho binding domain but lacking the amino-terminal kinase domain . Further analyses in yeast indicated that xROK-C bound to the putative PTB domain of xIRS-1 . Binding of xROK-C to xIRS-1 was confirmed in Xenopus oocytes after microinjection of mRNA corresponding to xROK-C . Furthermore, microinjection of xROK-C mRNA inhibited insulin-induced mitogen-activated protein kinase activation with a concomitant inhibition of oocyte maturation . In contrast, microinjection of xROK-C mRNA did not inhibit mitogen-activated protein kinase activation or oocyte maturation induced by progesterone or by microinjection of viral Ras (v-Ras) mRNA . These results suggest that xROKalpha may play a role in insulin signaling via a direct interaction with xIRS-1.

J Biol Chem, 1998 Feb 20, 273(8), 4616 - 21
Role of activating protein-1 in the regulation of the vascular cell adhesion molecule-1 gene expression by tumor necrosis factor-alpha; Ahmad M et al.; Endothelial cell surface expression of VCAM-1 is one of the initial steps in the pathogenesis of atherosclerosis . The inflammatory response transcription factor nuclear factor (NF)-kappaB plays an important role in the regulation of VCAM-1 expression by various stimuli including tumor necrosis factor (TNF)-alpha . Other transcription factors may modulate this response through interaction with NF-kappaB factors . Since c-Fos/c-Jun (activating protein-1 (AP-1)) are expressed in vascular endothelium during proinflammatory conditions, we investigated the role of AP-1 proteins in the expression of VCAM-1 by TNF-alpha in SV40 immortalized human microvascular endothelial cells (HMEC) . TNF-alpha induced expression of both early protooncogenes, c-fos and c-jun . The ability of TNF-alpha to activate the kappaB-motif (kappaL-kappaR)-dependent VCAM-1 promoter-chloramphenicol acetyltransferase (CAT) reporter gene lacking a consensus AP-1 element was markedly inhibited by co-transfection of the expression vector encoding c-fos ribozyme, which decreases the level of c-fos by degrading c-fos mRNA, or c-fos or c-jun oligonucleotides . Conversely, co-transfection of c-Fos and c-Jun encoding expression vectors potentiated the p65/NF-kappaB-mediated transactivation of the VCAM-1 promoter-CAT reporter gene . Furthermore the c-Fos encoding expression vector potentiated by 2-fold the transactivation activity of a chimeric transcriptional factor Gal/p65 (containing the transactivation domain of p65 and the DNA binding domain of the yeast transcriptional factor Gal-4) . Consistent with the promoter studies, curcumin and NDGA, inhibitors of AP-1 activation, markedly inhibited the ability of TNF-alpha to activate the expression of VCAM-1 mRNA levels at concentrations that did not inhibit the activation of NF-kappaB . In gel mobility supershift assays, the antibodies to c-Fos or c-Jun inhibited the binding of TNF-alpha-activated nuclear NF-kappaB to the kappaL-kappaR, suggesting that both c-Fos and c-Jun interacted with NF-kappaB . These results suggest that AP-1 proteins may mediate the effect of TNF-alpha in the regulation of VCAM-1 expression through interaction with NF-kappaB factors in endothelial cells.

J Biol Chem, 1998 Feb 20, 273(8), 4563 - 8
The TATA element and its context affect the cooperative interaction of TATA-binding protein with the TFIIB-related factor, TFIIIB70; Librizzi MD et al.; We have conducted a quantitative thermodynamic study of the effects of the TATA element and TATA-flanking sequences on the assembly of complexes containing TATA-binding protein (TBP) and the TFIIB-related factor, TFIIIB70 . TBP binds to the sequence TATAAAAG in the context of the yeast U6 gene (yU6 hybrid TATA) or the adenovirus major late promoter (AdMLP) with different affinities demonstrating that the sequence context of a TATA element contributes to TBP binding . We also determined the cooperative free energies of formation of TBP.TFIIIB70.DNA complexes on the yU6 TATA element, the yU6 hybrid TATA element and a nonconsensus TATA element . The yU6 hybrid TATA displayed a moderate, less than 5-fold, increase in TBP affinity similar to the 3-fold increase observed for the AdMLP . In contrast, the nonconsensus and yU6 TATAs increased the affinity of TBP for DNA 12- and 17-fold, respectively . Since the TBP-TFIIIB70 cooperativity is greater on lower affinity TATA boxes and most polymerase III genes contain low affinity "TATA boxes," we conclude that the cooperative binding of TFIIIB70 and TBP to DNA represents an important driving force in the assembly of polymerase III-specific transcription complexes . An effect of the sequences surrounding the TATA box was also observed on TBP-TFIIIB70 cooperativity . The mechanistic implications of the thermodynamic linkage between DNA sequence and binding cooperativity are discussed.

Curr Biol, 1998 Jan 15, 8(2), 121 - 4
Ubc9p and the conjugation of SUMO-1 to RanGAP1 and RanBP2; Saitoh H et al.; The yeast UBC9 gene encodes a protein with homology to the E2 ubiquitin-conjugating enzymes that mediate the attachment of ubiquitin to substrate proteins {1} . Depletion of Ubc9p arrests cells in G2 or early M phase and stabilizes B-type cyclins {1} . p18(Ubc9), the Xenopus homolog of Ubc9p, associates specifically with p88(RanGAP1) and p340(RanBP2) {2} . Ran-binding protein 2 (p340(RanBP2)) is a nuclear pore protein {3} {4}, and p88(RanGAP1) is a modified form of RanGAP1, a GTPase-activating protein for the small GTPase Ran {2} . It has recently been shown that mammalian RanGAP1 can be conjugated with SUMO-1, a small ubiquitin-related modifier {5-7}, and that SUMO-1 conjugation promotes RanGAP1's interaction with RanBP2 {2,5,6} . Here we show that p18(Ubc9) acts as an E2-like enzyme for SUMO-1 conjugation, but not for ubiquitin conjugation . This suggests that the SUMO-1 conjugation pathway is biochemically similar to the ubiquitin conjugation pathway but uses a distinct set of enzymes and regulatory mechanisms . We also show that p18(Ubc9) interacts specifically with the internal repeat domain of RanBP2, which is a substrate for SUMO-1 conjugation in Xenopus egg extracts.

Curr Biol, 1998 Jan 15, 8(2), 117 - 20
An oncogenic mutation uncouples the v-Jun oncoprotein from positive regulation by the SAPK/JNK pathway in vivo; May GH et al.; Stimulation of c-Jun transcriptional activity via phosphorylation mediated by the stress-activated or c-Jun amino-terminal (SAPK/JNK) subgroup of mitogen-activated protein kinases (MAP kinases) is thought to depend on a kinase-docking site (the delta region) within the amino-terminal activation domain, which is deleted from the oncogenic derivative, v-Jun {1} {2} {3} . This mutation markedly enhances v-Jun oncogenicity {4} {5}; however, its transcriptional consequences have not been resolved . In part, this reflects uncertainty as to whether binding of SAPK/JNK inhibits c-Jun function directly {6} {7} or, alternatively, serves to facilitate and maintain the specificity of positive regulatory phosphorylation {8} . Using a two-hybrid approach, we show that SAPK/JNK stimulates c-Jun transactivation in yeast and that this depends on both catalytic activity and physical interaction between the kinase and its substrate . Furthermore, c-Jun is active when tethered to DNA via SAPK/JNK, demonstrating that kinase binding does not preclude transactivation . Taken together, these results suggest that SAPK/JNK acts primarily as a positive regulator of c-Jun transactivation in situ, and that loss of the docking site physically uncouples v-Jun from this control . This loss-of-function model accounts for the deficit of v-Jun regulatory phosphorylation and repression of TPA response element (TRE)-dependent transcription observed in v-Jun-transformed cells and predicts that an important property of the oncoprotein is to antagonise SAPK/JNK-dependent gene expression.

Chromosoma, 1997 Dec, 106(8), 503 - 12
Organization of DNA sequences near the centromere of the Drosophila melanogaster Y chromosome; Losada A et al.; The structural analysis of a yeast artificial chromosome clone from Drosophila melanogaster enriched in dodecasatellite sequences has led us to find a new retrotransposon that we have called Circe . Moreover, this retrotransposon has allowed the isolation of a contig encompassing approximately 200 kb near the centromere of the Y chromosome, providing an entry point into a region from which very little sequence information has been obtained to date . The molecular characterization of the contig has shown the presence of HeT-A telomeric retrotransposons close to the centromere of the Y chromosome, suggesting a telocentric origin for this submetacentric chromosome.

Hum Mol Genet, 1998 Feb, 7(2), 299 - 305
The human FXY gene is located within Xp22.3: implications for evolution of the mammalian X chromosome; Perry J et al.; It has been proposed that the pseudoautosomal region of mammals has evolved by sequential addition of autosomal material onto the X and Y chromosomes followed by movement of the pseudoautosomal boundary to create X-unique regions . We have previously described a gene, Fxy , that spans the pseudoautosomal boundary in mice such that the first three exons of the gene are located on the X chromosome, but the remainder of the gene is located on both X and Y chromosomes . Therefore, this gene might be in a state of transition between pseudoautosomal and X-unique locations . In support of this theory we show here that the human FXY gene is located in Xp22.3 in humans, proximal to the pseudoautosomal boundary.

Hum Mol Genet, 1998 Feb, 7(2), 177 - 86
Molecular genetic analysis of autosomal dominant cerebellar ataxia with retinal degeneration (ADCA type II) caused by CAG triplet repeat expansion; Del-Favero J et al.; Autosomal dominant cerebellar ataxia with retinal degeneration (ADCAII) was previously mapped by linkage analysis studies to chromosome 3p12-p21.1 (SCA7) . Positional cloning efforts have recently identified a novel gene, SCA7 , containing a translated CAG repeat, expanded in SCA7 patients . We cloned the SCA7 gene from a yeast artificial chromosome (YAC) clone contig spanning the SCA7 candidate region . Using a combination of genomic sequencing and cosmid-based exon trapping, two expressed sequence tags were identified . Sequencing of the corresponding cDNA clones and RT-PCR analysis identified the full-length SCA7 cDNA . Together, our sequence data defined the intron/exon boundaries of the first two coding exons of the SCA7 gene, with the first exon containing the expanded CAG repeat . Further, sequence comparison with the published SCA7 cDNA identified one additional putative exon in the 5'-UTR region of the SCA7 gene . The SCA7 gene was mapped on the YAC contig in the 2.5 cM interval between D3S1600 and D3S1287 . In one extended Belgian SCA7 pedigree the expanded alleles ranged from 38 to at least 55 repeats with allele lengths being inversely correlated with onset age of ADCAII symptoms . The SCA7 repeats increased in length in successive generations . Normal alleles had from four to 18 repeats, with 10 repeats being the most common allele.

Hum Mol Genet, 1998 Feb, 7(2), 157 - 64
Comparative analysis of the phosphomannomutase genes PMM1, PMM2 and PMM2psi: the sequence variation in the processed pseudogene is a reflection of the mutations found in the functional gene; Schollen E et al.; The search for the carbohydrate-deficient glycoprotein syndrome type I (CDG1) gene has revealed the existence of a family of phosphomannomutase (PMM) genes in humans . Two expressed PMM genes, PMM1 and PMM2 , are located on chromosome bands 22q13 and 16p13, respectively, and a processed pseudogene PMM2 psi is located on chromosome 18p . Mutations in PMM2 are the cause of CDG type IA whereas no disorder has been associated with defects in PMM1 as yet . Here, we describe the genomic organization of these paralogous genes . There is a 65% identity of the coding sequence, and all intron/exon boundaries have been conserved . The processed pseudogene is more closely related to PMM2 . Remarkably, several base substitutions in PMM2 that are associated with disease are also present at the corresponding positions in the pseudogene . Thus, mutations that occur at a slow rate in the active gene in the population have also accumulated in the pseudogene.

Immunopharmacology, 1998 Jan, 38(3), 223 - 8
Mannan decelerates the clearance of human red blood cells in SCID mouse; Ishihara C et al.; Mannans and its related compounds decelerated human (Hu) red blood cell (RBC)-clearance in severe combined immunodeficiency (SCID) mice by inhibiting erythro-phagocytosis of macrophages . Chimeric SCID mice for Hu-RBC which are generated by repeated transfusions with mature Hu-RBCs are described recently as a model for Plasmodium falciparum infection, though the Hu-RBC clearance in the mice at present is very rapid and the parasitemia in the mice is only erratic . Here, we aimed to study the method to decelerate Hu-RBC clearance in SCID mice, to establish a suitable mouse model for malaria parasites . Yeast and Candida mannans as well as lactoferrin, a glycoprotein containing both oligomannoside- and N-acetyllactosamine-type glycans, decelerated Hu-RBC clearance, but instead other saccharides such as carboxymethyl chitin, N-acetylglucosamine, and D-glucose did not . Yeast mannan and lactoferrin interfered significantly with in vitro Hu-RBC-phagocytosis which was also inhibited by mannopentaose and mannotoriose . D-mannose exhibited a moderate inhibitory activity . N-acetyl-D-glucosamine, however, showed only a slight inhibitory activity, but D-glucose had no inhibitory activity on Hu-RBC phagocytosis . These results may postulate that Hu-RBC clearance in SCID mouse might be mediated by receptor-ligand binding by a macrophage lectin like receptor with mannose specificity.

Biochem Biophys Res Commun, 1998 Feb 24, 243(3), 688 - 93
CYP86A1 from Arabidopsis thaliana encodes a cytochrome P450-dependent fatty acid omega-hydroxylase; Benveniste I et al.; The A . thaliana EST database was screened using consensus motifs derived from P450 families CYP52 and CYP4 catalyzing the omega-hydroxylation of fatty acids and alkanes in Candida and in mammals . One EST cDNA fragment was detected in this way and the corresponding full-length cDNA was cloned from a cDNA library of A . thaliana . This cDNA coded the first member of a new plant P450 family and was termed CYP86A1 . The deduced peptide sequence showed highest homology with P450s from families 4 and 52 . To confirm the catalytic function, CYP86A1 was expressed in a yeast overexpressing its own NADPH-P450 reductase . Efficient expression was evidenced by spectrophotometry, SDS-PAGE and catalytic activity . CYP86A1 was found to catalyze the omega-hydroxylation of saturated and unsaturated fatty acids with chain lengths from C12 to C18 but not of hexadecane . Genomic organization analyzed by Southern blot suggested a single gene encoding CYP86A1 in A . thaliana.

J Virol, 1998 Mar, 72(3), 2213 - 23
Insect virus proteins (FALPE and p10) self-associate to form filaments in infected cells; Alaoui-Ismaili MH et al.; Entomopoxviruses and baculoviruses are pathogens of insects which replicate in the cytoplasm and nuclei of their host cells, respectively . During the late stages of infection, both groups of viruses produce occlusion bodies which serve to protect virions from the external environment . Immunofluorescence and electron microscopy studies have shown that large bundles of filaments are associated with these occlusion bodies . Entomopoxviruses produce cytoplasmic fibrils which appear to be composed of the filament-associated late protein of entomopoxviruses (FALPE) . Baculoviruses, on the other hand, yield filaments in the nuclei and cytoplasm of the infected cell which are composed of a protein called p10 . Despite significant differences in their sequences, FALPE and p10 have similar hydrophilicity profiles, and each has a proline-rich stretch of amino acids at its carboxyl terminus . Evidence that FALPE and p10 could produce filaments in the absence of other viral proteins is presented . When FALPE was expressed in insect cells from a recombinant baculovirus, filaments similar to those produced by the wild-type Amsacta moorei entomopoxvirus were observed . In addition, when expression plasmids containing FALPE or p10 genes were transfected into Vero monkey kidney cells, filament structures similar to those found in infected insect cells were produced . The manner in which FALPE and p10 subunits interact to form polymers was investigated through deletion and site-specific mutagenesis in conjunction with immunofluorescence microscopy, yeast two-hybrid protein interaction analysis, and chemical cross-linking of adjacent molecules . These studies indicated that the amino termini of FALPE and p10 were essential for subunit interaction . Although deletion of the carboxy termini did not affect this interaction, it did inhibit filament formation . In addition, modification of several potential sites for phosphorylation also abolished filament assembly . We concluded that although the sequences of FALPE and p10 were different, the structural and functional properties of the two polypeptides appeared to be similar.

J Virol, 1998 Mar, 72(3), 1737 - 43
Cloning and characterization of a novel hepatitis B virus x binding protein that inhibits viral replication; Melegari M et al.; The hepatitis B virus and the mammalian hepadnavirus genomes encode for a short open reading frame called x . Expression of the protein product (HBx) appears necessary for establishment of natural infection . However, in vitro studies have suggested a multifunctional role for HBx as an indirect transcriptional transactivator of a variety of different viral and cellular promoters . Indeed, HBx has no known direct DNA binding properties but may interact with transcription factors as well as activate intracellular signaling pathways associated with cell growth . To further address the possible functional role of HBx in the life cycle of hepatitis B virus, we performed an analysis using the yeast two-hybrid system to screen a cDNA library derived from a hepatocellular carcinoma cell line with a HBx fusion bait in an attempt to identify cellular partners that may bind to and alter the biologic properties of HBx . A HBx-interacting protein that specifically complexes with the carboxy terminus of wild-type HBx was identified and designated XIP . This 9.6-kDa protein is capable of binding to HBx in vitro, and transient and stable expression in hepatocellular carcinoma cells abolishes the transactivation properties of HBx on luciferase constructs driven by AP-1 and endogenous hepatitis B virus enhancer/promoter elements . Investigation of the role of XIP in hepatitis B virus replication in differentiated hepatocellular carcinoma cells revealed that XIP expression reduces wild-type hepatitis B virus replication to levels observed following transfection with an HBx-minus virus . In contrast, the replication levels of the duck hepatitis B virus, a hepadnavirus that lacks the x open reading frame, were unchanged in the context of XIP expression . We propose that one of the physiologic functions of the cellular protein XIP is to negatively regulate HBx activity and thus to alter the replication life cycle of the virus.

Exp Anim, 1998 Jan, 47(1), 69 - 73
Candida guilliermondii infection in SCID mice in association with the acceleration of the elimination of transfused human red blood cells; Ishihara C et al.; An acceleration of the elimination of transfused human (Hu) red blood cells (RBC) was found in C.B-17 scid (SCID) mice that were kept in our facility . Yeast like organisms were isolated from their tap water just as a pure culture and the two isolates SW5 and SW6 were assigned to be Candida guilliermondii by analysing their generic small subunit ribosomal RNA sequences . To test whether the isolates are infectious in mice, we inoculated SCID and BALB/c mice orally with SW5 and observed them for 63 and 48 days, respectively . The yeasts were frequently recovered from oral swabs, feces and their tap water throughout the experiment . Although none of the mice developed clinical signs or histopathological changes, a positive sero-conversion was confirmed in 4 of 5 SW5-inoculated BALB/c mice . Moreover, a significant acceleration of Hu-RBC elimination in all of the SW5-infected SCID mice was demonstrated . We believe this to be the first report of an inapparent but significant outbreak of C . guilliermondii infection in mice.

J Neurosci, 1998 Feb 15, 18(4), 1261 - 9
Interaction of huntingtin-associated protein with dynactin P150Glued; Li SH et al.; Huntingtin is the protein product of the gene for Huntington's disease (HD) and carries a polyglutamine repeat that is expanded in HD (>36 units) . Huntingtin-associated protein (HAP1) is a neuronal protein and binds to huntingtin in association with the polyglutamine repeat . Like huntingtin, HAP1 has been found to be a cytoplasmic protein associated with membranous organelles, suggesting the existence of a protein complex including HAP1, huntingtin, and other proteins . Using the yeast two-hybrid system, we found that HAP1 also binds to dynactin P150(Glued) (P150), an accessory protein for cytoplasmic dynein that participates in microtubule-dependent retrograde transport of membranous organelles . An in vitro binding assay showed that both huntingtin and P150 selectively bound to a glutathione transferase (GST)-HAP1 fusion protein . An immunoprecipitation assay demonstrated that P150 and huntingtin coprecipitated with HAP1 from rat brain cytosol . Western blot analysis revealed that HAP1 was enriched in rat brain microtubules and comigrated with P150 and huntingtin in sucrose gradients . Immunofluorescence showed that transfected HAP1 colocalized with P150 and huntingtin in human embryonic kidney (HEK) 293 cells . We propose that HAP1, P150, and huntingtin are present in a protein complex that may participate in dynein-dynactin-associated intracellular transport.

EMBO J, 1998 Feb 2, 17(3), 827 - 37
A helix-turn-helix structure unit in human centromere protein B (CENP-B); Iwahara J et al.; CENP-B has been suggested to organize arrays of centromere satellite DNA into a higher order structure which then directs centromere formation and kinetochore assembly in mammalian chromosomes . The N-terminal portion of CENP-B is a 15 kDa DNA binding domain (DBD) consisting of two repeating units, RP1 and RP2 . The DBD specifically binds to the CENP-B box sequence (17 bp) in centromere DNA . We determined the solution structure of human CENP-B DBD RP1 by multi-dimensional 1H, 13C and 15N NMR methods . The CENP-B DBD RP1 structure consists of four helices and has a helix-turn-helix structure . The overall folding is similar to those of some other eukaryotic DBDs, although significant sequence homology with these proteins was not found . The DBD of yeast RAP1, a telomere binding protein, is most similar to CENP-B DBD RP1 . We studied the interaction between CENP-B DBD RP1 and the CENP-B box by the use of NMR chemical shift perturbation . The results suggest that CENP-B DBD RP1 interacts with one of the essential regions of the CENP-B box DNA, mainly at the N-terminal basic region, the N-terminal portion of helix 2 and helix 3.

Nucleic Acids Res, 1998 Feb 1, 26(3), 847 - 53
Factors associated with the mammalian RNA polymerase II holoenzyme; Neish AS et al.; The RNA polymerase II (Pol II) holoenzyme in yeast is an essential transcriptional regulatory complex which has been defined by genetic and biochemical approaches . The mammalian counterpart to this complex, however, is less well defined . Experiments herein demonstrate that, along with Pol II and SRB proteins, proteins associated with transcriptional regulation as cofactors are associated with the Pol II holoenzyme . Earlier experiments have demonstrated that the breast cancer-associated tumor suppressor BRCA1 and the CREB binding protein (CBP) were associated with the holoenzyme complex . The protein related to CBP, the E1A-associated p300 protein, is shown in these experiments to be associated with the holoenzyme complex as well as the BRG1 subunit of the chromatin remodeling SWI/SNF complex . Importantly, the Pol II holoenzyme complex does not contain some factors previously reported as stoichiometric components of the holoenzyme complex, most notably the proteins which function in repair of damaged DNA, such as PCNA, RFC and RPA . The presence of the p300 coactivator and the chromatin-modifying BRG1 protein support a role for the Pol II holoenzyme as a key target for regulation by enhancer binding proteins.

Mol Biol Cell, 1998 Jan, 9(1), 63 - 73
Negative regulation of Cdc18 DNA replication protein by Cdc2; Lopez-Girona A et al.; Fission yeast Cdc18, a homologue of Cdc6 in budding yeast and metazoans, is periodically expressed during the S phase and required for activation of replication origins . Cdc18 overexpression induces DNA rereplication without mitosis, as does elimination of Cdc2-Cdc13 kinase during G2 phase . These findings suggest that illegitimate activation of origins may be prevented through inhibition of Cdc18 by Cdc2 . Consistent with this hypothesis, we report that Cdc18 interacts with Cdc2 in association with Cdc13 and Cig2 B-type cyclins in vivo . Cdc18 is phosphorylated by the associated Cdc2 in vitro . Mutation of a single phosphorylation site, T104A, activates Cdc18 in the rereplication assay . The cdc18-K9 mutation is suppressed by a cig2 mutation, providing genetic evidence that Cdc2-Cig2 kinase inhibits Cdc18 . Moreover, constitutive expression of Cig2 prevents rereplication in cells lacking Cdc13 . These findings identify Cdc18 as a key target of Cdc2-Cdc13 and Cdc2-Cig2 kinases in the mechanism that limits chromosomal DNA replication to once per cell cycle.

J Dent Res, 1998 Mar, 77(3), 496 - 502
Protein-to-protein interactions: criteria defining the assembly of the enamel organic matrix; Paine ML et al.; Enamel crystallites form in a protein matrix located proximal to the ameloblast cell layer . This unique organic extracellular matrix is constructed from structural protein components biosynthesized and secreted by ameloblasts . To date, three distinct classes of enamel matrix proteins have been cloned . These are the amelogenins, tuftelin, and ameloblastin, with recent data implicating ameloblastin gene expression during cementogenesis . The organic enamel extracellular matrix undergoes assembly to provide a three-dimensional array of protein domains that carry out the physiologic function of guiding enamel hydroxyapatite crystallite formation . Using the yeast two-hybrid system, we have surveyed these three known enamel gene products for their ability to direct self-assembly . We measured the capacity of the enamel gene products to direct protein-to-protein interactions, a characteristic of enamel proteins predicated to be required for self-assembly . We provide additional evidence for the self-assembly nature of amelogenin and tuftelin . Ameloblastin self-assembly could not be demonstrated, nor were protein-to-protein interactions observed between ameloblastin and either amelogenin or tuftelin . Within the limits of the yeast two-hybrid assay, these findings constrain the emerging model of enamel matrix assembly by helping to define the limits of enamel matrix protein-protein interactions that are believed to guide enamel mineral crystallite formation.

Scand J Immunol, 1998 Feb, 47(2), 152 - 8
Pityrosporum orbiculare-reactive T-cell lines in atopic dermatitis patients and healthy individuals; Tengvall Linder M et al.; The yeast Pityrosporum orbiculare is one of the factors that may contribute to atopic dermatitis (AD) . In the present study we compared the T-cell response to P . orbiculare in 12 AD patients with specific immunoglobulin (Ig)E antibodies (Ab) in serum against P . orbiculare with that of six non-atopic healthy controls . Freshly isolated peripheral blood mononuclear cells (PBMC) were cultured for 3 days in the presence of P . orbiculare extract . The proliferative response as measured by {3H}-thymidine incorporation was significantly higher in the AD patients than in the healthy controls (P < 0.05) . Furthermore, significantly higher levels of interleukin (IL)-5 (P < 0.05), as analyzed by ELISA, were produced by PBMC from the AD patients compared to the healthy controls . Pityrosporum orbiculare-reactive T-cell lines (TCL) established by P . orbiculare stimulation of PBMC for 11 days produced significantly higher levels of IL-4 and IL-5 after stimulation with anti-CD3 Ab and showed a higher IL-4/interferon (IFN)-gamma ratio (P < 0.05) in the AD patients compared to the healthy controls . The higher proliferative PBMC response to P . orbiculare and the Th2-like cytokine production by P . orbiculare-stimulated TCL from AD patients indicate that P . orbiculare may play a role in maintaining skin inflammation in AD.

J Endocrinol, 1998 Jan, 156(1), 77 - 82
Follistatin has a biphasic response but follicle-stimulating hormone is unchanged during an inflammatory episode in growing lambs; Phillips DJ et al.; The effects on plasma follistatin concentrations of an inflammatory episode, induced by the intrathoracic injection of yeast, were examined in growing lambs; this model results in acute loss of appetite, food intake and liveweight and the activation of the acute-phase pathway for several weeks as adjudged by the production of haptoglobin and other acute-phase proteins . In these animals (n = 8) there was a biphasic response in follistatin concentrations, with an initial 200% increase (P < 0.001) in follistatin within 24 h of injection of yeast . Thereafter, follistatin concentrations were depressed to 70% of pretreatment levels 48 h after injection (P < 0.01), followed by a gradual recovery of concentrations to pretreatment values . In another group of lambs (n = 16) that were feed-restricted to mimic the reduced food intakes and liveweight changes in the yeast-injected group, plasma follistatin was also reduced to around 70% of pretreatment levels (P < 0.01) within 1 day of the dietary regimen being implemented, followed by a gradual return to pretreatment values as food intakes were increased . Plasma follistatin correlated significantly (r = 0.57, P < 0.0001) with food intake, but not with liveweight changes . Plasma follistatin concentrations were unchanged in a third group fed ad libitum (n = 8), except during two periods when food intakes were significantly (P < 0.05) reduced, when follistatin concentrations also decreased (P < 0.01) . Plasma follicle-stimulating hormone (FSH) concentrations in the three groups of lambs were not significantly affected by the treatment regimes or changes in follistatin concentrations . These findings indicate that peripheral follistatin concentrations are modulated by both inflammatory and nutritional mechanisms, and that significant fluctuations in follistatin levels can occur without detectable perturbations in FSH secretion.

J Eukaryot Microbiol, 1998 Jan-Feb, 45(1), 105 - 11
Fibrillarin, a conserved pre-ribosomal RNA processing protein of Giardia; Narcisi EM et al.; The flagellated protozoan Giardia has been shown by 16S rRNA sequence analysis to be one of the most primitive of the eukaryotes . A gene encoding the protein fibrillarin, a pre-rRNA processing protein implicated in rRNA methylation and ribosome assembly, has been isolated . A genomic DNA fragment 1,240 base pairs long containing an open reading frame of 981 base pairs (327 amino acids) was sequenced . The deduced protein sequence of 35.3 kDa is similar to other known fibrillarin sequences . The Giardia sequence includes the amino terminal glycine/arginine rich domain characteristic of eukaryotic fibrillarins but is unique in having a large number of acidic residues in this domain . Phylogenetic analysis of the available fibrillarin sequences is consistent with the assignment of Giardia to a position close to the most primitive of the eukaryotes . A monoclonal antibody to yeast fibrillarin crossreacts with a 36 kDa polypeptide from Giardia on western blots and diffusely stains both nuclei of the organism by immunofluorescence microscopy . This result is consistent with the absence of well defined nucleoli in this organism . The evolutionary conservation of fibrillarin suggests an important function for this protein in ribosome biosynthesis, and this function appears to be maintained from the archaebacteria, which lack a nucleus, to Giardia, which contains a nucleus but lacks a prominent nucleolus, to higher mammals, which have both nucleus and nucleolus.

Ann Acad Med Singapore, 1997 Sep, 26(5), 671 - 81
Prevention and control of hepatitis B virus infection in Singapore; Goh KT; Hepatitis B virus (HBV) accounted for 24% to 54% of the reported acute viral hepatitis cases in Singapore from 1982 to 1996 . The prevalence of HBV infection, as indicated by the presence of markers of HBV, increased from 9.3% in children below 5 years of age to 54.6% in adults above 55 years . The overall hepatitis B surface antigen (HBsAg) prevalence was 5.7% for males and 3.4% for females, with the highest rate among the Chinese . About 39% of the HBsAg carriers were hepatitis B 'e' antigen positive . The main mode of transmission during the first year of life was perinatal, with 43% of the babies born to HBsAg-positive mothers developing the carrier state . Horizontal transmission within the infected household was significantly associated with sharing of personal and household articles . Based on the findings of seroprevalence surveys in various population groups and clinical trials on the safety, immunogenicity and efficacy of various doses and schedules with the plasma-based and yeast-derived hepatitis B vaccines in newborn babies, a national childhood hepatitis B vaccination programme was formulated and implemented in phases, starting with babies born to carrier mothers on 1 October 1985 and finally extending to all newborns on 1 September 1987 . The hepatitis B prevention and control programme has been successful . During the period 1994 to 1996, more than 90% of children completed the full schedule of immunisation by below one year of age, and 85% had evidence of vaccination at school entry at age six . Follow-up of 2 cohorts of vaccinated children showed that perinatal transmission has been reduced by 80% to 100% . Horizontal transmission has also declined through other public health measures . The efficacy of the hepatitis B vaccine and the adequacy of reduced doses in the long-term protection of chronic carrier state have been shown in children and adults . The incidence of acute hepatitis B has declined from 10.4 per 100,000 in 1985 to 4.8 per 100,000 in 1996 . There is a noticeable reduction in HBsAg prevalence in selected population (school children, national servicemen and antenatal women) . The age-standardised incidence rate of primary liver cancer among males had also dropped from 27.8 per 100,000 per year during 1978 to 1982 to 19.0 per 100,000 per year during 1988 to 1992.

J Exp Zool, 1998 Mar 1, 280(4), 288 - 303
The M31 gene has a complex developmentally regulated expression profile and may encode alternative protein products that possess diverse subcellular localisation patterns; Peterson K et al.; HP1-like chromobox genes comprise an evolutionarily conserved family of genes that encode components of centromeric heterochromatin . In order to investigate the role of the murine HP1-like gene, M31, in heterochromatin formation we have isolated its gene and characterised its transcripts and protein products . PCR products that represent M31 transcripts were detected at the one-cell stage and were maternal in origin . Maternal provision of M31 transcripts may reflect a need for M31 in the formation of a functional centromere in order that there is proper segregation of chromosomes during the early cleavage divisions; studies in fission yeast and Drosophila have suggested a crucial role for HP1-like genes in centromere function . There are three protein products encoded by the M31 gene . Surprisingly, the two smaller products are found almost exclusively in the cytoplasm.

Structure, 1998 Jan 15, 6(1), 39 - 50
The mechanism of regulation of hexokinase: new insights from the crystal structure of recombinant human brain hexokinase complexed with glucose and glucose-6-phosphate; Aleshin AE et al.; BACKGROUND: Hexokinase I is the pacemaker of glycolysis in brain tissue . The type I isozyme exhibits unique regulatory properties in that physiological levels of phosphate relieve potent inhibition by the product, glucose-6-phosphate (Gluc-6-P) . The 100 kDa polypeptide chain of hexokinase I consists of a C-terminal (catalytic) domain and an N-terminal (regulatory) domain . Structures of ligated hexokinase I should provide a basis for understanding mechanisms of catalysis and regulation at an atomic level . RESULTS: The complex of human hexokinase I with glucose and Gluc-6-P (determined to 2.8 A resolution) is a dimer with twofold molecular symmetry . The N- and C-terminal domains of one monomer interact with the C- and N-terminal domains, respectively, of the symmetry-related monomer . The two domains of a monomer are connected by a single alpha helix and each have the fold of yeast hexokinase . Salt links between a possible cation-binding loop of the N-terminal domain and a loop of the C-terminal domain may be important to regulation . Each domain binds single glucose and Gluc-6-P molecules in proximity to each other . The 6-phosphoryl group of bound Gluc-6-P at the C-terminal domain occupies the putative binding site for ATP, whereas the 6-phosphoryl group at the N-terminal domain may overlap the binding site for phosphate . CONCLUSIONS: The binding synergism of glucose and Gluc-6-P probably arises out of the mutual stabilization of a common (glucose-bound) conformation of hexokinase I . Conformational changes in the N-terminal domain in response to glucose, phosphate, and/or Gluc-6-P may influence the binding of ATP to the C-terminal domain.

Essays Biochem, 1997, 32, 1 - 16
Mammalian MAP kinase modules: how to transduce specific signals; Brunet A et al.; MAPK modules are composed of a cascade of three intracellular protein kinases (MKKK, MKK and MAPK) which are activated successively by phosphorylation events . They are used to transduce a variety of information in organisms as diverse as yeasts, worms, flies or mammals . MAPK modules integrate signals coming from membrane receptor activation and, by the ability of MAPK to translocate into the nucleus and phosphorylate nuclear targets such as transcription factors, they relay extracellular signals into a genomic response . Since several MAPK modules transducing different information are expressed in the same cell, in yeast or in mammals, the question arises as to how fidelity is maintained between the distinct MAPK modules of a single cell . Two levels of specificity have been documented: the molecular selectivity of each enzyme for its substrate, which is particularly evident for the MKK-MAPK couple, permits specificity within one particular module; exogenous proteins, such as the yeast Ste5 protein, may serve as 'chaperone' proteins to tether all the members of a module and restrict signal transduction to this module . In mammalian cells, the MAPK modules are not strictly independent and one pathway may interfere with another . It remains to be determined whether this interference is of physiological relevance.

Genes Chromosomes Cancer, 1998 Feb, 21(2), 160 - 5
Characterization of the breakpoints in a t(8;13)(p11;q12) translocation from a patient with myeloproliferative disease using fluorescence in situ hybridization; Chernova O et al.; We used fluorescence in situ hybridization to characterize the molecular position of the breakpoints in a t(8;13)(p11;q12) reciprocal translocation from a patient with an atypical myeloproliferative disorder . This structural chromosome abnormality is characteristic of this specific disease and occurs often as the only chromosome abnormality in the malignant cells . Yeast artificial chromosome (YAC) analysis has demonstrated that the 8p11 breakpoint lies within a region defined by YAC 959A4 and that the 13q12 breakpoint is spanned by YAC 769F9 . Identifying the position of the breakpoints in this rearrangement provides the means to search for candidate genes rearranged by this highly specific structural chromosome abnormality.

Genes Chromosomes Cancer, 1998 Feb, 21(2), 152 - 9
Fish mapping of YAC clones at human chromosomal band 7q31.2: identification of YACS spanning FRA7G within the common region of LOH in breast and prostate cancer; Huang H et al.; Loss of DNA sequences within human chromosomal band 7q31.2 is frequently observed in a number of different solid tumors including breast, prostate, and ovarian cancer . This chromosomal band also contains the common fragile site, FRA7G . Many of the common fragile sites occur within chromosomal regions that are frequently deleted during tumor formation but their precise position, relative to the chromosome breakpoints and deletions, has not been defined for the majority of the fragile sites . Because the frequency of expression of FRA7G is low, we analyzed the expression of FRA7G in a chromosome 7-only somatic cell hybrid (hamster-human) . YAC clones defining a contig spanning 7q31.2 were then used as FISH probes against metaphase spreads prepared from the hybrid cells after aphidicolin induction . This analysis quickly revealed whether a specific YAC clone mapped proximal, distal, or actually spanned the region of decondensation/breakage of FRA7G . By using this approach, we have identified several overlapping YAC clones that clearly span FRA7G . Interestingly, these clones map precisely to the common region of LOH in breast cancer and prostate cancer . In addition, the MET oncogene is contained within the three YACs that span FRA7G.

Genes Chromosomes Cancer, 1998 Feb, 21(2), 113 - 8
Deletion 10q23.2-q23.33 in a patient with gastrointestinal juvenile polyposis and other features of a Cowden-like syndrome; Tsuchiya KD et al.; A cytogenetically visible interstitial deletion of chromosome band 10q23 was found in a 6-year-old boy with mental retardation, dysmorphic features, and juvenile polyposis coli . In order to map this patient's deletion physically, we performed fluorescence in situ hybridization by using yeast artificial chromosomes (YACs) in the vicinity of the deletion . Five YACs that span an 11-15 cM region within the deletion were identified . This patient's deletion contains the putative locus for Cowden syndrome and a recently discovered candidate tumor suppressor gene (MMAC1 or PTEN) that has been implicated in the progression of a variety of human malignancies . Furthermore, the deletion is near and possibly overlaps a locus associated with juvenile polyposis . The findings in this patient with a constitutional 10q23 deletion raise the issue of whether there are separate genes in this region that are involved in Cowden syndrome, Bannayan-Riley-Ruvalcaba syndrome, juvenile polyposis, and tumor progression, or whether all of these entities could be due to a single gene.

Genes Chromosomes Cancer, 1998 Feb, 21(2), 75 - 81
Refinement of the commonly deleted segment in myeloid leukemias with a del(20q); Wang PW et al.; A deletion of the long arm of chromosome 20 {del(20q)} is a recurring abnormality in a wide spectrum of myeloid disorders . Loss of genetic material from 20q may confer a proliferative advantage to myeloid cells, possibly through loss of function of a tumor suppressor gene . Previously, we analyzed leukemia cells from 19 patients with a del(20q) by fluorescence in situ hybridization (FISH) and identified a segment that was deleted in 95% of all patients examined . The deleted interval extended from 20q11.2 to q12, spanned approximately 13 Mb, and was flanked proximally by RPN2 and distally by D20S17 . To narrow the commonly deleted segment and facilitate the identification of candidate genes, we have employed molecular approaches in combination with FISH . By using 21 microsatellite markers positioned in a recently generated physical map of 20q, we performed allele loss studies in myeloid leukemia cells from 23 patients with a del(20q) . The results of these studies allowed us to delineate a new proximal border, flanked by marker D20S206 . By FISH analysis of additional leukemia samples from patients with a del(20q), we have also delineated a new distal boundary between markers D20S119 and UT654 . As a result of the redesignation of both the proximal and distal boundaries, we have successfully narrowed the commonly deleted segment within 20q12 to a region spanning approximately 8 Mb . Identification of the smallest deleted segment will facilitate the eventual cloning of a candidate myeloid tumor suppressor gene.

Dev Dyn, 1998 Feb, 211(2), 123 - 30
Characterization of cellular nucleic acid binding protein from Xenopus laevis: expression in all three germ layers during early development; Flink IL et al.; The Xenopus CNBP homologue (XCNBP) has been cloned from stage 14 neurula . XCNBP encodes a 18.4-kDa protein containing seven highly conserved zinc finger (Zn-finger) repeats (CX2CX4HX4CX2), with sequence similarity to human, mouse, rat, and yeast CNBP . A unique feature of XCNBP is that it contains a 10 amino acid (aa) deletion in the linker region between Zn-fingers 1 and 2, immediately downstream from an alternatively spliced exon of human CNBP isoforms . A similar deletion is found in mouse and yeast CNBP proteins . The deleted region lacks potential PEST and casein kinase II phosphorylation sites . Because CNBP proteins from a variety of species contain deletions in a similar region, these results suggest that the pattern of alternative processing of CNBP isoforms is highly conserved among metazoa and unicellular eukaryotes . XCNBP RNA is initially maternally derived and is widely expressed throughout early development at the gastrula, neurula, and tailbud stages . At the early gastrula stage, XCNBP is expressed in ectodermal, endodermal, and mesodermal germ layers . Previous data have demonstrated the presence of CNBP in the cytoplasm and nucleus . The interactions of CNBP with single-stranded DNA and RNA suggest that CNBP may serve dual functions in transcriptional and translational regulation in a wide variety of tissues during development.

Tsitologiia, 1997, 39(7), 552 - 9
{The effect of biologically active compounds on lysosome fusion with phagosomes and the F-actin content in mouse peritoneal macrophages and on the status of the lysosomal membranes in mouse hepatocytes}; Mozhenok TP et al.; Effects of biologically active compounds bilirubin (BR, 0.1 and 0.2 mM), chelerythrine (CR, 0.1 and 0.5 mM) and farmorubicin (FR, 0.6 and 6.0 mM) on the phagosome-lysosome fusion (P-LF) were studied using fluorescent dye acridine orange for lysosomal labelling and yeast cells as a target . To investigate mechanisms of these effects, changes in fluidity of lysosomal membranes from murine liver were studied by measuring of fluorescence intensity, lifetime and polarization of the fluorescent membrane probes: DPH (1,6-diphenyl-1,3,5-hexatriene) and TMA-DPH {1-(4-triphenylamino)-6-phenyl-1,3,5-hexatriene} incorporated in isolated murine liver lysosomes . In order to characterize the induced cytoskeleton changes, the F-actin content in murine peritoneal macrophages was determined . It was found that all three compounds tested enhanced P-LF . Our results demonstrate that BR induces a decrease in DPH and TMA-DPH fluorescence polarization, FR increases DPH and TMA-DPH fluorescence polarization, and CR causes an increase in TMA-DPH fluorescence polarization in lysosomal membranes . All the three compounds increase F-actin content in peritoneal macrophages . Thus, the action of BR extended on P-LF is associated with increasing lysosomal membranes fluidity and cytoskeleton changes . The enhancement of P-LF under the action of FR and CR can be most likely explained by changes of cytoskeleton.

Hum Genet, 1998 Jan, 102(1), 63 - 8
Cloning and tissue expression of cDNAs from chromosome 5q21-22 which is frequently deleted in advanced lung cancer; Ueno K et al.; Previously, we have reported that the inactivation of putative tumor-suppressor gene(s) on chromosome 5q21-22 may play an important role in the progression of lung cancer . Here, we describe the establishment of a yeast artificial chromosome (YAC) contig that spans 8-10 Mb at the 5q21-22 region . Six cosmid contigs have also been established in this YAC contig . About 35 exon-like fragments have been detected by exon-amplification, direct screening, cross-species hybridization, and searches of a database . Thus far, 14 cDNAs have been isolated, and two of them coincide with known genes, viz., cysteine dioxygenase I and geranylgeranyltransferase I . The other 12 cDNAs are considered to be novel genes . Two of these novel cDNA show partial homology to known genes, viz., semaphorin CD100 and the 28S rRNA gene . In addition, four known genes, including APC (adenomatous polyposis coli), MCC (mutated in colorectal cancer), proto-oncogene tyrosine kinase FER, and genomic imprinted gene U2AF1-RS1, have also been mapped in this contig . This large contig and expression map should prove crucial in the identification of susceptibility gene(s) related to the progression of lung cancer.

Hum Genet, 1998 Jan, 102(1), 57 - 62
Molecular screening of Batten disease: identification of a missense mutation (E295K) in the CLN3 gene; Zhong N et al.; Batten disease, the juvenile form of neuronal ceroid lipofuscinosis, is a prevalent neuron degenerative disorder of childhood . A 1.02-kb genomic deletion in the Batten disease gene CLN3 has been determined to be a common mutation . We developed a PCR method to screen for this deletion and tested 43 Batten disease probands . We found 36% (31/86) of Batten disease chromosomes did not carry the 1.02-kb deletion . Of the three heterozygotes for the 1.02-kb deletion, a novel G-to-A missense mutation at nucleotide 1020 of the CLN3 cDNA sequence was found on two of the non-1.02-kb deletion chromosomes . The missense mutation resulted in a substitution of glutamic acid (E) by lysine (K) at position 295 (E295 K) . The E295 K mutation causes a change in predicted local protein conformation . This glutamic acid is a highly conserved acidic amino acid, being present in human, mouse, dog and yeast, which suggests it may play an important role in the function of the Batten disease protein.

Hum Genet, 1998 Jan, 102(1), 103 - 6
Juvenile open angle glaucoma: fine mapping of the TIGR gene to 1q24.3-q25.2 and mutation analysis; Michels-Rautenstrauss KG et al.; Autosomal dominant juvenile open angle glaucoma (JOAG) is an early-onset form of primary open angle glaucoma (POAG), which has been linked to chromosome 1q21-q31 . Recently, mutations in the trabecular meshwork inducible glucocorticoid response gene (TIGR), one of the candidate genes mapped in this region, were identified in glaucoma patients of several families . We screened for mutations of the TIGR gene in two German families with JOAG and in 100 unselected sporadic cases of POAG . In the first family we identified a Pro370Leu mutation and in the second family a Gly367Arg mutation cosegregating with the glaucoma phenotype . No pathogenic mutation was found in 100 sporadic cases but a Tyr347Tyr polymorphism was found in two patients . Furthermore, fluorescence in situ hybridization (FISH) analysis was used to map a TIGR-specific yeast artificial chromosome to 1q24.3-q25.2.

Cancer Immunol Immunother, 1998 Feb, 45(6), 321 - 6
Parameters for using mannan-MUC1 fusion protein to induce cellular immunity; Pietersz GA et al.; We have previously reported preclinical studies in mice of the human mucin 1 (MUC1) antigen covalently linked to the yeast cell-wall mannan polysaccharide (MFP), and shown strong cellular responses of the T1 type using mice . We now describe the optimum parameters for administration of MFP to obtain cellular immunity {as measured by the cytotoxic T cell precursor (CTLp) frequency} . In dose/response studies, in which 1 microg-150 microg was given by the i.p . route, it was clear that doses of 1-7 microg led to cellular and not humoral immunity; at doses above 7 microg humoral immunity prevailed with little cellular immunity increasing doses giving greater amounts of antibody . The most favoured routes of administration were intraperitoneal or intradermal immunisation, which were substantially better than i.m., i.v.; s.c . administration was the worst . Three immunisations were necessary for a maximum cellular response, further immunisation decreasing the CTLp frequency . Six different adjuvants were used with MFP {complete and incomplete Freund's adjuvant (CFA, IFA) Alum, Adjuprime, muramyl dipeptide (MDP) and glutaminyl-muramyl dipeptide (GMDP)}; Alum, GMDP, MDP and IFA moderately increased the CTLp frequency, IFA being the best . Even though preclinical studies of the immunogen in mice may not necessarily mirror the behaviour of the immunogen in humans, these studies demonstrate the factors to be taken into account for phase I/II clinical trials.

Eur J Biochem, 1998 Feb 1, 251(3), 583 - 9
The PC motif: a novel and evolutionarily conserved sequence involved in interaction between p40phox and p67phox, SH3 domain-containing cytosolic factors of the phagocyte NADPH oxidase; Nakamura R et al.; The superoxide-generating NADPH oxidase, dormant in resting phagocytes, is activated during phagocytosis following assembly of the membrane-integrated protein cytochrome b558 and cytosolic factors . Among the latter are the three proteins containing Src homology 3 (SH3) domains, p67phox, p47phox and p40phox . While the first two factors are indispensable for the activity, p40phox is tightly associated with p67phox in resting cells and is suggested to have some modulatory role . Here we describe a systematic analysis of the interaction between p40phox and p67phox using the yeast two-hybrid system and in vitro binding assays with recombinant proteins . Both methods unequivocally showed that the minimum requirements for stable interaction are the C-terminal region of p40phox and the region between the two SH3 domains of p67phox . This interaction is maintained even in the presence of anionic amphiphiles used for the activation of the NADPH oxidase, raising a possibility that it mediates constitutive association of the two factors in both resting and activated cells . The C-terminal region of p40phox responsible for the interaction contains a characteristic stretch of amino acids designated as the PC motif, that also exists in other signal-transducing proteins from yeast to human . Intensive site-directed mutagenesis to the motif in p40phox revealed that it plays a critical role in the binding to p67phox . Thus the PC motif appears to represent a novel module for protein-protein interaction used in a variety of signaling pathways.

Cell, 1998 Jan 9, 92(1), 51 - 61
A novel CNS gene required for neuronal migration and involved in X-linked subcortical laminar heterotopia and lissencephaly syndrome; des Portes V et al.; X-SCLH/LIS syndrome is a neuronal migration disorder with disruption of the six-layered neocortex . It consists of subcortical laminar heterotopia (SCLH, band heterotopia, or double cortex) in females and lissencephaly (LIS) in males, leading to epilepsy and cognitive impairment . We report the characterization of a novel CNS gene encoding a 40 kDa predicted protein that we named Doublecortin and the identification of mutations in four unrelated X-SCLH/LIS cases . The predicted protein shares significant homology with the N-terminal segment of a protein containing a protein kinase domain at its C-terminal part . This novel gene is highly expressed during brain development, mainly in fetal neurons including precursors . The complete disorganization observed in lissencephaly and heterotopia thus seems to reflect a failure of early events associated with neuron dispersion.

Mol Microbiol, 1998 Feb, 27(3), 541 - 51
Heat shock inducibility of an archaeal TATA-like promoter is controlled by adjacent sequence elements; Thompson DK et al.; The expression of a heat-inducible cct1 (chaperonin-containing Tcp-1) family member gene is regulated at the transcription level in the archaeon Haloferax volcanii . Transcriptional fusions of the cct1 promoter region with a yeast proline tRNA reporter gene were constructed to analyse the functional domains of this archaeal heat shock promoter . Both basal and heat-induced transcription of the reporter gene was directed by an archaeal consensus TATA element (5'-TTTATA-3') centred 25bp upstream of the transcription start site . Deletion mutagenesis indicated that the 5' boundary of the cct1 regulatory region mapped to position -37 . Nucleotide alignment with the 5' flanking regions of two additional cct-related genes identified in H . volcanii showed a high degree of sequence conservation between positions +1 and -37, especially in and immediately surrounding the TATA element of the putative core promoter . Mutational analysis of conserved sequences demonstrated that basal and heat-induced transcription required sequence elements located upstream and downstream of the TATA-box . These findings indicate that the regulatory sequences involved in heat-induced transcription lie within the core promoter region and suggest that the mechanism controlling heat shock gene expression in H . volcanii differs from the bacterial and eukaryal strategies.

Blood, 1998 Feb 15, 91(4), 1382 - 90
Detailed molecular delineation of 13q14.3 loss in B-cell chronic lymphocytic leukemia; Corcoran MM et al.; A region of chromosome 13q14.3, telomeric to the Retinoblastoma gene RB-1 is frequently deleted in patients with B-cell chronic lymphocytic leukemia (B-CLL) . A cosmid and P1-derived artificial chromosome (PAC) contig spanning over 600 kb has been constructed, which encompasses this locus . The contig clones have been used to order a number of markers along the minimally deleted region and to localize a series of CpG islands corresponding to possible candidate genes . A novel polymorphic dinucleotide repeat, 6E3.2, present in one of the ordered cosmid clones has been isolated for use in deletion mapping studies of patient DNA . Leukemic samples from 229 CLL patients have been screened for loss of heterozygosity using microsatellite markers and analyzed for hemizygous and homozygous deletions by Southern blot techniques using genomic probes selected from cosmids across the region . Hemizygous deletions were found in 31% of cases with an additional 10% showing homozygous loss . The use of these probes has defined the commonly deleted area to less than 130 kb, centromeric to the locus D13S272.

Blood, 1998 Feb 15, 91(4), 1295 - 303
Human signaling protein 14-3-3zeta interacts with platelet glycoprotein Ib subunits Ibalpha and Ibbeta; Calverley DC et al.; The initiation of primary hemostasis is mediated by interaction of the platelet glycoprotein Ib (GPIb) surface receptor and its arterial subendothelial von Willebrand factor (vWF) ligand . The intracellular signaling immediately following GPIb receptor occupancy connecting the adhesive event to platelet activation and aggregation has not been well characterized . The 14-3-3 proteins are a 27- to 30-kD ubiquitous protein family with diverse biologic roles, including functional modulation of several prominent signaling proteins . We used the yeast two-hybrid system and confocal microscopy to characterize the recently described interaction between GPIb and platelet 14-3-3zeta, and provide evidence for the potential signaling role of this protein . Two-hybrid interactions suggest that platelet 14-3-3zeta associates with the cytoplasmic domain of GPIb subunits Ibalpha and Ibbeta in transformed yeast cells . The 14-3-3 interaction with GPIbbeta may be partly mediated through the latter's phosphorylated serine 166 residue as its mutagenesis results in 20% to 40% reduced interaction . There was 51% to 59% reduced interaction between GPIb and three 14-3-3zeta deletion mutants compared with full-length 14-3-3zeta, suggesting that either the N-terminal dimerization or membrane-binding domains or more than one noncontiguous 14-3-3zeta element may be required for optimal GPIb interaction . Confocal studies of platelets and a megakaryocyte cell line provided additional evidence for interaction of 14-3-3zeta with GPIbalpha and GPIbbeta . We also found that, similar to the signaling mediators phosphatidylinositol 3-kinase and Src, platelet cytoskeletal 14-3-3zeta content is increased following vWF and ristocetin stimulation . We suggest that platelet 14-3-3zeta interacts with GPIbalpha and Ibbeta, that this interaction may be partly mediated through phosphoserine recognition, and that 14-3-3zeta cytoskeletal translocation may serve as a GPIb post-receptor occupancy signaling event.

J Mol Evol, 1998 Feb, 46(2), 240 - 4
An alphoid-like satellite DNA sequence is present in the genome of a lacertid lizard; Capriglione T et al.; A PstI DNA family was isolated from the genome of a lacertid, Lacerta graeca . The 185-bp monomeric unit (pGPS) was cloned and hybridized to DNAs and chromosomes of several lacertid species . The data showed that pGPS hybridizes to the (1) centromeric or pericentromeric heterochromatin of almost all the chromosomes of L . graeca and (2) genomic DNA of species phylogenetically related and unrelated to L . graeca . The presence of pGPS even in species immunologically apart more than 30 million years suggests that this repeated family might be either very ancient or have been conserved during evolution due to its functional role . The latter hypothesis might be supported by the results of sequence analysis which showed some homology with both several alphoid sequences of primates and the CDEIII centromeric sequence of yeast . Segments of the satellite sequence are similar to the mammalian CENP-B box . These observations suggest that pGPS might have a role in determining the centromeric function in lacertid lizards.

J Biol Chem, 1998 Feb 6, 273(6), 3679 - 86
Hop modulates Hsp70/Hsp90 interactions in protein folding; Johnson BD et al.; Hop is a 60-kDa protein characterized by its ability to bind the two chaperones, hsp70 and hsp90 . We have tested the function of Hop using an assay for the refolding of denatured firefly luciferase . We show that Hop is involved in the process of refolding thermally denatured firefly luciferase in rabbit reticulocyte lysate . Hop also stimulates refolding by hsp70 and Ydj-1 in a purified refolding system . Hsp90 can also stimulate refolding, and optimal refolding is observed in the presence of both Hop and hsp90 . Similar stimulation was observed when Hop was replaced by its yeast homolog Sti1 . In assays of the binding of Hop to hsp70 and hsp90, Hop preferentially forms a complex with ADP-bound hsp70, and this process is unaffected by the presence of hsp90 . Hop does not alter the ATPase activity or the rate of ADP dissociation of hsp70 . Hop also appears to bind to the ADP-bound form of hsp90, blocking the ATP-dependent conversion of hsp90 to a form capable of interacting with p23 . Conversely, once p23 is bound to hsp90, Hop binding is diminished . These results confirm that Hop provides a physical link between hsp70 and hsp90 and also indicate that Hop modulates the activities of both of these chaperone proteins.

J Biol Chem, 1998 Feb 6, 273(6), 3517 - 27
Characterization of the human class Mu glutathione S-transferase gene cluster and the GSTM1 deletion; Xu S et al.; A partial physical map has been constructed of the human class Mu glutathione S-transferase genes on chromosome 1p13.3 . The glutathione S-transferase genes in this cluster are spaced about 20 kilobase pairs (kb) apart, and arranged as 5'-GSTM4-GSTM2-GSTM1-GSTM5-3' . This map has been used to localize the end points of the polymorphic GSTM1 deletion . The left repeated region is 5 kb downstream from the 3'-end of the GSTM2 gene and 5 kb upstream from the beginning of the GSTM1 gene; the right repeated region is 5 kb downstream from the 3'-end of the GSTM1 and 10 kb upstream from the 5'-end of the GSTM5 gene . The GSTM1-0 deletion produces a novel 7.4-kb HindIII fragment with the loss of 10.3- and 11.4-kb HindIII fragments . The same novel fragment was seen in 13 unrelated individuals (20 null alleles), suggesting that most GSTM1-0 deletions involve recombinations between the same two regions . We have cloned and sequenced the deletion junction that is produced at the GSTM1-null locus; the 5'- and 3'-flanking regions are more than 99% identical to each other and to the deletion junction sequence over 2.3 kb . Because of the high sequence identity between the left repeat, right repeat, and deletion junction regions, the crossing over cannot be localized within the 2.3-kb region . The 2.3-kb repeated region contains a reverse class IV Alu repetitive element near one end of the repeat.

J Biol Chem, 1998 Feb 6, 273(6), 3212 - 5
IkappaBbeta interacts with the retinoid X receptor and inhibits retinoid-dependent transactivation in lipopolysaccharide-treated cells; Na SY et al.; To elucidate the molecular action of the NFkappaB inhibitor IkappaBbeta, we isolated a number of IkappaBbeta interactors using the yeast two-hybrid system . These include the retinoid X receptor (RXR), whose interaction with IkappaBbeta is significantly stimulated by the RXR ligand 9-cis-retinoic acid, as shown in the yeast system as well as the glutathione S-transferase pull down assays . RXR is a nuclear protein, whereas IkappaBbeta accumulates in the nucleus only in cells stimulated with lipopolysaccharide or other inducers that result in prolonged activation of NFkappaB . Consistent with this, cotransfection with IkappaBbeta specifically repressed the 9-cis-RA-induced transcriptional activities of RXR in an lipopolysaccharide-dependent manner . These results suggest a novel IkappaBbeta-mediated antagonism between the signaling pathways of NFkappaB and RXR.

J Biol Chem, 1998 Feb 6, 273(6), 3136 - 9
Insulin-like growth factor-I receptor and insulin receptor association with a Src homology-2 domain-containing putative adapter; Wang J et al.; Insulin receptor (IR) and the related insulin-like growth factor-I (IGF-I) receptor (IGF-IR) mediate a variety of metabolic and mitogenic cellular responses, some of which may involve unidentified receptor targets . A Src homology-2 (SH2) domain-coding region of a mouse protein was cloned based on its interaction with IR . It was designated mSH2-B based on its high similarity to an earlier reported rat sequence SH2-B . A role of mSH2-B in IGF-I and insulin action was suggested by the interaction of the SH2 domain with activated IGF-IR and IR catalytic fragments but not with an inactive IR catalytic fragment in the yeast two-hybrid system in vivo and by the hormone-dependent association of a glutathione S-transferase (GST) SH2 domain fusion protein of mSH2-B with both receptors in cell extracts . A comparison of IGF-IR and IR mutants lacking individual Tyr autophosphorylation sites for association with GST mSH2-B showed that homologous juxtamembrane (IR960/IGF-IR950) and C-terminal (IR1322/IGF-IR1316) receptor motifs were required . Synthetic phosphopeptides representing IR960 and IR1322 competed for GST mSH2-B binding to the receptor, suggesting that both motifs participate in the association with mSH2-B . Antibodies raised against GST mSH2-B identified a cellular protein of 92 kDa that was not found to be phosphorylated on Tyr . It co-immunoprecipitated with IGF-IR or IR, which was strictly dependent on receptor activation . IR and IGF-IR Tyr phosphorylation motifs were not identified in the complete SH2-B primary structure, suggesting that it may participate as an adapter rather than a substrate in the IGF-I and insulin signaling pathways.

Biochem J, 1998 Feb 1, 329 ( Pt 3), 615 - 21
tRNA is entrapped in similar, but distinct, nuclear and cytoplasmic ribonucleoprotein complexes, both of which contain vigilin and elongation factor 1 alpha; Kruse C et al.; Vigilin, which is found predominantly in cells and tissues with high levels of protein biosynthesis, was isolated in its native form from human HEp-2 cells (A.T.C.C . CCL23) by immunoaffinity chromatography . Here we demonstrate that vigilin is part of a novel large tRNA-binding ribonucleoprotein complex (tRNP), found not only in the cytoplasm, but also in the nuclei of human cells . Compositional differences in the protein pattern were detected between the nuclear and cytoplasmic tRNPs, although some properties of the purified nuclear tRNP, such as tRNA protection against nuclease attack, were identical with those of the cytoplasmic tRNP . By using either a pool of total human nuclear RNA or radioactively labelled yeast tRNAAsp in rebinding experiments, we could show that tRNA is specifically recaptured by the RNA-depleted, vigilin-containing nuclear complex . We could also show that vigilin is capable of binding tRNA in vitro . Another tRNA-binding protein is elongation factor 1 alpha, which appears to be enriched in the cytoplasmic and nuclear tRNP complexes . This suggests that the cytoplasmic tRNP may be involved in the channelled tRNA cycle in the cytoplasm of eukaryotic cells . Our results also suggest that the nuclear vigilin-containing tRNP may be related to the nuclear export of tRNA.

Genes Dev, 1998 Jan 15, 12(2), 175 - 85
HIV-1 Vpr interacts with the nuclear transport pathway to promote macrophage infection; Vodicka MA et al.; HIV-1 Vpr promotes nuclear entry of viral nucleic acids in nondividing macrophages and also causes a G2 cell-cycle arrest . Consistent with its role in nuclear transport, we show Vpr localizes to the nuclear envelope in both human and yeast cells . Like the importin-beta subunit of the nuclear import receptor, Vpr also interacts with the yeast importin-alpha subunit and nucleoporins . Moreover, overexpression of either Vpr or importin-beta in yeast blocks nuclear transport of mRNAs . A mutant form of Vpr (Vpr F34I) that does not localize at the nuclear envelope, or bind to importin-alpha and nucleoporins, renders HIV-1 incapable of infecting macrophages efficiently . Vpr F34I, however, still causes a G2 arrest, demonstrating that the dual functions of Vpr are genetically separable . Our data suggest Vpr functionally resembles importin-beta in nuclear import of the HIV-1 pre-integration complex and this function is essential for the role of Vpr in macrophage infection, but not G2 arrest.

Structure, 1997 Nov 15, 5(11), 1511 - 23
Distinct phosphorylation signals converge at the catalytic center in glycogen phosphorylases; Lin K et al.; BACKGROUND: Glycogen phosphorylases (GPs) catalyze the conversion of the storage form of carbohydrate (glycogen) to the readily usable form (glucose-1-phosphate) to provide cellular energy . Members of this enzyme family have evolved diverse regulatory mechanisms that control a conserved catalytic function . The mammalian and yeast GPs are expressed as inactive forms requiring phosphorylation for activation . Phosphorylation of yeast GP occurs at a distinct site from that of mammalian GP . This work addresses the structural basis by which distinct activation signals relay to the conserved catalytic site in yeast and mammalian GPs . Such knowledge may help understand the principles by which diverse biological regulation evolves . RESULTS: We have compared the crystal structures of the unphosphorylated and phosphorylated forms of yeast GP and propose a relay which links phosphorylation to enzyme activation . Structural components along the activation relay becomes more conserved within the GP family downstream along the relay, towards the catalytic center . Despite distinct upstream activation signals, a response element downstream of the relay leading to the catalytic center is conserved in all GPs . The response element consists of ten hydrophobic residues dispersed over two subunits of the homodimer . Phosphorylation induces hydrophobic condensation of these residues via structural rearrangement, which triggers conformation change of the active site GATE loop, leading to enzyme activation . CONCLUSIONS: Members of the GP family with diverse activation mechanisms have evolved from a constitutively active ancestral enzyme which has the TOWER hydrophobic response element in the active position . Diverse regulation evolved as a result of evolutionary constraint on the downstream response element in the active state, coupled with flexibility and variability in elements of the upstream relays.

Mol Cell Biol, 1998 Mar, 18(3), 1757 - 62
Formation and function of the Rbl2p-beta-tubulin complex; Archer JE et al.; The yeast protein Rbl2p suppresses the deleterious effects of excess beta-tubulin as efficiently as does alpha-tubulin . Both in vivo and in vitro, Rbl2p forms a complex with beta-tubulin that does not contain alpha-tubulin, thus defining a second pool of beta-tubulin in the cell . Formation of the complex depends upon the conformation of beta-tubulin . Newly synthesized beta-tubulin can bind to Rbl2p before it binds to alpha-tubulin . Rbl2p can also bind beta-tubulin from the alpha/beta-tubulin heterodimer, apparently by competing with alpha-tubulin . The Rbl2p-beta-tubulin complex has a half-life of approximately 2.5 h and is less stable than the alpha/beta-tubulin heterodimer . The results of our experiments explain both how excess Rbl2p can rescue cells overexpressing beta-tubulin and how it can be deleterious in a wild-type background . They also suggest that the Rbl2p-beta-tubulin complex is part of a cellular mechanism for regulating the levels and dimerization of tubulin chains.

Mol Cell Biol, 1998 Mar, 18(3), 1444 - 8
I-SceI-induced gene replacement at a natural locus in embryonic stem cells; Cohen-Tannoudji M et al.; Gene targeting is a very powerful tool for studying mammalian development and physiology and for creating models of human diseases . In many instances, however, it is desirable to study different modifications of a target gene, but this is limited by the generally low frequency of homologous recombination in mammalian cells . We have developed a novel gene-targeting strategy in mouse embryonic stem cells that is based on the induction of endogenous gap repair processes at a defined location within the genome by induction of a double-strand break (DSB) in the gene to be mutated . This strategy was used to knock in an NH2-ezrin mutant in the villin gene, which encodes an actin-binding protein expressed in the brush border of the intestine and the kidney . To induce the DSB, an I-SceI yeast meganuclease restriction site was first introduced by gene targeting to the villin gene, followed by transient expression of I-SceI . The repair of the ensuing DSB was achieved with high efficiency (6 x 10{-6}) by a repair shuttle vector sharing only a 2.8-kb region of homology with the villin gene and no negative selection marker . Compared to conventional gene-targeting experiments at the villin locus, this represents a 100-fold stimulation of gene-targeting frequency, notwithstanding a much lower length of homology . This strategy will be very helpful in facilitating the targeted introduction of several types of mutations within a gene of interest.

Mol Cell Biol, 1998 Mar, 18(3), 1436 - 43
DNA mismatch repair catalyzed by extracts of mitotic, postmitotic, and senescent Drosophila tissues and involvement of mei-9 gene function for full activity; Bhui-Kaur A et al.; Extracts of Drosophila embryos and adults have been found to catalyze highly efficient DNA mismatch repair, as well as repair of 1- and 5-bp loops . For mispairs T.G and G.G, repair is nick dependent and is specific for the nicked strand of heteroduplex DNA . In contrast, repair of A.A, C.A, G.A, C.T, T.T, and C.C is not nick dependent, suggesting the presence of glycosylase activities . For nick-dependent repair, the specific activity of embryo extracts was similar to that of extracts derived from the entirely postmitotic cells of young and senescent adults . Thus, DNA mismatch repair activity is expressed in Drosophila cells during both development and aging, suggesting that there may be a function or requirement for mismatch repair throughout the Drosophila life span . Nick-dependent repair was reduced in extracts of animals mutant for the mei-9 gene . mei-9 has been shown to be required in vivo for certain types of DNA mismatch repair, nucleotide excision repair (NER), and meiotic crossing over and is the Drosophila homolog of the yeast NER gene rad1.

Nat Biotechnol, 1998 Feb, 16(2), 177 - 80
An ethanol inducible gene switch for plants used to manipulate carbon metabolism; Caddick MX et al.; Many transgenic plant studies use constitutive promoters to express transgenes . For certain genes, deleterious effects arise from constant expression in all tissues throughout development . We describe a chemically inducible plant gene expression system, with negligible background activity, that obviates this problem . We demonstrate its potential by showing inducible manipulation of carbon metabolism in transgenic plants . Upon rapid induction of yeast cytosolic invertase, a marked phenotype appears in developing leaves that is absent from leaves that developed before induction or after it has ceased.

Cell Mol Life Sci, 1998 Jan, 54(1), 80 - 93
SET domain proteins modulate chromatin domains in eu- and heterochromatin; Jenuwein T et al.; The SET domain is a 130-amino acid, evolutionarily conserved sequence motif present in chromosomal proteins that function in modulating gene activities from yeast to mammals . Initially identified as members of the Polycomb- and trithorax-group (Pc-G and trx-G) gene families, which are required to maintain expression boundaries of homeotic selector (HOM-C) genes, SET domain proteins are also involved in position-effect-variegation (PEV), telomeric and centromeric gene silencing, and possibly in determining chromosome architecture . These observations implicate SET domain proteins as multifunctional chromatin regulators with activities in both eu- and heterochromatin--a role consistent with their modular structure, which combines the SET domain with additional sequence motifs of either a cysteine-rich region/zinc-finger type or the chromo domain . Multiple functions for chromatin regulators are not restricted to the SET protein family, since many trx-G (but only very few Pc-G) genes are also modifiers of PEV . Together, these data establish a model in which the modulation of chromatin domains is mechanistically linked with the regulation of key developmental loci (e.g . HOM-C).

Mech Dev, 1997 Dec, 69(1-2), 115 - 24
A basic-helix-loop-helix protein expressed in precursors of Drosophila longitudinal visceral muscles; Georgias C et al.; Basic-helix-loop-helix (bHLH) transcription factors are involved in the control of many developmental processes in vertebrates and invertebrates . The HLH domain mediates formation of homo- or heterodimers . We have taken advantage of these dimerisation properties to identify a novel Drosophila HLH protein using the yeast two-hybrid system . Expression of bHLH54F at the blastoderm stage is restricted to a small subpopulation of mesodermal cells near the posterior pole . During germ band retraction these cells spread along the future midgut region . Later bHLH54F-expressing cells make up the longitudinal portion of the visceral musculature . Characterisation of this expression pattern demonstrates that precursors of the outer, longitudinal muscles of the midgut are distinct in origin and morphology from precursors of the inner, circular muscles.

Immunology, 1997 Nov, 92(3), 362 - 8
Autoantibodies to evolutionarily conserved epitopes of enolase in a patient with discoid lupus erythematosus; Gitlits VM et al.; Although the pathology of discoid lupus erythematosus is well documented the causative agents are not known . Here, we report the identity of the target antigen of an autoantibody present in high titre in the serum of a patient with discoid lupus erythematosus . We have demonstrated that the antigen is enolase; first, because it has properties consistent with this glycolytic enzyme (47,000 MW, cytosolic localization and ubiquitous tissue distribution) . Secondly, limited amino acid sequence determination after trypsin digestion shows identity with alpha-enolase . Finally, the autoimmune serum immunoblots rabbit and yeast enolase and predominantly one isoelectric form of enolase (PI approximately 6.1) . These results indicate that the reactive autoepitopes are highly conserved from man to yeast . The results also suggest that the autoantibodies are most reactive to the alpha-isoform of enolase, although it is possible that they may also be reactive with gamma-enolase, and have least reactivity to beta-enolase . The anti-enolase autoantibodies belong to the immunoglobulin G1 (IgG1) isotype . This is the first report of IgG1 autoantibodies to evolutionarily conserved autoepitopes of enolase in the serum of a patient with discoid lupus erythematosus . Previous reports of autoantibodies to enolase have suggested associations with autoimmune polyglandular syndrome type I and cancer-associated retinopathy . This report and an earlier report of what is likely to be enolase autoantibodies in two patients without systemic disease suggest that enolase autoantibodies have a broad association and are not restricted to any particular disease.

Cancer Res, 1998 Feb 15, 58(4), 698 - 703
Independent pathways of p53 induction by cisplatin and X-rays in a cisplatin-resistant ovarian tumor cell line; Siddik ZH et al.; The p53 tumor suppressor gene is critical in regulating cell proliferation following DNA damage, and disruption of p53 protein function by mutation has been implicated as a factor responsible for resistance of tumor cells to chemotherapeutic agents . Our studies were initiated by asking whether the translational product of the p53 gene is associated with cisplatin resistance in the 2780CP human ovarian tumor model . We have demonstrated by single-strand conformation polymorphism analysis and sequencing that p53 in parental cisplatin-sensitive A2780 cells was wild type . In 2780CP cells, however, a mutation was found in exon 5 at codon 172 (Val to Phe) . Interestingly, exposure to X-rays resulted in p53 induction in both A2780 and 2780CP tumor models . The p53 increases by the ionizing radiation were accompanied by concomitant increases in levels of the p53-regulated p21Waf1/Cip1 protein and led to arrest of cells in G1 phase of the cell cycle . A yeast functional assay confirmed that p53 in A2780 was wild type, but, more importantly, it provided evidence that the p53 mutation in 2780CP cells was temperature sensitive and heterozygous . These experiments demonstrate that sensitive and resistant cells have normal p53 functions, despite the presence of p53 mutation in the 2780CP model . In parallel investigations using the Western technique, exposure of A2780 cells to clinically relevant concentrations of cisplatin (1-20 microM) resulted in time- and dose-dependent increases in p53, together with coordinate increases in p21Waf1/Cip1 . In contrast, cisplatin did not induce these proteins in 2780CP cells to any significant degree . The results indicate that a defect exists in the signal transduction pathway for p53 induction following cisplatin-induced DNA damage in 2780CP cells, and this may represent a significant mechanism of cisplatin resistance . Furthermore, induction of p53 in 2780CP cells by X-rays, but not cisplatin, strongly suggests that independent pathways are involved in p53 regulation for the two DNA-damaging agents.

Plant Mol Biol, 1998 Feb, 36(3), 463 - 71
The Arabidopsis AUX1 gene: a model system to study mRNA processing in plants; Marchant A et al.; It is advantageous for an organism to be able to remove aberrant mRNAs that have either been incorrectly transcribed or processed in order to prevent the accumulation of potentially harmful proteins . The selective degradation of nonsense containing transcripts has been described in yeast . Caenorhabiditis elegans and plants . The ease of identification of new mutant alleles in the AUX1 gene of Arabidopsis thaliana has provided a useful system to study novel mutations affecting mRNA stability and pre-mRNA splicing . To date 50 alleles of AUX1 have been identified of which 14 have been characterised at the sequence level . Eight of the characterised alleles encode missense mutations while the others cause nonsense mutations or splicing defects . The 2 splicing mutants identified affect the 5' or 3' splice sites and lead to cryptic splicing events resulting in premature stop codons . The AUX1 mRNA levels of the nonsense containing mutants are reduced compared to the wild-type or missense mutants whereas those of a control transcript (SecY) are unaltered . This provides further evidence for a nonsense-mediated mRNA degradation mechanism in plants and provides a system to study the phenomenon further in Arabidopsis.

Plant Mol Biol, 1998 Jan, 36(1), 113 - 23
Implication of 5'-flanking sequence elements in expression of a plant tRNA(Leu) gene; Choisne N et al.; A comparison of 5'-flanking sequences from 68 different nuclear plant tRNA genes was analyzed to find consensus sequences . Three conserved features stood out, all of which are present in the tRNA(Leu) gene used in this study: (1) a high proportion of A and T residues upstream of all tRNA genes; (2) a region of low duplex stability about 30-35 bp before the coding sequence, often containing a TATA-box like motif; (3) a CAA triplet in the region of the presumed transcription start . The effect of replacement of the AT-rich upstream sequences with GC-rich sequences or unrelated AT-rich sequences was tested by progressive deletions and by inserting randomly cloned sequences upstream of the tRNA gene . GC-rich 5'-flanking sequences were found to be generally incompatible with high levels of expression . The TATA-box like motifs and the CAA triplet were removed or altered by deletion or directed mutagenesis . Mutation of the CAA triplet significantly decreased expression of the tRNA(Leu) gene, suggesting that this CAA triplet is important for transcription efficiency, but mutation or elimination of the TATA-box like motifs generally had little effect . The presence or absence of each of these features in tRNA genes from other organisms is discussed; there are clear and interesting differences between plant tRNA genes and those of yeast and mammals.

Plant Mol Biol, 1998 Mar, 36(4), 499 - 508
Impairment of tapetum and mitochondria in engineered male-sterile tobacco plants; Hernould M et al.; Flowers of tobacco transformed with an unedited copy of the mitochondrial atp9 gene sequence fused to the yeast coxIV mitochondrial targeting presequence, showed several anther abnormalities leading to pollen abortion . The gene was expressed in vegetative and reproductive tissues of the plant . Cytological analysis revealed that tapetum development was impaired . Mitochondria of the tapetum cells were severely affected showing characteristic signs of degeneration: loss of cristae and swelling . These mitochondrial modifications were correlated with the presence of the transcript and translated product of the 'unedited' atp9 and a significant decrease in oxygen consumption in non-photosynthetic tissues . The main effect of the unedited atp9 expression in transgenic plants was male sterility.

Biochem J, 1998 Mar 1, 330 ( Pt 2), 777 - 84
A dehydrogenase-mediated recycling system of NADPH in plant peroxisomes; Corpas FJ et al.; The presence of the two NADP-dependent dehydrogenases of the pentose phosphate pathway has been investigated in plant peroxisomes from pea (Pisum sativum L.) leaves . Both enzymes, glucose-6-phosphate dehydrogenase (G6PDH; EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (6PGDH; EC 1.1.1.44), were present in the matrix of leaf peroxisomes, and their kinetic properties were studied . G6PDH and 6PGDH showed a typical Michaelis-Menten kinetic saturation curve, and had specific activities of 12.4 and 29.6 mU/mg protein, respectively . The Km values of G6PDH and 6PGDH for glucose 6-phosphate and for 6-phosphogluconate were 107.3 and 10.2 microM, respectively . Dithiothreitol did not inhibit G6PDH activity . By isoelectric focusing of peroxisomal matrices, the G6PDH activity was resolved into three isoforms with isoelectric points of 5.55, 5.30 and 4.85 . The isoelectric point of peroxisomal 6PGDH was 5.10 . Immunoblot analyses of peroxisomal matrix with an antibody against yeast G6PDH revealed a single cross-reactive band of 56 kDa . Post-embedment, EM immunogold labelling of G6PDH confirmed that this enzyme was localized in the peroxisomal matrices, the thylakoid membrane and matrix of chloroplasts, and the cytosol . The presence of the two oxidative enzymes of the pentose phosphate pathway in plant peroxisomes implies that these organelles have the capacity to reduce NADP+ to NADPH for its re-utilization in the peroxisomal metabolism . NADPH is particularly required for the ascorbate-glutathione cycle, which has been recently demonstrated in plant peroxisomes {Jimenez, Hernandez, del Rio and Sevilla (1997) Plant Physiol . 114, 275-284} and represents an important antioxidant protection system against H2O2 generated in peroxisomes.

J Biolumin Chemilumin, 1997 Jul-Aug, 12(4), 193 - 7
Evaluation of some immune functions in a patient affected by common variable immunodeficiency using luminescent techniques; Di Renzo M et al.; Common variable immunodeficiency is a primary immunodeficiency characterized by a failure of antibody synthesis, whose fundamental immunologic abnormality is still unknown . In our study, we evaluated some immune functions using chemiluminescence in a 32-year-old woman affected by common variable immunodeficiency . In particular, we showed an impairment of her lymphomonocyte proliferative response which was evaluated using a method based on the bioluminescent measurement of ATP . Besides, we found a reduction of her lymphomonocyte IL2 and IL4 production: the IL4 production was evaluated through an ELISA method, whereas the IL2 activity was determined by its ability to support the IL2-dependent murine T-cell line (CTLL) proliferation which was established through a method based on the bioluminescent measurement of ATP . Finally, we evaluated both yeast-induced and fMLP-induced polymorphonuclear and monocyte oxidative metabolism through a luminol-amplified chemiluminescence; these functions were within normal values . Therefore, in our patient affected by common variable immunodeficiency, we demonstrated an impairment of cellular immunity, which might contribute to the pathogenesis of the disease.

Biochem Biophys Res Commun, 1998 Feb 13, 243(2), 368 - 71
Peroxisome biogenesis disorders: identification of a new complementation group distinct from peroxisome-deficient CHO mutants and not complemented by human PEX 13; Shimozawa N et al.; Ten complementation groups of generalized peroxisome biogenesis disorders (PBD), (excluding rhizomelic chondrodysplasia punctata) have been identified using complementation analysis . Four of the genes involved have been identified using two different methods of (1) genetic functional complementation of peroxisome deficient CHO cell mutants and (2) homology searches for human dbEST, based on yeast genes involved in peroxisome biogenesis (PEX genes) . We report here the first identification of a new complementation group which is genetically different from peroxisome deficient CHO mutants . There were no complementations by the human PEX 13 gene . The nature of the related gene is being investigated.

Biochem Biophys Res Commun, 1998 Feb 13, 243(2), 342 - 8
Association of nucleoside diphosphate kinase nm23-H2 with human telomeres; Nosaka K et al.; Telomeres, the ends of eukaryotic chromosomes, are essential structures formed by specific protein-DNA complexes that protect chromosomes from degradation and end-to-end fusion . TRF1, a double-stranded telomeric TTAGGG-repeat binding protein, is associated with mammalian telomeres and controls telomere length by inhibiting the action of telomerase . We identified human nucleoside diphosphate kinase nm23-H2 as a human TRF1-interacting protein by yeast two-hybrid screening . In vitro-binding assays using different recombinant nucleoside diphosphate kinases showed that TRF1 predominantly binds the nm23-H2 isoform rather than nm23-H1 . Electrophoretic mobility shift analysis revealed that the recombinant nm23-H2 protein can bind the single-stranded telomeric TTAGGG-repeat while it cannot bind the double-stranded telomeric repeat . The synthetic 20 base oligoribonucleotide, which consists of the template sequence CUAACCCUAAC and the adjacent region of the RNA component of human telomerase, was also found to form the complex with the recombinant nm23-H2 protein . Furthermore, the affinity of telomerase for its substrate was increased in vitro by presence of the plentiful nm23-H2 protein . These findings indicate a close relationship between nm23-H2 and human telomeres and suggest a new biological role for nucleoside diphosphate kinase.

Genomics, 1998 Jan 15, 47(2), 314 - 8
An expression map from human chromosome 14q24.3; Sharma V et al.; We have constructed an expression map of chromosome 14q24.3 between markers D14S42 and D14S63 . cDNA selection with YACs from 14q24.3 was used to generate expressed sequence tags (ESTs) . The localization of ESTs was confirmed on a YAC contig . PCR products of ESTs were used as probes to screen cDNA libraries leading to the isolation of transcripts for known and unknown genes . In total, the expression map contains 7 known genes previously mapped to 14q24.3, 6 cDNA transcripts, and 15 anonymous ESTs . The addition of 21 unique transcribed loci from an approximately 5- to 7-Mb region of chromosome 14q24.3 will facilitate future efforts to identify human disease genes from this region.

J Cell Biol, 1998 Jan 26, 140(2), 283 - 93
A presumptive developmental role for a sea urchin cyclin B splice variant; Lozano JC et al.; We show that a splice variant-derived cyclin B is produced in sea urchin oocytes and embryos . This splice variant protein lacks highly conserved sequences in the COOH terminus of the protein . It is found strikingly abundant in growing oocytes and cells committed to differentiation during embryogenesis . Cyclin B splice variant (CBsv) protein associates weakly in the cell with Xenopus cdc2 and with budding yeast CDC28p . In contrast to classical cyclin B, CBsv very poorly complements a triple CLN deletion in budding yeast, and its microinjection prevents an initial step in MPF activation, leading to an important delay in oocyte meiosis reinitiation . CBsv microinjection in fertilized eggs induces cell cycle delay and abnormal development . We assume that CBsv is produced in growing oocytes to keep them in prophase, and during embryogenesis to slow down cell cycle in cells that will be committed to differentiation.

EMBO J, 1998 Jan 15, 17(2), 598 - 608
Rad51-deficient vertebrate cells accumulate chromosomal breaks prior to cell death; Sonoda E et al.; Yeast rad51 mutants are viable, but extremely sensitive to gamma-rays due to defective repair of double-strand breaks . In contrast, disruption of the murine RAD51 homologue is lethal, indicating an essential role of Rad51 in vertebrate cells . We generated clones of the chicken B lymphocyte line DT40 carrying a human RAD51 transgene under the control of a repressible promoter and subsequently disrupted the endogenous RAD51 loci . Upon inhibition of the RAD51 transgene, Rad51- cells accumulated in the G2/M phase of the cell cycle before dying . Chromosome analysis revealed that most metaphase-arrested Rad51- cells carried isochromatid-type breaks . In conclusion, Rad51 fulfils an essential role in the repair of spontaneously occurring chromosome breaks in proliferating cells of higher eukaryotes.

EMBO J, 1998 Jan 15, 17(2), 590 - 7
An actively retrotransposing, novel subfamily of mouse L1 elements; Naas TP et al.; Retrotransposition of LINEs and other retroelements increases repetition in mammalian genomes and can cause deleterious mutations . Recent insertions of two full-length L1s, L1spa and L1Orl, caused the disease phenotypes of the spastic and Orleans reeler mice respectively . Here we show that these two recently retrotransposed L1s are nearly identical in sequence, have two open reading frames and belong to a novel subfamily related to the ancient F subfamily . We have named this new subfamily TF (for transposable) and show that many full-length members of this family are present in the mouse genome . The TF 5' untranslated region has promoter activity, and TF-type RNA is abundant in cytoplasmic ribonucleoprotein particles, which are likely intermediates in retrotransposition . Both L1spa and L1Orl have reverse transcriptase activity in a yeast-based assay and retrotranspose at high frequency in cultured cells . Together, our data indicate that the TF subfamily of L1s contains a major class of mobile elements that is expanding in the mouse genome.

EMBO J, 1998 Jan 15, 17(2), 482 - 97
Regulation of the G1 phase of the cell cycle by periodic stabilization and degradation of the p25rum1 CDK inhibitor; Benito J et al.; In fission yeast, the cyclin-dependent kinase (CDK) inhibitor p25(rum1) is a key regulator of progression through the G1 phase of the cell cycle . We show here that p25(rum1) protein levels are sharply periodic . p25(rum1) begins to accumulate at anaphase, persists in G1 and is destroyed during S phase . p25(rum1 )is stabilized and polyubiquitinated in a mutant defective in the 26S proteasome, suggesting that its degradation normally occurs through the ubiquitin-dependent 26S proteasome pathway . Phosphorylation of p25(rum1 )by cdc2-cyclin complexes at residues T58 and T62 is important to target the protein for degradation . Mutation of one or both of these residues to alanine causes stabilization of p25(rum1) and induces a cell cycle delay in G1 and polyploidization due to occasional re-initiation of DNA replication before mitosis . The CDK-cyclin complex cdc2-cig1, which is insensitive to p25(rum1 )inhibition, seems to be the main kinase that phosphorylates p25(rum1) . Phosphorylation of p25(rum1) in S phase and G2 serves as the trigger for p25(rum1) proteolysis . Thus, periodic accumulation and degradation of the CDK inhibitor p25(rum1 )in G1 plays a role in setting a threshold of cyclin levels important in determining the length of the pre-Start G1 phase and in ensuring the correct order of cell cycle events.

Nucleic Acids Res, 1997 Dec 15, 25(24), 4861 - 5
A multiplicity of mediators: alternative forms of transcription complexes communicate with transcriptional regulators; Chang M et al.; The already complex process of transcription by RNA polymerase II has become even more complicated in the last few years with the identification of auxiliary factors in addition to the essential general initiation factors . In many cases these factors, which have been termed mediators or co-activators, are only required for activated or repressed transcription . In some cases the effects are specific for certain activators and repressors . Recently some of these auxiliary factors have been found in large complexes with either TBP, as TBP-associated factors (TAFs) in the general factor TFIID, or with pol II and a subset of the general factors, referred to as the 'holoenzyme' . Although the exact composition of these huge assemblies is still a matter of some debate, it is becoming clear that the complexes themselves come in more than one form . In particular, at least four forms of TFIID have been described, including one that contains a tissue-specific TAF and another with a cell type-specific form of TBP . In addition, in yeast there are at least two forms of the 'holoenzyme' distinguished by their mediator composition and by the spectrum of transcripts whose expression they affect . Genetic and biochemical analyses have begun to identify the interactions between the components of these complexes and the ever increasing family of DNA binding regulatory factors . These studies are complicated by the fact that individual regulatory factors often appear to have redundant interactions with multiple mediators . The existence of these different forms of transcription complexes defines a new target for regulation of subsets of eukaryotic genes.

Curr Biol, 1997 Nov 1, 7(11), 901 - 4
Immunolocalization of CENP-A suggests a distinct nucleosome structure at the inner kinetochore plate of active centromeres; Warburton PE et al.; The trilaminar kinetochore directs the segregation of chromosomes in mitosis and meiosis . Despite its importance, the molecular architecture of this structure remains poorly understood {1} . The best known component of the kinetochore plates is CENP-C, a protein that is required for kinetochore assembly {2}, but whose molecular role in kinetochore structure and function is unknown . Here we have raised for the first time monospecific antisera to CENP-A {3}, a 17 kD centromere-specific histone variant that is 62% identical to the carboxy-terminal domain of histone H3 {4,5} and that resembles the yeast centromeric component CSE4 {6} . We have found by simultaneous immunofluorescence with centromere antigens of known ultrastructural location that CENP-A is concentrated in the region of the inner kinetochore plate at active centromeres . Because CENP-A was previously shown to co-purify with nucleosomes {7}, our data suggest a specific nucleosomal substructure for the kinetochore . In human cells, these kinetochore-specific nucleosomes are enriched in alpha-satellite DNA {8} . However, the association of CENP-A with neocentromeres lacking detectable alpha-satellite DNA, and the lack of CENP-A association with alpha-satellite-rich inactive centromeres of dicentric chromosomes together suggest that CENP-A association with kinetochores is unlikely to be determined solely by DNA sequence recognition . We speculate that CENP-A binding could be a consequence of epigenetic tagging of mammalian centromeres.

Curr Biol, 1997 Nov 1, 7(11), 827 - 35
Extrinsic cues, intrinsic cues and microfilaments regulate asymmetric protein localization in Drosophila neuroblasts; Broadus J et al.; BACKGROUND: The Drosophila central nervous system develops from stem cell like precursors called neuroblasts, which divide unequally to bud off a series of smaller daughter cells called ganglion mother cells . Neuroblasts show cell-cycle-specific asymmetric localization of both RNA and proteins: at late interphase, prospero RNA and Inscuteable, Prospero and Staufen proteins are all apically localized; at mitosis, Inscuteable remains apical whereas prospero RNA, Prospero protein and Staufen protein form basal cortical crescents . Here we use in vitro culture of neuroblasts to investigate the role of intrinsic and extrinsic cues and the cytoskeleton in asymmetric localization of Inscuteable, Prospero and Staufen proteins . RESULTS: Neuroblast cytokinesis is normal in vitro, producing a larger neuroblast and a smaller ganglion mother cell . Apical localization of Inscuteable, Prospero and Staufen in interphase neuroblasts is reduced or eliminated in vitro, but all three proteins are localized normally during mitosis (apical Inscuteable, basal Prospero and Staufen) . Microfilament inhibitors result in delocalization of all three proteins . Inscuteable becomes uniform at the cortex, whereas Prospero and Staufen become cytoplasmic; inhibitor washout leads to recovery of microfilaments and asymmetric localization of all three proteins . Microtubule disruption has no effect on protein localization, but disruption of both microtubules and microfilaments results in cytoplasmic localization of Inscuteable . CONCLUSIONS: Both extrinsic and intrinsic cues regulate protein localization in neuroblasts . Microfilaments, but not microtubules, are essential for asymmetric protein anchoring (and possibly localization) in mitotic neuroblasts . Our results highlight the similarity between Drosophila, Caenorhabditis elegans, vertebrates, plants and yeast: in all organisms, asymmetric protein or RNA localization and/or anchoring requires microfilaments.

Curr Biol, 1997 Nov 1, 7(11), R723 - 5
Dictyostelium development: lower STATs; Kay RR; The discovery of a STAT protein in Dictyostelium indicates that this organism uses phosphotyrosine-SH2-domain signalling during development . Such signalling is lacking in yeast and its appearance may therefore be an early step in the evolution of multicellularity.

Front Biosci, 1998 Jan 01, 3, c1 - 7
A regulatory system for target gene expression; Burcin MM et al.; Temporally-regulated expression of endogenous genes is a desirable goal in stable cell line and transgenic animal systems, as well as in clinical gene therapy . Protocols for introducing genes into stable cell lines and experimental animals are often unsatisfactory due to the constitutive expression of such transgenes . To circumvent this problem we have demonstrated specific and temporally regulable expression of a target gene in vivo effected by a chimeric regulator in response to an orally-administered, non-toxic chemical . This regulatory system utilizes a chimeric regulator GLVP, consisting of a mutated human progesterone receptor ligand binding domain (PRLBD-delta) fused to the yeast GAL4 DNA binding domain (DBD) and the HSV VP16 transcriptional activation domain and whose activity is solely regulable by non-physiological doses of RU486 but not by progesterone or other endogenous progestins . Replacing the activation domain of the chimeric regulator with a transcriptional repression domain results in inducible repression of target gene expression in vitro . Our regulatory system functions in transient and stable transfections as well as in transgenic animals, and will have a wide variety of potential applications.

Mycoses, 1997 Oct, 40(5-6), 147 - 52
Comparative studies on the detection of antibodies and delayed hypersensitivity responses with 10 Blastomyces dermatitidis lysate antigens; Wakamoto A et al.; Yeast-phase lysate antigens were prepared from 10 different isolates of Blastomyces dermatitidis . Comparative studies were performed using the lysate antigens in an enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies in sera from dogs with blastomycosis and histoplasmosis . In order to evaluate the ability of the lysate reagents to elicit delayed dermal hypersensitivity (DTH) responses, the lysates were compared as skin-testing antigens in hairless guinea pigs that were previously sensitized with B . dermatitidis or Histoplasma capsulatum killed whole yeast cells . All ten of the lysate reagents were able to detect antibody with the ELISA in the serum specimens from dogs with blastomycosis (absorbance values ranged from 0.184 to 0.272; mean value 0.235) . In contrast, when the lysates were assayed against sera from dogs with histoplasmosis, the absorbance values ranged from 0.053 to 0.151, with a mean value of 0.092 . All ten lysate antigens were able to elicit a DTH response in the B . dermatitidis-immunized animals (mean axes of induration values ranged from 7.0 to 14.4 mm; mean value 8.6 mm) . On the other hand, only minimal reactivity was evidenced in the guinea pigs immunized with H . capsulatum (mean axes of induration values ranged from 0.8 to 2.9 mm; mean value 1.8 mm).

J Med Genet, 1998 Jan, 35(1), 78 - 80
Intrachromosomal triplication of distal 7p; Rivera H et al.; A female infant who died at 2 years of age with growth and psychomotor retardation, wide anterior fontanelle, downward slanting palpebral fissures, large, simple ears, joint dislocation/contractures, recurrent infections, and severe pulmonary hypertension was found to have a de novo 7p+ chromosome . The G banding pattern was suggestive of a triplication of 7p21.3 and 7p22; results of fluorescence in situ hybridisation studies using a chromosome 7 specific library, a subtelomeric 7p repeat (109A6), and yeast artificial chromosome clones 786g1 and 850a1, which are respectively associated with the (CA)n repeat markers D7S517 and D7S513, supported the cytogenetic interpretation and showed that the middle repeat was inverted . The patient's phenotype was consistent with the 7p duplication syndrome, allowing for the effects of the extra burden introduced by the partial tetrasomy . The present rearrangement may have resulted from several meiotic events occurring at the four chromatid stage, namely an unequal crossover or interhomologue translocation with points of exchange at 7p22 and 7p15 followed by the inverted insertion of 7p21.3-->p21.2 at the former breakpoint junction; moreover, a further duplication including D7S517 within the terminal 7p22 band is also required.

Cytotechnology, 1997, 25(1-3), 115 - 26
Antigen-specific inhibition of CD4+ T-cell responses to beta-lactoglobulin by its single amino acid-substituted mutant form through T-cell receptor antagonism; Totsuka M et al.; T-cell responses can be antagonized by some single amino acid-substituted analogs of a peptide ligand for T-cell receptors (TCR), and these are called TCR antagonists . In this study, we addressed the question of whether TCR antagonism can be elicited by a whole protein antigen carrying a mutated T-cell determinant region corresponding to a TCR antagonist peptide . To clarify this, we examined the ability of a single amino acid-substituted mutant form of bovine beta-lactoglobulin (beta-Lg) to inhibit three CD4+ T-cell clones recognizing a peptide corresponding to an immunodominant determinant region 119-133 of beta-Lg (p119-133) . First, we identified pD129A, an analog of p119-133 with a substitution of Ala for 129Asp, as an antagonist which can inhibit the response of two of the three T-cell clones . Then, using a yeast expression system, we prepared a mutant beta-Lg (mutD129A) with the same substitution of Ala for 129Asp as that in pD129A . This mutant protein could inhibit the proliferation of the two T-cell clones in a manner similar to the effect of pD129A . From these results we can demonstrate that TCR antagonism can be elicited by peptides naturally processed from a single-substituted mutant protein as well as by the corresponding peptides added exogenously.

Biochim Biophys Acta, 1998 Jan 21, 1395(2), 159 - 64
A Drosophila homologue of oxysterol binding protein (OSBP)--implications for the role of OSBP; Alphey L et al.; The identification of a Drosophila homologue (OSBP-Dm) of mammalian oxysterol binding protein (OSBP) is reported . OSBP-Dm was identified by its ability to overcome the cell cycle arrest induced by over-expression of Wee1p in fission yeast . OSBP-Dm has an overall sequence identity of 52% with mammalian OSBP, and shows a number of highly conserved regions of functional significance . Insects are unable to biosynthesize the steroid core, relying instead on dietary sterols to satisfy their requirements . It is therefore unlikely that OSBP-Dm is involved in feedback inhibition of the mevalonate pathway, as has previously been suggested for its mammalian homologues.

Biochem Biophys Res Commun, 1998 Feb 4, 243(1), 101 - 8
Cloning and characterization of two human cDNAs encoding the mRNA capping enzyme; Tsukamoto T et al.; Previous studies demonstrated that the mammalian mRNA capping enzyme is a bifunctional enzyme containing RNA 5'-triphosphatase and mRNA guanylyl-transferase activities in a single polypeptide . In yeast, both the above activities are separated into two different subunits, alpha and beta, the genes for which we have cloned recently . It is thus interesting to compare the structural and functional relationships between the mammalian and yeast capping enzymes . Here we isolated two human cDNAs encoding mRNA capping enzymes termed hCAP1a and hCAP1b which encode 597 and 541 amino acids, respectively . They are different only at the region coding for the C-terminal portion of the enzyme . Comparison of the deduced amino acid sequences with other cellular and viral capping enzymes showed that all the regions conserved among mRNA guanylyltransferases are observed in our clones except one conserved C-terminal region which was absent in the hCAP1b protein . The purified recombinant hCAP1a gene product, hCAP1a, exhibited both RNA 5'-triphosphatase and mRNA guanylyltransferase activities . Deletion mutant analysis of hCAP1a showed that the N-terminal 213 amino acid fragment containing a tyrosine specific protein phosphatase motif catalyzed the RNA 5'-triphosphatase activity and the C-terminal 369 amino acid fragment exhibited the mRNA guanylyltransferase activity . On the other hand, hCAP1b showed RNA 5'-triphosphatase activity, but neither enzyme-GMP covalent complex formation nor cap structure formation was detected.

J Gen Virol, 1998 Feb, 79 ( Pt 2), 371 - 4
The identification of a conserved binding motif within human papillomavirus type 16 E6 binding peptides, E6AP and E6BP; Elston RC et al.; A 16-mer peptide library was screened using the yeast two-hybrid system to identify peptides which specifically interact with the human papillomavirus type 16 (HPV-16) E6 protein . Four different peptides were identified, three of which contained an E-L-L/V-G motif . A fifth E6 binding peptide, derived from the putative tumour suppressor protein tuberin, was identified during a two-hybrid screen of a HeLa cDNA expression library . This peptide contained a D-I-L-G motif . Homology to the peptides was found within the E6 binding proteins E6-AP and E6-BP . A synthetic peptide containing the ELLG motif blocked the interaction of E6 with both E6-AP and E6-BP . The data suggest that E6 interacts through a structurally similar binding domain present within a number of cellular proteins.

J Gen Virol, 1998 Feb, 79 ( Pt 2), 363 - 70
Transcriptional repression by the Epstein-Barr virus EBNA3A protein tethered to DNA does not require RBP-Jkappa; Bourillot PY et al.; The Epstein-Barr virus (EBV) proteins EBNA1, EBNA2, EBNA3A, EBNA3C, LMP1 and EBNA-LP are essential for the in vitro immortalization of primary B lymphocytes by EBV . EBNA2 is a transcriptional activator of viral and cellular genes . Both EBNA3A and EBNA3C have been shown to specifically inhibit EBNA2-activated transcription by direct interaction with RBP-Jkappa, a cellular DNA-binding factor known to recruit EBNA2 to EBNA2-responsive genes . This interaction interferes with the binding of RBP-Jkappa to DNA in vitro, and this is probably the mechanism by which EBNA3A and EBNA3C repress EBNA2-activated transcription in vivo . EBNA3A and EBNA3C also directly repress transcription when tethered to a promoter via the DNA-binding domain of the yeast Gal4 protein . As RBP-Jkappa has been previously shown to be a repressor in mammalian cells, this repression could be due to the recruitment of RBP-Jkappa by Gal4-EBNA3A and 3C . In this study, we have precisely mapped the domain of EBNA3A involved in the interaction with RBP-Jkappa and we have shown that interaction with RBP-Jkappa is not required for the Gal4-EBNA3A-mediated repression . Furthermore, we have characterized in EBNA3A a domain of 143 amino acids which is necessary and sufficient for EBNA3A-dependent repression.

Genome Res, 1998 Jan, 8(1), 57 - 68
Contig maps and genomic sequencing identify candidate genes in the usher 1C locus; Higgins MJ et al.; Usher syndrome 1C (USH1C) is a congenital condition manifesting profound hearing loss, the absence of vestibular function, and eventual retinal degeneration . The USH1C locus has been mapped genetically to a 2- to 3-cM interval in 11p14-15.1 between D11S899 and D11S861 . In an effort to identify the USH1C disease gene we have isolated the region between these markers in yeast artificial chromosomes (YACs) using a combination of STS content mapping and Alu-PCR hybridization . The YAC contig is approximately 3.5 Mb and has located several other loci within this interval, resulting in the order CEN-LDHA-SAA1-TPH-D11S1310-(D11S1888/KCNC1 )-MYOD1-D11S902D11S921-D11S 1890-TEL . Subsequent haplotyping and homozygosity analysis refined the location of the disease gene to a 400-kb interval between D11S902 and D11S1890 with all affected individuals being homozygous for the internal marker D11S921 . To facilitate gene identification, the critical region has been converted into P1 artificial chromosome (PAC) clones using sequence-tagged sites (STSs) mapped to the YAC contig, Alu-PCR products generated from the YACs, and PAC end probes . A contig of >50 PAC clones has been assembled between D11S1310 and D11S1890, confirming the order of markers used in haplotyping . Three PAC clones representing nearly two-thirds of the USH1C critical region have been sequenced . PowerBLAST analysis identified six clusters of expressed sequence tags (ESTs), two known genes (BIR, SUR1) mapped previously to this region, and a previously characterized but unmapped gene NEFA (DNA binding/EF hand/acidic amino-acid-rich) . GRAIL analysis identified 11 CpG islands and 73 exons of excellent quality . These data allowed the construction of a transcription map for the USH1C critical region, consisting of three known genes and six or more novel transcripts . Based on their map location, these loci represent candidate disease loci for USH1C . The NEFA gene was assessed as the USH1C locus by the sequencing of an amplified NEFA cDNA from an USH1C patient; however, no mutations were detected.

Proc Natl Acad Sci U S A, 1998 Jan 6, 95(1), 167 - 71
Creation of monosomic derivatives of human cultured cell lines; Clarke DJ et al.; Monosomic mammalian cell lines would be ideal for studying gene dosage effects, including gene imprinting, and for systematic isolation of recessive somatic mutants parallel to the invaluable mutants derived from haploid yeast . But autosomal monosomies are lethal in early development; although monosomies appear in tumors, deriving cell lines from these tumors is difficult and cannot provide several syngenic lines . We have developed a strategy for generating stable monosomic human cells, based on random autosomal integration of the gpt plasmid, partial inhibition of DNA topoisomerase II during mitosis to promote chromatid nondisjunction, and selection against retention of gpt . These are likely to be valuable as a source of otherwise inaccessible mutants . The strategy can also be used to generate partial mammalian monosomies, which are desirable as a source of information on recessive genes and gene imprinting.

Proc Natl Acad Sci U S A, 1998 Jan 6, 95(1), 144 - 9
Characterization of mammalian translocase of inner mitochondrial membrane (Tim44) isolated from diabetic newborn mouse kidney; Wada J et al.; Mammalian translocase of mitochondrial inner membrane (mTim44) was isolated during representational difference analysis of cDNA from diabetic mouse kidney . Streptozotocin-induced diabetic mouse kidney cDNA was prepared and subtracted by normal mouse kidney cDNA . By using one of the isolated cDNA fragments as a screening probe, full-length cDNA of mTim44 was isolated from lambdaZAP kidney cDNA library . At the nucleotide level, mTim44 did not exhibit significant homology with any known genes; however, at the amino acid level, it had 50% similarity and 29% identity with yeast Tim44 . C-terminal FLAG epitope-tagged mTim44 fusion protein was transiently expressed in COS7 cells . By using anti-FLAG epitope M2 monoclonal antibody, mTim44 was found to have its subcellular localization associated with mitochondria . By immunoelectron microscopy, mTim44 was seen in the paracrystalline structures within the mitochondria, as well as in their cristae . Mitochondrial import assay of in vitro translated mTim44 indicated that its precursor product ( approximately 50 kDa) was imported and proteolytically processed to a mature approximately 44-kDa protein, and its translocation was inner membrane potential (DeltaPsi)-dependent . Imported mTim44 was protected from protease digestion in which outer membranes were selectively permeabilized with digitonin . The mature form of mTim44 could be recovered in the supernatant of sonicated mitochondrial membrane fraction treated with 0.1 M Na2CO3, pH 11.5 . The data indicate that mTim44 is a mitochondrial inner membrane protein, one of the members of the mammalian TIM complex and up-regulated in hyperglycemic states.

Nucleic Acids Res, 1998 Jan 1, 26(1), 372 - 5
Histone Sequence Database: new histone fold family members; Baxevanis AD et al.; Searches of the major public protein databases with core and linker chicken and human histone sequences have resulted in the compilation of an annotated set of histone protein sequences . In addition, new database searches with two distinct motif search algorithms have identified several members of the histone fold family, including human DRAP1 and yeast CSE4 . Database resources include information on conflicts between similar sequence entries in different source databases, multiple sequence alignments, links to the Entrez integrated information retrieval system, structures for histone and histone fold proteins, and the ability to visualize structural data through Cn3D . The database currently contains >1000 protein sequences, which are searchable by protein type, accession number, organism name, or any other free text appearing in the definition line of the entry . All sequences and alignments in this database are available through the World Wide Web at gov/DIR/GTB/HISTONES or gov/Baxevani/HISTONES

Nucleic Acids Res, 1998 Jan 1, 26(1), 33 - 7
MIPS: a database for protein sequences and complete genomes; Mewes HW et al.; The MIPS group {Munich Information Center for Protein Sequences of the German National Center for Environment and Health (GSF)} at the Max-Planck-Institute for Biochemistry, Martinsried near Munich, Germany, is involved in a number of data collection activities, including a comprehensive database of the yeast genome, a database reflecting the progress in sequencing the Arabidopsis thaliana genome, the systematic analysis of other small genomes and the collection of protein sequence data within the framework of the PIR-International Protein Sequence Database (described elsewhere in this volume) . Through its WWW server MIPS provides access to a variety of generic databases, including a database of protein families as well as automatically generated data by the systematic application of sequence analysis algorithms . The yeast genome sequence and its related information was also compiled on CD-ROM to provide dynamic interactive access to the 16 chromosomes of the first eukaryotic genome unraveled.

Curr Biol, 1997 Dec 1, 7(12), 977 - 86
Atm-dependent interactions of a mammalian chk1 homolog with meiotic chromosomes; Flaggs G et al.; BACKGROUND: Checkpoint pathways prevent cell-cycle progression in the event of DNA lesions . Checkpoints are well defined in mitosis, where lesions can be the result of extrinsic damage, and they are critical in meiosis, where DNA breaks are a programmed step in meiotic recombination . In mitotic yeast cells, the Chk1 protein couples DNA repair to the cell-cycle machinery . The Atm and Atr proteins are mitotic cell-cycle proteins that also associate with chromatin during meiotic prophase I . The genetic and regulatory interaction between Atm and mammalian Chk1 appears to be important for integrating DNA-damage repair with cell-cycle arrest . RESULTS: We have identified structural homologs of yeast Chk1 in human and mouse . Chk1(Hu/Mo) has protein kinase activity and is expressed in the testis . Chk1 accumulates in late zygotene and pachytene spermatocytes and is present along synapsed meiotic chromosomes . Chk1 localizes along the unsynapsed axes of X and Y chromosomes in pachytene spermatocytes . The association of Chk1 with meiotic chromosomes and levels of Chk1 protein depend upon a functional Atm gene product, but Chk1 is not dependent upon p53 for meiosis I functions . Mapping of CHK1 to human chromosomes indicates that the gene is located at 11q22-23, a region marked by frequent deletions and loss of heterozygosity in human tumors . CONCLUSIONS: The Atm-dependent presence of Chk1 in mouse cells and along meiotic chromosomes, and the late pachynema co-localization of Atr and Chk1 on the unsynapsed axes of the paired X and Y chromosomes, suggest that Chk1 acts as an integrator for Atm and Atr signals and may be involved in monitoring the processing of meiotic recombination . Furthermore, mapping of the CHK1 gene to a region of frequent loss of heterozygosity in human tumors at 11q22-23 indicates that the CHK1 gene is a candidate tumor suppressor gene.

Curr Biol, 1997 Dec 1, 7(12), 987 - 90
Association of a phosphatidylinositol-specific 3-kinase with a human trans-Golgi network resident protein; Hickinson DM et al.; The eukaryotic trans-Golgi network (TGN) is a key site for the formation of transport vesicles destined for different intracellular compartments {1} . A key marker for the mammalian TGN is TGN38/46 {2} . This integral membrane glycoprotein cycles between the TGN and the cell surface and is implicated in recruitment of cytosolic factors and regulation of at least one type of vesicle formation at the mammalian TGN {2} {3} . In this study, we have identified a phosphatidylinositol (PtdIns)-specific 3-kinase activity associated with the human orthologue (TGN46), which is sensitive to lipid kinase inhibitors . Treatment of HeLa cells with low levels of these inhibitors reveals subtle morphological changes in TGN46-positive compartments . Our findings suggest a role for PtdIns 3-kinases and presumably for the product, PtdIns 3-phosphate (PtdIns3P), in the formation of secretory transport vesicles by mechanisms conserved in yeast and mammals.

Curr Biol, 1997 Dec 1, 7(12), R754 - 6
Cytoskeleton: anatomy of an organizing center; Marschall LG et al.; One component of the yeast spindle pole body, Spc42p, has been found to form a crystalline array within one of the central layers of the structure; the Spc42p crystal might provide a scaffold around which the spindle pole body is assembled, and could be involved in regulating the size of the spindle pole body.

J Cell Sci, 1997 Dec, 110 ( Pt 24), 3083 - 90
A casein kinase I isoform is required for proper cell cycle progression in the fertilized mouse oocyte; Gross SD et al.; Casein kinase I is a family of serine/threonine protein kinases common to all eukaryotes . In yeast, casein kinase I homologues have been linked to the regulation of growth, DNA repair and cell division . In addition, their subcellular localization to membraneous structures and the nucleus is essential for function . In higher eukaryotes, there exist seven genetically distinct isoforms: (alpha), ss, (gamma)1, (gamma)2, (gamma)3, (delta) and (epsilon) . Casein kinase I(alpha) exhibits a cell cycle-dependent subcellular localization including an association with cytosolic vesicular structures and the nucleus during interphase, and the spindle during mitosis . casein kinase I has also been shown to modulate critical regulators of growth and DNA synthesis/repair in mammalian cells such as SV40 large T antigen and p53 . These results suggest that casein kinase I may be involved in processes similar to those ascribed to the yeast casein kinase I homologues . To define a role for casein kinase I(alpha) in cell cycle regulation, the mouse oocyte was utilized because of its well-defined cell cycle and ease of micromanipulation . Immunofluorescence studies from meiosis I of maturation to the first zygotic cleavage demonstrated that the kinase was associated with structures similar to those previously reported . Microinjection of casein kinase I(alpha) antibodies at metaphase II-arrest and G2 phase, had no effect on the completion of second meiosis or first division . However, microinjection of these antibodies during the early pronucleate phase prior to S-phase onset blocked uptake of the kinase into pronuclei and interfered with proper and timely cell cycle progression to first cleavage . These results suggest that the kinase regulates the progression from interphase to mitosis during the first cell cycle.

Curr Opin Genet Dev, 1997 Dec, 7(6), 784 - 91
Comparative painting of mammalian chromosomes; Wienberg J et al.; Comparative chromosome painting has shown that synteny has been conserved for large segments of the genome in various placental mammals . Advances such as spectral karyotyping and multicolour 'bar coding' lend speed and precision to comparative molecular cytogenetics . Reciprocal chromosome painting and hybridizations with probes such as yeast artificial chromosomes, cosmids, and fibre fluorescence in situ hybridisation allow subchromosomal assignments of chromosome regions and can identify breakpoints of rearranged chromosomes . Advances in molecular cytogenetics can now be used to test the hypothesis that chromosome rearrangement breakpoints in human pathology and in evolution are correlated.

Microbiology, 1998 Jan, 144 ( Pt 1), 37 - 43
Differential expression of glucose-regulated (grp78) and heat-shock-inducible (hsp70) genes during asexual development of Neurospora crassa; Hafker T et al.; The expression of a glucose-regulated gene (grp78) changes significantly during the vegetative life cycle of Neurospora crassa: the amounts of grp78 mRNA are low in dormant conidia, increase during germination and exponential growth, decline in young aerial hyphae and reach a maximum in late (15-18 h) aerial hyphae . Heat shock (30 min at 45 degrees C) elevated the mRNA level of this gene especially in early aerial hyphae, whereas no increase above the high constitutive amount was found after heat treatment of late aerial hyphae . The expression of the inducible hsp70 gene after heat shock also varied with the state of development and showed the highest inducibility in late aerial hyphae . Surface mycelium, from which aerial hyphae emerge, showed a similar increase in the amounts of both mRNA species . A developmental mutant (acon-2), which is defective in minor constriction budding of aerial hyphae, showed lower levels of con-2 mRNA as well as of grp78 and hsp70 mRNA (after heat shock) in late aerial hyphae . The acon-2 mutant did not form conidia at this stage . It is concluded that the high constitutive and inducible expression of stress genes in late aerial hyphae is due to a developmental activation of their transcription or, alternatively, to a lower degradation rate of their mRNA during this stage.

Biochemistry (Mosc), 1997 Nov, 62(11), 1254 - 62
Immortalized cells with no detectable telomerase activity . A review; Reddel RR et al.; Immortalization of human cells in culture is usually associated with expression of telomerase activity . In some cases, however, no telomerase activity is detectable even though comparison of the terminal restriction fragment (TRF) pattern before and after immortalization shows that lengthening of telomeres has occurred . The extreme heterogeneity in telomere length and the differences in the dynamics of telomere maintenance in telomerase-negative cell lines compared to telomerase-positive cell lines indicate that these cells have utilized one or more alternative mechanisms for lengthening of telomeres (ALT) . All telomerase-negative immortalized cell lines examined to date show evidence of ALT activity, consistent with the hypothesis that telomere maintenance either by telomerase or by ALT is required for immortalization . The nature of the ALT mechanism(s) is currently unknown, but studies of telomere dynamics in an ALT cell line containing a marker just proximal to the telomeric sequences show gradual shortening of the telomere followed by rapid elongation . This is consistent with a non-reciprocal recombinational mechanism similar to that found in telomerase-defective mutant yeast strains.

J Med Vet Mycol, 1997 Nov-Dec, 35(6), 379 - 88
Importance of calcium to the regulation of polymorphism in Wangiella (Exophiala) dermatitidis; Karuppayil SM et al.; Critical steps implicated in the polymorphism of Wangiella dermatitidis were found to be sensitive to calcium ion availability . When grown in a defined, synthetic medium under various pH and temperature conditions, two thresholds of calcium ion concentrations were identified: a lower concentration favouring non-polarized growth leading to multicellular form development and a higher concentration promoting polarized growth characterized by yeast budding or pseudo/true hyphal growth . The phenotypic transition of yeasts to multicellular forms or to hyphae was induced at both 25 and 37 degrees C in the wild-type strain by the addition of calcium to the synthetic medium adjusted to pH 2.5, which was otherwise not conducive to the production of either growth form . However, the calcium additions did not allow maintenance of polarized growth of yeasts or hyphae in a temperature-sensitive, cell-division-cycle mutant (wdcdc2) derived from the same strain and grown at 37 degrees C in the same medium adjusted to either pH 2.5 or 6.5 . Instead these conditions allowed only the nonpolarized, multicellular form development associated with this conditional mutant cultured in rich media at the 37 degree C restrictive temperature for yeast bud formation . Results from experiments using the calcium chelator EGTA added to the synthetic medium supported these conclusions at neutral pH with both the wild type and the wdcdc2 mutant cultured at 37 degrees C . The results suggested that during infection different concentrations of calcium may be encountered by W . dermatitidis in different tissues, which might directly regulate its growth and polymorphism and indirectly its virulence depending on host conditions.

J Colloid Interface Sci, 1998 Jan 1, 197(1), 29 - 35
Reverse Micelle Systems Composed of Water, Triton X-100, and Phospholipids in Organic Solvents
Fernandez-Velasco DA, Rodriguez R, Vargas S, de Gomez-Puyou MT, Gomez-Puyou A.
Catalysis, stability, and thermostability of yeast hexokinase were determined in the microenvironments of two organic solvent/Triton X-100/phospholipids systems . In the abscence of enzyme, phase diagrams showed two transparent/turbid transitions, and reverse micelles were only observed in the second region of transparency (T2), where particle size as a function of water content shows a minima (see previous paper in this issue) . In the present work, enzyme activity was detected throughout the four regions of the phase diagrams of these systems . Catalysis increased with water content; nevertheless, the maximum activities that were reached in the toluene and propylbenzene systems were 30 and 1.6%, respectively, of the activity in all aqueous media . Because in the T2 region in the propylbenzene system, micelles are much smaller than in toluene (see preceding paper), it would appear that expression of catalysis depends on the size of the micelles . However, a comparison of the dimensions of hexokinase and those of reverse micelles in the T2 region, suggests that in this region, hexokinase entrapment increases the inner volume of the micelle . High enzyme thermostability was only observed in the first transparent region (T1) of the system that contained phospholipids . In this region, hexokinase induced the formation of reverse micelles from dispersed surfactant monomers . There is a striking similarity in the dimensions of hexokinase entrapped in reverse micelles as determined by dynamic light scattering measurements in the T1 region with those of hexokinase as obtained from X ray diffraction studies of the enzyme in a crystalline environment . This suggest that high thermostability, and low catalytic rates result from restrictions in mobility imposed by a low water environment .

Arch Biochem Biophys, 1998 Feb 1, 350(1), 109 - 17
Further studies on the coupling of mitochondrially bound hexokinase to intramitochondrially compartmented ATP, generated by oxidative phosphorylation; Cesar Mde C et al.; Hexokinase, bound to nonphosphorylating rat brain mitochondria, exhibits Michaelis-Menten kinetic behavior, with an apparent K(m) for ATP of 0.44 +/- 0.08 mM . After initiation of oxidative phosphorylation, a steady-state rate of Glc phosphorylation is maintained despite the fact that extramitochondrial {ATP} continues to increase but remains well below saturating levels (i.e., < 0.4 mM) . This independence from extramitochondrial {ATP} is taken to indicate that hexokinase is not utilizing extramitochondrial ATP as substrate, but rather draws substrate ATP from an intramitochondrial compartment supplied by oxidative phosphorylation . The steady-state rate of Glc phosphorylation by hexokinase bound to phosphorylating mitochondria is not altered by increase in total rate of ATP production resulting from addition of hexokinase-depleted mitochondria to the system . In contrast, the steady-state rate of Glc phosphorylation by yeast hexokinase, which does not bind to mitochondria, is directly related to the total rate of ATP production in the system . These results are also consistent with the view that, during oxidative phosphorylation, mitochondrially bound hexokinase is selectively using intramitochondrially compartmented ATP; such substrate selectivity would be expected to require physical association of hexokinase with the mitochondria and be dependent solely on the oxidative phosphorylation activity of the hexokinase-bearing organelles . The K(m) for Glc is only modestly affected by the binding of hexokinase to mitochondria and not further altered upon induction of active oxidative phosphorylation, suggesting that neither binding nor oxidative phosphorylation greatly affects the conformation of the Glc binding site . The reliance on intramitochondrial ATP is suggested to result from oxidative phosphorylation-dependent changes in the interaction between the mitochondrial surface and the regions of the hexokinase molecule involved in binding ATP.

Cytogenet Cell Genet, 1997, 78(3-4), 247 - 52
Molecular studies of an ependymoma-associated constitutional t(1;22)(p22;q11.2); Rhodes CH et al.; We previously described a patient with a de novo constitutional translocation, t(1;22)(p22;q11.2), who developed a malignant ependymoma at age 5, and we proposed that the translocation predisposed the child to the development of the tumor . As a step toward isolation of a putative cancer gene, we have characterized the breakpoints of the (1;22) translocation at the molecular level . The chromosome 22 breakpoint has been narrowed to a region between ARVCF and D22S264 . The chromosome 1 breakpoint has been mapped onto a doubly-linked Whitehead YAC contig by PCR analysis of the STS contents of the patient's derivative chromosomes isolated in somatic cell hybrids . Loss-of-heterozygosity (LOH) studies of the patient's ependymoma and of sporadic ependymomas showed no evidence of consistent loss in the breakpoint regions, suggesting that activation of an oncogene, rather than inactivation of a tumor suppressor gene, is the more likely molecular mechanism involved in this case . The gene for Edg-1, a neurally expressed, seven-segment transmembrane receptor, maps to the region of the chromosome 1 breakpoint but does not appear to be interrupted by the translocation . Molecular characterization of the breakpoint regions reported here represents an important step in the identification of the gene(s) affected by this translocation.

Cytogenet Cell Genet, 1997, 78(3-4), 240 - 6
Novel COL4A5/COL4A6 deletions and further characterization of the diffuse leiomyomatosis-Alport syndrome (DL-AS) locus define the DL critical region; Heidet L et al.; Diffuse leiomyomatosis (DL) with Alport syndrome (AS) has been shown to be associated with contiguous gene deletions of the COL4A5 and COL4A6 genes, with the COL4A6 breakpoint of the deletions invariably located in the large intron 2 of the gene . We describe four YAC clones covering the locus and a refined restriction map of the entire COL4A6 gene . These resources have allowed us to make a precise estimate of the size of COL4A6 introns 2 and 3, as well as the size of the gene itself . We also describe five novel deletions which, in conjunction with previous reports, allow the definition of a 90-kb critical region in which to search for a gene or other entity involved in the pathogenesis of DL.

FEMS Microbiol Lett, 1998 Jan 15, 158(2), 255 - 60
Effect of peroxisomicine A2 and T 544 of the genus Karwinskia on peroxisomes of Candida boidinii; Salazar-Aranda R et al.; Dimeric anthracenones have been isolated from toxic plants of the genus Karwinskia (Rhamnaceae) . T 514 or peroxisomicine A1 is one of the anthracenonic compounds which produce irreversible and selective damage on the peroxisomes of yeast cells in vivo . In this paper we describe the effect of two structurally related anthracenones on cell viability and on the peroxisomes of the methylotrophic yeast Candida boidinii . As has been described for peroxisomicine A1, peroxisomicine A2 and T 544 caused a decrease in the viability of C . boidinii at all concentrations tested, and disruption of the peroxisomal membrane, T 544 showing the strongest effect . In C . boidinii cell death and peroxisomal damage seem to be independent events.

Genomics, 1998 Jan 1, 47(1), 143 - 5
The human glutamate receptor delta 2 gene (GRID2) maps to chromosome 4q22; Hu W et al.; We isolated the human glutamate receptor delta 2 (GRID2) gene, which has 97.0% identity in amino acid sequence to the mouse glutamate receptor delta 2 (Grid2) gene . We subsequently mapped this gene to human chromosome 4q22 by radiation hybrid mapping and by hybridization to two overlapping human yeast artificial chromosomes that are located in 4q22 . The Grid2 gene, which is mutated in lurcher (Lc) mice, maps to mouse chromosome 6 . Thus, the mapping of the GRID2 gene to human chromosome 4q22 confirms and refines a region of synteny between mouse and human genomes.

Genomics, 1998 Jan 1, 47(1), 125 - 30
Sequence characterization of a newly identified human alpha-tubulin gene (TUBA2); Dode C et al.; We report on the isolation and initial characterization of a human alpha-tubulin gene named TUBA2 . This gene is located in the 13q11 region and has been considered a candidate gene for two nonsyndromic deafnesses, DFNB1 and DFNA3 . The gene, with a minimum size of 6.5 kb, contains five exons and four introns starting at codon positions 1, 76, 125, and 352, one of which is inserted between the initiation methionine codon and the codon specifying the second amino acid, arginine 2 . Neither rearrangement nor point mutation was found in the coding region of the gene in DFNB1- and DFNA3-affected patients . The gene was therefore unlikely to be responsible for either of these deafnesses . During the characterization of TUBA2, the gene encoding connexin 26 was proven to be responsible for both DFNB1 and DFNA3 (D . P . Kelsell et al., 1997, Nature 387: 80-83) . However, the present data offer the possibility of testing the involvement of the TUBA2 gene in the Clouston hidrotic ectodermal dysplasia and the Kabuki syndrome, two genetic diseases that have recently been mapped to the 13q11 region.

Genomics, 1998 Jan 1, 47(1), 115 - 20
Mapping of the human genes encoding cyclin H (CCNH) and the CDK-activating kinase (CAK) assembly factor MAT1 (MNAT1) to chromosome bands 5q13.3-q14 and 14q23, respectively; Eki T et al.; Cyclin-dependent kinases (CDKs), which play a key role in cell cycle control, are activated by the CDK-activating kinase (CAK), which activates cyclin-bound CDKs by phosphorylation at the specific threonine residue . Mammalian CAK contains three components: CDK7, cyclin H, and an assembly factor called MAT1 . The CDK7-cyclin H-MAT1 complex is tightly associated with a multiprotein complex TFIIH, which plays a dual role in transcription and DNA repair . Here, we have determined chromosomal localizations of the human genes encoding cyclin H (CCNH) and MAT1 (HGMW-approved symbol MNAT1) to chromosome bands 5q13.3-q14 and 14q23, respectively, by using fluorescence in situ hybridization, somatic cell hybrid analyses, and mapping to the human YAC contigs.

Genomics, 1998 Jan 1, 47(1), 26 - 43
High-resolution YAC-cosmid-STS map of human chromosome 13; Cayanis E et al.; We have assembled a high-resolution physical map of human chromosome 13 DNA (approximately 114 Mb) from hybridization, PCR, and FISH mapping data using a specifically designed set of computer programs . Although the mapping of 13p is limited, 13q (approximately 98 Mb) is covered by an almost continuous contig of 736 YACs aligned to 597 contigs of cosmids . Of a total of 10,789 cosmids initially selected from a chromosome 13-specific cosmid library (16,896 colonies) using inter-Alu PCR probes from the YACs and probes for markers mapped to chromosome 13, 511 were assembled in contigs that were established from cross-hybridization relationships between the cosmids . The 13q YAC-cosmid map was annotated with 655 sequence tagged sites (STSs) with an average spacing of 1 STS per 150 kb . This set of STSs, each identified by a D number and cytogenetic location, includes database markers (198), expressed sequence tags (93), and STSs generated by sequencing of the ends of cosmid inserts (364) . Additional annotation has been provided by positioning 197 cosmids mapped by FISH on 13q . The final (comprehensive) map, a list of STS primers, and raw data used in map assembly are available at our Web site (genome1.ccc.columbia.edu/ approximately genome/) and can serve as a resource to facilitate accurate localization of additional markers, provide substrates for sequencing, and assist in the discovery of chromosome 13 genes associated with hereditary diseases.

Appl Environ Microbiol, 1998 Feb, 64(2), 669 - 77
Reverse transcriptase (RT) inhibition of PCR at low concentrations of template and its implications for quantitative RT-PCR; Chandler DP et al.; Numerous instances of reverse transcriptase (RT) inhibition of the PCR were observed while developing nonquantitative uncoupled RT-PCR techniques for detecting nitrogenase and ammonia monooxygenase gene expression in situ . The inhibitory effect of RT on the PCR was removed with increasing template concentrations beyond 10(5) to 10(6) copies . Including T4 gene 32 protein during the reverse transcription phase of the RT-PCR reaction increased the RT-PCR product yield by as much as 483%; if gene 32 protein was introduced after reverse transcription but prior to the PCR phase, no improvement in product yield was observed . Addition of 1 microgram of exogenous calf thymus DNA or yeast tRNA did little to relieve RT inhibition of the PCR on both genomic DNA and mRNA templates . These results suggest that RT inhibition of the PCR is mediated through direct interaction with the specific primer-template combination (DNA and RNA) and point to specific assay modifications for estimating the extent of RT inhibition and counteracting some of the inhibitory effect . Furthermore, the working hypothesis of RT inhibition below a 10(5) to 10(6) copy threshold has important implications for quantitative RT-PCR studies . In particular, competitive, quantitative RT-PCR systems will consistently underestimate the actual RNA concentration . Hence, enumerations of RNA templates below 10(5) to 10(6) copies will be relative to an internal standard and will not be an absolute measure of RNA abundance in situ.

Oncogene, 1998 Jan 15, 16(2), 227 - 36
Interaction of the pRB-family proteins with factors containing paired-like homeodomains; Wiggan O et al.; The specific loss of pRB or p107 together with p130 disrupts the normal development of only a very limited spectrum of tissues . These developmental defects have been attributed primarily to deregulation of E2F activity and consequent uncontrolled proliferation . We hypothesized, however, that the tissue-specific nature of these defects may also reflect deregulation of pRB-family associated factors that are specifically involved in determining cell fate . We report here that the pRB-family members interact with transcription factors which contain paired-like homeodomains such as MHox, Chx10 and Pax-3 . The interaction between the pRB-family and the paired-like homeodomain proteins was initially identified in a yeast two-hybrid screen where the N-terminal portion of p130 was used to isolate interacting factors from an embryonic mouse library . This interaction was confirmed by in vitro binding and co-immunoprecipitation assays . We show further that co-expression of Pax-3 dependent pRB, p107 or p130 with Pax-3 causes repression of activated transcription from the c-met promoter . These data demonstrate that the pRB-family proteins can modulate the activity of factors which specifically control cell fate and/or differentiation as well as controlling cell cycle regulators.

Biochem Biophys Res Commun, 1998 Jan 26, 242(3), 648 - 52
Differential display cloning of a novel human histone deacetylase (HDAC3) cDNA from PHA-activated immune cells; Dangond F et al.; The nucleosomal histones can be modified through reversible acetylation by histone acetyltransferases (HATs) and deacetylases (HDACs) . HATs induce nucleosomal relaxation and allow DNA-binding by transcriptional activators . HDACs from corepressor complexes which negatively regulate cell growth . However, the HDAC inhibitors butyrate and Trichostatin A block T cell proliferation, suggesting that not all effects of HDACs lead to repression . Using mRNA differential display and 5'RACE we isolated human HDAC3, a novel gene that is upregulated in PHA-activated T cell clones . HDAC3 is homologous to other human HDACs and yeast RPD3 . In peripheral blood mononuclear cells (PBMCs), activation by PHA, PMA and alpha-CD3 increased HDAC mRNA but no effect was seen with IFN-gamma, LPS, or IL-4 . In contrast, GMCSF downregulated PBMC levels of HDAC3 mRNA . All HDACs were found to be ubiquitously expressed in immune and non-immune tissues . In human myeloid leukemia THP-1 cells, HDAC3 transfection resulted in increased size, aberrant nuclear morphology and cell cycle G2/M cell accumulation . Functional activity of the expressed HDAC3 protein was confirmed in alpha-HDAC3 antibody immunoprecipitates by a histone deacetylase assay . Our study suggests the participation of HDACs in cell cycle progression and activation.

Nature, 1998 Jan 29, 391(6666), 493 - 6
Regulation of the Hedgehog and Wingless signalling pathways by the F-box/WD40-repeat protein Slimb; Jiang J et al.; Members of the Hedgehog (Hh) and Wnt/Wingless (Wg) families of secreted proteins control many aspects of growth and patterning during animal development . Hh signal transduction leads to increased stability of a transcription factor, Cubitus interruptus (Ci), whereas Wg signal transduction causes increased stability of Armadillo (Arm/beta-catenin), a possible co-factor for the transcriptional regulator Lef1/TCF . Here we describe a new gene, slimb (for supernumerary limbs), which negatively regulates both of these signal transduction pathways . Loss of function of slimb results in a cell-autonomous accumulation of high levels of both Ci and Arm, and the ectopic expression of both Hh- and Wg- responsive genes . The slimb gene encodes a conserved F-box/WD40-repeat protein related to Cdc4p, a protein in budding yeast that targets cell-cycle regulators for degradation by the ubiquitin/proteasome pathway . We propose that Slimb protein normally targets Ci and Arm for processing or degradation by the ubiquitin/proteasome pathway, and that Hh and Wg regulate gene expression at least in part by inducing changes in Ci and Arm, which protect them from Slimb-mediated proteolysis.

Nature, 1998 Jan 29, 391(6666), 485 - 8
Analysis of 1.9 Mb of contiguous sequence from chromosome 4 of Arabidopsis thaliana; Bevan M et al.; The plant Arabidopsis thaliana (Arabidopsis) has become an important model species for the study of many aspects of plant biology . The relatively small size of the nuclear genome and the availability of extensive physical maps of the five chromosomes provide a feasible basis for initiating sequencing of the five chromosomes . The YAC (yeast artificial chromosome)-based physical map of chromosome 4 was used to construct a sequence-ready map of cosmid and BAC (bacterial artificial chromosome) clones covering a 1.9-megabase (Mb) contiguous region, and the sequence of this region is reported here . Analysis of the sequence revealed an average gene density of one gene every 4.8 kilobases (kb), and 54% of the predicted genes had significant similarity to known genes . Other interesting features were found, such as the sequence of a disease-resistance gene locus, the distribution of retroelements, the frequent occurrence of clustered gene families, and the sequence of several classes of genes not previously encountered in plants.

Toxicol Ind Health, 1998 Jan-Apr, 14(1-2), 261 - 73
Cellular bioavailability of natural hormones and environmental contaminants as a function of serum and cytosolic binding factors; Crain DA et al.; Environmental contaminants have been reported to function as hormone mimics in various wildlife species . To investigate a potential mechanism for the interaction of contaminants with the endocrine system, we evaluated the cellular bioavailability of numerous chemicals . Hormone binding proteins from oviductal cytosol of the American alligator (Alligator mississippiensis) and yellow-bellied turtle (Trachemys scripta) were used in competitive binding assays with {3H} 17 beta-estradiol . Most of the environmental contaminants, and the potent, synthetic estrogen diethylstilbestrol (DES), did not interact with the cytosolic binding proteins . Among the compounds tested, o,p'-DDT and toxaphene exhibited the greatest affinity for the binding proteins . The functional consequence of the apparent lack of interaction of most contaminants with binding proteins was studied in a strain of yeast containing the human estrogen receptor (YES assay) . The activation of YES with estradiol was reduced 30% in the presence of a physiological concentration (0.01 mg/mL) of human sex hormone binding globulin (SHBG), a hormone binding protein found in the blood . In contrast, the activity of DES was not inhibited by 0.01 mg/mL SHBG . Interestingly, ethinyl estradiol, a major component of contraceptives, did not appear to appreciably interact with SHBG in the YES system . Together, these data suggest that cytosolic and circulating binding proteins bind many environmental contaminants with much less affinity than native steroids . Therefore, such contaminants may be more hormonally active than previously hypothesized.

J Nat Prod, 1997 Dec, 60(12), 1281 - 6
Bioactive and other sesquiterpenes from Chiloscyphus rivularis; Wu C et al.; Bioassay-directed fractionation of the methyl ethyl ketone extract of Chiloscyphus rivularis yielded five new sesquiterpenes, 12-hydroxychiloscyphone (2), chiloscypha-2,7-dione (3), 12-hydroxychiloscypha-2,7-dione (4), chiloscypha-2,7,9-trione (5), and rivulalactone (6) in addition to the known sesquiterpenes, 4-hydroxyoppositan-7-one (7), chiloscyphone (1), and isointermedeol (8) . The structure and stereochemistry of rivulalactone, a novel trinorsesquiterpene, was confirmed by its synthesis starting from 1 . Compound 2 showed selective bioactivity in our yeast-based DNA-damaging assay and cytotoxicity to human lung carcinoma cells.

Biol Chem, 1997 Dec, 378(12), 1521 - 30
Long non-stop reading frames on the antisense strand of heat shock protein 70 genes and prion protein (PrP) genes are conserved between species; Rother KI et al.; Several mammalian genes, including heat shock protein (Hsp70) and prion protein (PrP) genes, have been reported to have long open reading frames (ORFs) or non-stop reading frames (NRFs) in the antisense direction . A simple explanation would be that these long antisense reading frames, which are usually in the same triplet frame as the coding strand, are the fortuitous byproduct of a high overall {G+C} content with concomitant preference for G/C over A/T in the third codon position, a preference for RNY type codons (purine/any nucleotide/pyrimidine), and/or a bias against serine and leucine, the only amino acids with codons that can be read as stop codons in the antisense direction . The PrP genes and most heat shock genes with long antisense NRFs (aNRFs) are indeed relatively {G+C} rich but do not show a bias against serine and leucine . In several vertebrates investigated, at least one of the Hsp70 genes has a long antisense reading frame, and we found that some, though not all, putative stop codons in long Hsp70 antisense reading frames were due to sequencing errors . The PrP gene contains an extended antisense open reading frame in all 45 eutherian mammals tested, but not in a marsupial and in a bird . In the PrP gene, the long, protein-coding exon also harbors the antisense nonstop reading frame . In both Hsp70 and PrP genes, the putative antisense protein sequence is well conserved . Even though there is no clear evidence in Hsp70 or PrP genes for the existence of the respective antisense proteins, we speculate that such antisense proteins serve to regulate the genuine Hsp and PrP proteins under special circumstances . Alternatively, regulation might occur at the RNA level, and the antisense RNA would merely lack stop codons to prevent its rapid degradation by an mRNA quality control mechanism that is triggered by premature stop codons . We note that both Hsp and PrP are involved in physiological or pathological protein aggregation phenomena, that scrapie prions have been reported to modify the expression or localization of heat shock proteins, and that in yeast, propagation of a prion-like state (PSI+) depends on a heat shock (Hsp104) protein.

Lab Invest, 1998 Jan, 78(1), 117 - 25
Identification and partial characterization of PDZK1: a novel protein containing PDZ interaction domains; Kocher O et al.; We recently reported the isolation and partial characterization of a novel membrane-associated protein designated MAP17 . In normal tissues, MAP17 was expressed only in the apical brush border of proximal tubular epithelial cells of the human adult kidney . However, MAP17 was diffusely expressed in most carcinomas originating in the kidney, colon, lung, and breast . Transfection of MAP17 into the HT29 carcinoma cell line markedly decreased cell proliferation in vitro and tumor growth in vivo, suggesting that MAP17 plays a role, either direct or indirect, in the control of cell proliferation . In an attempt to elucidate the function of MAP17, we screened a human kidney cDNA library for interacting proteins using the yeast two-hybrid system and isolated a novel protein containing PDZ protein interaction domains, which we have named PDZK1 . PDZK1 is a 519-amino acid protein with a molecular weight of 63 kd; it is expressed in the kidney, pancreas, liver, gastrointestinal tract, and adrenal cortex . In situ hybridization experiments showed that the expression of PDZK1 was limited to epithelial cells . In the kidney, it colocalized with MAP17 in the brush border of proximal tubular epithelial cells . In addition, PDZK1 was overexpressed in selected tumors of epithelial origin . Although the function of PDZK1 has yet to be determined, proteins containing PDZ domains have been shown to play important roles as diverse as cell-cell interaction, cell differentiation, growth control, ion channels organization, and signal transduction . This is of particular interest because MAP17 is localized in areas either of cell-cell contact or where ion channels are localized, for example in the kidney . PDZK1 may represent the link between the cell membrane-where it interacts with MAP17-and other cytoplasmic proteins involved in biologic functions such as cell proliferation, differentiation, and ion transport.

Zh Mikrobiol Epidemiol Immunobiol, 1997 Nov-Dec, (6), 55 - 8
{The efficacy of recombinant vaccines against hepatitis B and the results of their use in a schedule for pediatric prophylactic inoculations}; Gorbunov MA et al.; The results of field clinical trials of Russian and American yeast vaccines against hepatitis B are presented . The study revealed that both vaccines were faintly reactogenic, safe and exhibited high immunological activity . After the full course of immunization following the schedule 0-1-2 months 92.5% and 97.5% of patients receiving, respectively . Russian vaccine "Combiotech" and American vaccine "H-B-Vax II" were found to have specific antibodies . The maximum effect was registered when the vaccines were introduced according to the schedule 0-1-6 months . Seroconversions were observed in 97.5% and 100% of the vaccinees receiving the Russian and American vaccines respectively, in the latter case the highest antibody level being observed . The use of the vaccines within the prophylactic immunization schedule showed that antibodies to hepatitis B appeared in immunized children in 93-100% of cases . Seroconversion indices and the levels of antibodies to diphtheria, tetanus, poliomyelitis and measles were statistically significant and were the same in children receiving only the vaccines according to the immunization schedule and in children immunized, in addition to these vaccines, against hepatitis B.

Psychiatr Genet, 1997 Winter, 7(4), 165 - 9
Physical mapping of a glutamate receptor gene in relation to a balanced translocation associated with schizophrenia in a large Scottish family; Devon RS et al.; The metabotropic glutamate receptor subtype 5a (mGluR5a) gene has been localised on the Gene Map of the Human Genome to chromosome 11q, approximately 1 cM from the genetic marker D11S931 . D11S931 has been shown to lie close to a translocation breakpoint associated with schizophrenia and other psychiatric disorders in a large Scottish family . Because glutamate receptor genes are excellent candidates for psychiatric disorders, we have investigated the physical distance of this gene from the translocation breakpoint on chromosome 11 . We have shown that the mGluR5a gene lies at least 850 kb from the breakpoint and, hence, cannot be directly disrupted in translocation carriers . However, a long range position effect of the translocation on this gene, or co-segregation of the translocation with a mutant allele of mGluR5a cannot be ruled out.

Cancer Genet Cytogenet, 1998 Feb, 101(1), 24 - 34
A distamycin A-inducible fragile site, FRA8E, located in the region of the hereditary multiple exostoses gene, is not involved in HPV16 DNA integration and amplification; Hori T et al.; The rare fragile site is a specific point on a chromosome that is expressed as an isochromatid gap or break under certain conditions of cell culture and is inherited in a Mendelian codominant fashion . Five folate-sensitive fragile sites were cloned, and the molecular basis of fragile site mutation was shown to be a new class of mutation, called dynamic mutation, resulting from an allelic expansion of (CCG)n repeats . The mechanism responsible for other types of rare fragile sites, i.e., distamycin A-inducible and BrdU-requiring, is unknown, although cytogenetic studies suggested that these fragile sites play a mechanistic role in breakage and recombination and may also be integration and modification sites of foreign viral DNA genomes . A distamycin A-inducible fragile site, FRA8E, is mapped to 8q24.1 in which various loci implicated in genomic instability are located . Here we identified a YAC clone spanning both FRA8E and the hereditary multiple exostosis (EXT1) gene, using fluorescence in situ hybridization (FISH) analysis of a yeast artificial chromosome (YAC) contig . By using P1 clones as probes, the FRA8E locus was further localized to a 400-kb region including the EXT1 gene . Furthermore, the integration and amplification site of human papillomavirus 16 DNA in the ASCC (argyrophil small cell carcinoma) cells were shown not to coincide with FRA8E, but to be involved in an extensively broad genomic region of 8q24.1, including the c-myc gene.

J Biol Chem, 1998 Jan 9, 273(2), 1044 - 51
Dymple, a novel dynamin-like high molecular weight GTPase lacking a proline-rich carboxyl-terminal domain in mammalian cells; Kamimoto T et al.; We have cloned human dymple, a novel dynamin family member . The full-length cDNA sequence encodes a protein composed of 736 amino acids with a molecular mass of 80 kDa . This amino acid sequence most resembles yeast DNM1P and VPS1P . Dymple lacks a proline-rich carboxyl-terminal domain through which dynamin binds to SH3 domains to be activated . Northern blot analysis revealed two transcript sizes of 2.5 and 4.2 kilobases with alternative polyadenylation at the highest levels in brain, skeletal muscle, and testis . It was further established that there are three patterns of alternative splicing producing in-frame deletions in the coding sequence of dymple in a tissue-specific manner . When overexpressed, wild-type dymple exhibited a punctate perinuclear cytoplasmic pattern, whereas an amino-terminal deletion mutant formed large aggregates bounded by a trans-Golgi network marker . Since dynamin participates in clathrin-mediated endocytosis through a well-characterized mechanism, the existence of a dynamin-like molecule in each specific vesicle transport pathway has been predicted . Our findings suggest that dymple may be the first example of such a subfamily in mammalian cells other than dynamin itself, although its precise role and membrane localization remain to be resolved.

J Biol Chem, 1998 Jan 9, 273(2), 814 - 21
Identification and characterization of a novel protein interacting with Ral-binding protein 1, a putative effector protein of Ral; Ikeda M et al.; Ral-binding protein 1 (RalBP1) is a putative effector protein of Ral and exhibits a GTPase activating activity for Rac and CDC42 . To clarify the function of RalBP1, we isolated a novel protein that interacts with RalBP1 by yeast two-hybrid screening and designated it POB1 (partner of RalBP1) . POB1 consists of 521 amino acids, shares a homology with Eps15, which has been identified as an epidermal growth factor (EGF) receptor substrate, and has two proline-rich motifs . The POB1 mRNA was expressed in cerebrum, cerebellum, lung, kidney, and testis . POB1 interacted with RalBP1 in COS cells and the C-terminal region of POB1 was responsible for this interaction . The binding domain of RalBP1 to POB1 was distinct from its binding domain to Ral . Ral and POB1 simultaneously interacted with RalBP1 in COS cells . The binding of POB1 to RalBP1 did not affect the GTPase activating activity of RalBP1 . Furthermore, POB1 bound to Grb2 but not to Nck or Crk . POB1 was tyrosine-phosphorylated in COS cells upon stimulation with EGF and made a complex with EGF receptor . These results suggest that RalBP1 makes a complex with POB1 and that this complex may provide a link between tyrosine kinase, Src homology 3 (SH3)-containing protein, and Ral.

J Biol Chem, 1998 Jan 9, 273(2), 720 - 8
Necdin, a postmitotic neuron-specific growth suppressor, interacts with viral transforming proteins and cellular transcription factor E2F1; Taniura H et al.; Necdin is a nuclear protein expressed in virtually all postmitotic neurons, and ectopic expression of this protein strongly suppresses the proliferation of NIH3T3 cells . Simian virus 40 large T antigen targets both p53 and the retinoblastoma protein (Rb) for cellular transformation . By analogy with the interactions of the large T antigen with these nuclear growth suppressors, we examined the ability of necdin to bind to the large T antigen . Necdin was co-immunoprecipitated with the large T antigen from the nuclear extract of necdin cDNA-transfected COS-1 cells . Yeast two-hybrid and in vitro binding analyses revealed that necdin bound to an amino-terminal region of the large T antigen, which encompasses the Rb-binding domain . Moreover, necdin bound to adenovirus E1A, another viral oncoprotein that forms a specific complex with Rb . We then examined the ability of necdin to bind to the transcription factor E2F1, a cellular Rb-binding factor involved in cell-cycle progression . Intriguingly, necdin, like Rb, bound to a carboxyl-terminal domain of E2F1, and repressed E2F-dependent transactivation in vivo . In addition, necdin suppressed the colony formation of Rb-deficient SAOS-2 osteosarcoma cells . These results suggest that necdin is a postmitotic neuron-specific growth suppressor that is functionally similar to Rb.

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