Mol Gen Genet, 1996 Mar 20, 250(5), 533 - 9 Characterization and subcellular localization of a small GTP-binding protein (Ara-4) from Arabidopsis: conditional expression under control of the promoter of the gene for heat-shock protein HSP81-1; Ueda T et al.; Small GTP-binding proteins belonging to the rab/YPT family play key roles at various steps in intracellular transport pathways in yeast and mammalian cells . Many members of rab/YPT family have been isolated from plants to date . However, detailed information about the localization and function of the gene products remains limited, even though intracellular transport is likely to be involved in important phenomena such as cell elongation, transport of storage proteins, determination and maintenance of cell polarity and intercellular signal transduction . We have attempted to establish transgenic Arabidopsis plants that overexpress ARA-4, a rab/YPT homologue in order to analyze the function and the localization of the gene product . For overexpression and also for regulation of the expression of this gene, the promoter of the gene for HSP81-1 was employed to drive the transcription of ARA-4 in transgenic plants . The response of the introduced genes to heat shock was analyzed . Upon heat-shock treatment, the ARA-4 gene was efficiently transcribed and translated . The induction of ARA-4 by heat shock was transient, and at least two distinct forms of this protein were found in membrane and cytosolic fractions from transgenic plants . Prolonged incubation after heat shock reduced the amount of the cytosolic form of the induced protein, and the cytosolic form of the protein thus probably represents the unprocessed precursor . Using transgenic plants, we determined the subcellular localization of the product of ARA-4 . The protein was predominantly localized on Golgi-derived vesicles, Golgi cisternae and the trans-Golgi network. Proc Natl Acad Sci U S A, 1996 Mar 19, 93(6), 2426 - 30 A long-range regulatory element of Hoxc8 identified by using the pClasper vector; Bradshaw MS et al.; Hox genes are located in highly conserved clusters . The significance of this organization is unclear, but one possibility is that regulatory regions for individual genes are dispersed throughout the cluster and shared with other Hox genes . This hypothesis is supported by studies on several Hox genes in which even large genomic regions immediately surrounding the gene fail to direct the complete expression pattern in transgenic mice . In particular, previous studies have identified proximal regulatory regions that are primarily responsible for early phases of mouse Hoxc8 expression . To locate additional regulatory regions governing expression during the later periods of development, a yeast homologous recombination-based strategy utilizing the pClasper vector was employed . Using homologous recombination into pClasper, we cloned a 27-kb region around the Hoxc8 gene from a yeast artificial chromosome . A reporter gene was introduced into the coding region of the isolated gene by homologous recombination in yeast . This large fragment recapitulates critical aspects of Hoxc8 expression in transgenic mice . We show that the regulatory elements that maintain the anterior boundaries of expression in the neural tube and paraxial mesoderm are located between 11 and 19 kb downstream of the gene. FEBS Lett, 1996 Mar 18, 382(3), 261 - 4 Induction of proteins in response to cold acclimation of rainbow trout cells; Yamashita M et al.; The in vitro translation of poly(A)-RNA isolated from control and cold-treated cells showed that low temperatures induced changes in the population of translatable mRNAs . When cellular proteins extracted from cold-treated cells were subjected to two-dimensional gel electrophoresis, the 70 kDa protein was found to be synthesized during the cold treatment . N-terminal sequence analysis showed that the 70 kDa cold-inducible protein was a homolog of the mammalian valosin-containing protein and yeast CDC48p . The changes in mRNA and protein contents during cold acclimation may result from the expression of genes involved in the adjustment of cellular metabolism to low temperature or the induced proteins may be directly involved in the cold acclimation. Genomics, 1996 Mar 15, 32(3), 447 - 54 YAC contigs of the Rab1 and wobbler (wr) spinal muscular atrophy gene region on proximal mouse chromosome 11 and of the homologous region on human chromosome 2p; Wedemeyer N et al.; Despite rapid progress in the physical characterization of murine and human genomes, little molecular information is available on certain regions, e.g., proximal mouse chromosome 11 (Chr 11) and human chromosome 2p (Chr 2p) . We have localized the wobbler spinal atrophy gene wr to proximal mouse Chr 11, tightly linked to Rab1, a gene coding for a small GTP-binding protein, and Glnsps1, an intronless pseudogene of the glutamine synthetase gene . We have now used these markers to construct a 1.3-Mb yeast artificial chromosome (YAC) contig of the Rab1 region on mouse Chr 11 . Four YAC clones isolated from two independent YAC libraries were characterized by rare-cutting analysis, fluorescence in situ hybridization (FISH), and sequence-tagged site (STS) isolation and mapping . Rab1 and Glns-ps1 were found to be only 200 kb apart . A potential CpG island near a methylated NarI site and a trapped exon, ETG1.1, were found between these loci, and a new STS, AHY1.1, was found over 250 kb from Rab1 . Two overlapping YACs were identified that contained a 150-kb region of human Chr 2p, comprising the RAB1 locus, AHY1.1, and the human homologue of ETG1.1, indicating a high degree of conservation of this region in the two species . We mapped AHY1.1 and thus human RAB1 on Chr 2p13.4-p14 using somatic cell hybrids and a radiation hybrid panel, thus extending a known region of conserved synteny between mouse Chr 11 and human Chr 2p . Recently, the gene LMGMD2B for a human recessive neuromuscular disease, limb girdle muscular dystrophy type 2B, has been mapped to 2p13-p16 . The conservation between the mouse Rab1 and human RAB1 regions will be helpful in identifying candidate genes for the wobbler spinal muscular atrophy and in clarifying a possible relationship between wr and LMGMD2B. Genomics, 1996 Mar 15, 32(3), 425 - 30 Walking, cloning, and mapping with YACs in 3q27: localization of five ESTs including three members of the cystatin gene family and identification of CpG islands; James LA et al.; Using yeast artificial chromosomes, we have generated a high-resolution physical map for 2.7 Mb of human chromosomal region 3q27 . The YAC clones group into three contigs, one of which has also been linked to the CEPH YAC contig map of human chromosome 3 . Fluorescence in sity hybridization has been used to order the contigs on the chromosome and to estimate the distance between them . Expressed sequence tags for five genes, including three members of the cystatin gene family and a gene thought to be involved in B-cell non-Hodgkin lymphoma, have been placed within the YAC contigs, and 12 putative CpG islands have been identified . These YACs provide a useful resource to complete the physical mapping of 3q27 and to begin identification and characterization of further genes that are located there. J Cell Biochem, 1996 Mar 15, 60(4), 584 - 99 Endogenously inhibited protein kinase C in transgenic Drosophila embryonic neuroblasts down regulates the outgrowth of type I and II processes of cultured mature neurons; Broughton SJ et al.; Embryonic neurons were cultured from transgenic Drosophila melanogaster expressing a highly specific pseudosubstrate inhibitor of protein kinase C (PKC) . Flies homozygous for this transgene, which is under the control of the yeast UAS promoter, were crossed to flies homozygous for the yeast heat shock inducible transcription factor GAL 4 . Following heat shock, the progeny express the pseudosubstrate inhibitor at high levels . This strategy, which has the advantage of avoiding the non-specific effects of drugs, was used to study the role of PKC in process growth of cultured, differentiating neuroblasts . An external gold particle labeling procedure using a cell surface antigen expressed by mature neurons and processes was used to visualize neuronal processes directly in the scanning electron microscope . We observed that cell cultures expressing a low concentration of the pseudosubstrate inhibitor showed a significant decrease in the number of type I and II processes as compared to control cultures, while the proportions of neuroblasts, ganglion mother cells (GMCs), and mature neurons in the clusters were little affected. Int J Cancer, 1996 Mar 15, 65(6), 762 - 7 Deletion mapping of chromosome region 9p21-p22 surrounding the CDKN2 locus in melanoma; Ohta M et al.; The cyclin-dependent kinase-4 inhibitor gene CDKN2, localized at chromosome region 9p21, has been shown to be a familial melanoma gene, though we found that mutations of it are rare in uncultured sporadic melanomas . To determine Whether the region of allelic loss at 9p21 frequently observed in sporadic melanomas includes the CDKN2 locus, new polymorphic microsatellite probes were isolated from the genomic segments surrounding the CDKN2 gene and used for the study of loss of heterozygosity (LOH) in melanoma . The LOH study of matched uncultured tumor-constitutional DNA pairs from 66 metastatic cutaneous and 19 primary uveal melanomas showed that 63% and 32% of the respective tumors suffered allelic loss in the 9p21 region . Two regions of common losses which did not include the CDKN2 locus were observed: in a region of common loss near the D9S157 locus, telomeric to the CDKN2 locus, deletions were observed in 51% of informative cases; in the other region of common loss, near the D9S171 locus, centromeric to the CDKN2 locus, deletions were observed in 47% of informative cases . At the D9S974 locus, located within 20 kb of the CDKN2 gene, deletions were observed in 43% of informative cases . Homozygous deletions of the CDKN2 locus were observed in 8 cases of cutaneous melanoma and 2 cases of uveal melanoma; mutations in CDKN2 exon 2 were found in 2 of the 46 cases with allelic deletion in 9p21 . Our results support the following conclusions: (i) somatic mutation of the CDKN2 gene is rare in sporadic melanomas with allelic loss at 9p21; (ii) homozygous loss is more frequent than mutation of the CDKN2 gene in sporadic melanomas; (iii) at 9p21-p23 genes other than CDKN2 may be involved in the development of sporadic melanomas. Blood, 1996 Mar 15, 87(6), 2496 - 505 Incidence and characterization of MLL gene (11q23) rearrangements in acute myeloid leukemia M1 and M5; Poirel H et al.; To determine the incidence of MLL rearrangement in acute myeloid leukemia (AML) French-American-British (FAB) type M1 and to evaluate optimal screening strategies for the characterization of such abnormalities, we analyzed specimens from 41 patients with AML by Southern blotting with two MLL genomic probes and compared the capacities of reverse transcription-polymerase chain reaction (RT-PCR) and fluorescent in situ hybridization (FISH) to identify the types of rearrangement found in AML M1 with those observed in AML M5 . MLL rearrangement was found in 6 of 29 (20%) AML M1 and 6 of 10 AML M5 cases . RT-PCR characterization of 11 cases showed four MLL self-fusions, four MLL-AF6, two MLL-AF9, including a novel AF9 breakpoint, and one uncharacterized t(11:19) . Only 5 of 10 MLL-rearranged cases tested demonstrated karyotypic 11q23 abnormalities . FISH analysis of nine cases with an MLL-specific yeast artificial chromosome (YAC) confirmed the cytogenetic abnormalities in two cases, clarified them in one, and did not detect six cases, including three MLL self-fusions, one case with a probable MLL-rearranged subclone not represented karyotypically, and twoMLL-AF6 . A whole chromosome 11 paint detected one of these MLL-AF6, and an AF6 cosmid demonstrated that the other was probably due to insertion of a submicroscopic portion of chromosome 6, including part of AF6, into an apparently normal chromosome 11 . We conclude that MLL rearrangements are common in adult AML M1, that MLL self-fusion and MLL-AF6 are the most frequent types of abnormalities, and that RT-PCR is preferable to 11q23 FISH analysis for their characterization. Blood, 1996 Mar 15, 87(6), 2459 - 63 The abnormal eosinophils are part of the leukemic cell population in acute myelomonocytic leukemia with abnormal eosinophils (AML M4Eo) and carry the pericentric inversion 16: a combination of May-Grünwald-Giemsa staining and fluorescence in situ hybridization; Haferlach T et al.; The French-American-British subtype acute myelomonocytic leukemia with abnormal eosinophils (FAB AML M4Eo) with pericentric inversion of chromosome 16 is cytomorphologically defined by a myelomonoblastic blast population and abnormal eosinophils . Until now, it remained an open question whether these abnormal eosinophils are part of the malignant clone or an epiphenomenon . We analyzed five cases of AML M4Eo with inv(16) and combined May-Grunwald-Giemsa staining with fluorescence in situ hybridization using yeast artificial chromosome clone 854E2, which spans the inv(16) breakpoint on 16p . In the case of inv(16), three instead of the normal two hybridization signals can be observed both on metaphase spreads and in interphase cells . With this approach, we were able to show inversion 16 in abnormal eosinophils and, therefore, identified them as a part of the leukemic cell population. J Biol Chem, 1996 Mar 15, 271(11), 6451 - 7 A consensus cAMP-dependent protein kinase (PK-A) site in place of the CcN motif casein kinase II site simian virus 40 large T-antigen confers PK-A-mediated regulation of nuclear import; Xiao CY et al.; The regulation of nuclear protein transport by phosphorylation plays a central role in gene expression in eukaryotic cells . We previously showed that nuclear import of SV40 large tumor antigen (T-ag) fusion proteins is regulated by the CcN motif, comprising phosphorylation sites for casein kinase II and the cyclin-dependent kinase cdc2, together with the nuclear localization signal . Regulation of nuclear uptake by CcN motif kinase sites also holds true for the yeast transcription factor SWI5 and the Xenopus nuclear phosphoprotein nucleoplasmin . To test directly whether a kinase site other than those of the CcN motif could regulate nuclear import of T-ag, the CcN motif casein kinase II site, which markedly increases the rate of T-ag nuclear import, was replaced by a consensus site for the cAMP-dependent protein kinase (PK-A) using site-directed mutagenesis . The resultant fusion protein could be specifically phosphorylated by PK-A in vitro and in cell extracts . Nuclear import of the fluorescently labeled protein was analyzed in the HTC rat hepatoma cell line both in vivo (microinjected cells) and in vitro (mechanically perforated cells) in the presence and the absence of cAMP and/or PK-A catalytic subunit using confocal laser scanning microscopy . In vitro PK-A-prephosphorylated protein was also tested . All results indicated that the rate of nuclear import was increased by phosphorylation at the PK-A site (2-5-fold), demonstrating that kinases other than those of the CcN motif can regulate nuclear import in response to stimulatory signals . The phosphorylation-regulated nuclear localization signal derived here represents an important first step toward developing a signal conferring inducible nuclear targeting of molecules of interest. Biochim Biophys Acta, 1996 Mar 13, 1279(2), 214 - 8 Regulation of plasma membrane H+-ATPase from corn-root by Mg2+ and pH; Costa MS et al.; The plasma membrane H+-ATPase of corn-root is activated by free Mg2+ at pH 6.0 (Ks = 2.9 mM) but not at pH 7.0 . As a result, the pH dependence of the enzyme varies depending on the Mg2+ concentration of the medium . The activation by Mg2+ is promoted by an increase in Vmax with no effect on the apparent Km for Mg.ATP . Different from Mg2+, free Mn2+, Co2+, and Ni2+ do not activate at pH 6.0 and inhibit the H+-ATPase at pH 7.0 . The effects of divalent cations on the corn ATPase observed in this report are different from those previously described for the yeast enzyme (Brooker, R.J . and Slayman, C.W . (1983) J . Biol . Chem . 258, 8833-8838), suggesting different mechanisms of regulation for the isoforms of yeast and corn H+-ATPase. Gene, 1996 Mar 9, 169(2), 263 - 7 Isolation and characterization of a cDNA encoding a novel human transcription factor TFIID subunit containing similarities with histones H2B and H3; Choi BI et al.; Using the yeast two-hybrid system, we isolated a human cDNA that encodes a protein (hp22) interacting with TATA box-binding factor TFIID subunit p80 containing similarity with histone H4 . Sequence analysis showed that the open reading frame (ORF) specifies a 161-amino-acid (aa) polypeptide homologous to Drosophila melanogaster TFIID subunit p22 (dp22) . Comparison of the aa sequence of human TFIID subunit p22 (hp22) with that of dp22 revealed that p22 is composed of two distinct regions; the less conserved N-terminal (20% identity) and the highly conserved C-terminal (65% identity) regions . Additionally, the C-terminal region was found to contain similarities with histones H2B and H3 . Northern blot analysis showed mRNA corresponding to hp22 to be expressed in all tissues examined. Gene, 1996 Mar 9, 169(2), 197 - 201 Cell-cycle-dependent expression of the STK-1 gene encoding a novel murine putative protein kinase; Niwa H et al.; We have cloned a novel putative serine/threonine kinase-encoding gene, designed STK-1, from murine embryonic stem (ES) cell and testis cDNA libraries . The kinase most closely related to STK-1 is Xenopus laevis XLP46 protein kinase which shows 71% amino-acid identity to STK-1 between their kinase domains . Nevertheless, STK-1 is conserved throughout phylogeny with hybridizing sequences being detected in DNA from mammals, amphibians, insects and yeast . STK-1 mRNA is detected in testis, intestine and spleen, tissues that contain a large number of proliferating cells, but not in other tissues . All cell lines tested expressed STK-1 mRNA with levels being dependent upon proliferation rates . In NIH 3T3 cells, STK-1 is expressed in a cell-cycle-dependent fashion . These findings suggest a role for STK-1 in cell growth. Science, 1996 Mar 8, 271(5254), 1423 - 7 Friedreich's ataxia: autosomal recessive disease caused by an intronic GAA triplet repeat expansion; Campuzano V et al.; Friedreich's ataxia (FRDA) is an autosomal recessive, degenerative disease that involves the central and peripheral nervous systems and the heart . A gene, X25, was identified in the critical region for the FRDA locus on chromosome 9q13 . This gene encodes a 210-amino acid protein, frataxin, that has homologs in distant species such as Caenorhabditis elegans and yeast . A few FRDA patients were found to have point mutations in X25, but the majority were homozygous for an unstable GAA trinucleotide expansion in the first X25 intron. Oncogene, 1996 Mar 7, 12(5), 1165 - 72 Mouse Sin3A interacts with and can functionally substitute for the amino-terminal repression of the Myc antagonist Mxi1; Rao G et al.; Mxi1 is a basic region helix-loop-helix leucine zipper (bHLH/LZ) protein that, in association with Max, antagonizes Myc oncogenic activities . A possible mechanistic basis for Mxi1-mediated repression was provided by the recent demonstration that the repressive potential of Mxi1 correlates with its ability to physically associate with mSin3B, one of two mammalian homologues of the yeast transcriptional repressor SIN3 . Here, we sought to characterize more fully the physical properties of the second homologue, mSin3A and to determine whether the recruitment of mSin3A by Mxi1 is indeed required for anti-Myc activity . Transient transfection of mammalian cells showed that the mSin3A protein can associate with the strong repressive isoform of Mxi1 (Mxi1-SR) and that, like other Myc superfamily members, both mSin3A and Mxi1-SR localize to the nucleus . From a developmental standpoint, a comparative analysis of Myc, Mxi1-SR and Sin3A expression during postnatal mouse development and in differentiating mouse erythroleukemia (MEL) cells revealed that dramatic and reciprocal changes in Myc and Mxi1-SR mRNA levels are accompanied by minimal stage-specific changes in mSin3A gene expression . This constant expression profile, coupled with the observation that over-expression of mSin3A does not augment the anti-Myc activity of Mxi1-SR in the rat embryo fibroblast (REF) transformation assay, suggests that mSin3A is not a limiting factor in the regulation of Myc superfamily function . Finally, a mSin3A-Mxi1 fusion protein, in which the amino terminal mSin3-interacting domain of Mxi1-SR was replaced with the full-length mSin3A, exhibited a level of repression activity equivalent to, or greater than, the level of repression obtained with Mxi1-SR . Taken together, these observations directly demonstrate that the amino-terminal repression domain of Mxi1-SR functions solely to recruit mSin3A and possibly other proteins like mSin3A and this association is necessary for the anti-Myc activity of Mxi1-SR. Proc Natl Acad Sci U S A, 1996 Mar 5, 93(5), 1902 - 6 Nonclathrin coat protein gamma, a subunit of coatomer, binds to the cytoplasmic dilysine motif of membrane proteins of the early secretory pathway; Harter C et al.; Coatomer, a cytosolic heterooligomeric protein complex that consists of seven subunits {alpha-, beta-, beta'-, gamma-, delta-, epsilon-, and zeta-COP (nonclathrin coat protein)}, has been shown to interact with dilysine motifs typically found in the cytoplasmic domains of various endoplasmic-reticulum-resident membrane proteins {Cosson, P . & Letourneur, F . (1994) Science 263, 1629-1631} . We have used a photo-cross-linking approach to identify the site of coatomer that is involved in binding to the dilysine motifs . An octapeptide corresponding to the C-terminal tail of Wbp1p, a component of the yeast N-oligosaccharyltransferase complex, has been synthesized with a photoreactive phenylalanine at position -5 and was radioactively labeled with {125I}iodine at a tyrosine residue introduced at the N terminus of the peptide . Photolysis of isolated coatomer in the presence of this peptide and immunoprecipitation of coatomer from photo-cross-linked cell lysates reveal that gamma-COP is the predominantly labeled protein . From these results, we conclude that coatomer is able to bind to the cytoplasmic dilysine motifs of membrane proteins of the early secretory pathway via its gamma-COP subunit, whose complete cDNA-derived amino acid sequence is also presented. Proc Natl Acad Sci U S A, 1996 Mar 5, 93(5), 1759 - 63 A quantitative test to estimate neutralizing antibodies to the hepatitis C virus: cytofluorimetric assessment of envelope glycoprotein 2 binding to target cells; Rosa D et al.; Hepatitis C virus (HCV) is a major cause of chronic hepatitis . The virus does not replicate efficiently in cell cultures, and it is therefore difficult to assess infection-neutralizing antibodies and to evaluate protective immunity in vitro . To study the binding of the HCV envelope to cell-surface receptors, we developed an assay to assess specific binding of recombinant envelope proteins to human cells and neutralization thereof . HCV recombinant envelope proteins expressed in various systems were incubated with human cells, and binding was assessed by flow cytometry using anti-envelope antibodies . Envelope glycoprotein 2 (E2) expressed in mammalian cells, but not in yeast or insect cells, binds human cells with high affinity (Kd approximately 10(-8) M) . We then assessed antibodies able to neutralize E2 binding in the sera of both vaccinated and carrier chimpanzees, as well as in the sera of humans infected with various HCV genotypes . Vaccination with recombinant envelope proteins expressed in mammalian cells elicited high titers of neutralizing antibodies that correlated with protection from HCV challenge . HCV infection does not elicit neutralizing antibodies in most chimpanzees and humans, although low titers of neutralizing antibodies were detectable in a minority of infections . The ability to neutralize binding of E2 derived from the HCV-1 genotype was equally distributed among sera from patients infected with HCV genotypes 1, 2, and 3, demonstrating that binding of E2 is partly independent of E2 hypervariable regions . However, a mouse monoclonal antibody raised against the E2 hypervariable region 1 can partially neutralize binding of E2, indicating that at least two neutralizing epitopes, one of which is hypervariable, should exist on the E2 protein . The neutralization-of-binding assay described will be useful to study protective immunity to HCV infection and for vaccine development. Ukr Biokhim Zh, 1996 Mar-Apr, 68(2), 58 - 63 {The interaction of pyruvate decarboxylase with the quinone-ferricyanide oxidative system}; Vovk AI et al.; Inactivation of yeast pyruvate decarboxylase in the presence of substrate and oxidative system containing substituted quinone and ferricyanide has been investigated . It was established that ferricyanide at pH 5.2-6.4 can prevent irreversible inactivation of the pyruvate decarboxylase caused by the concerted action of pyruvate and substituted quinone . The influence of ferricyanide which depends on the redox potential of the substituted quinone is decreasing in a series tetramethyl-p-benzoquinone, trimethyl-p-benzoquinone, 2-methyl-5-isopropyl-p-benzoquinone . It is supposed that the effect of the oxidative system partially converting the nonoxidative to oxidative function of pyruvate decarboxylase is attributed to the oxidation of active acetaldehyde by substituted quinone and reaction of resultant semiquinone radical with ferricyanide. Genome Res, 1996 Mar, 6(3), 202 - 10 Positional candidate genes for congenital chloride diarrhea suggested by high-resolution physical mapping in chromosome region 7q31; Hoglund P et al.; Congenital chloride diarrhea affects intestinal transportation of electrolytes, resulting in potentially fatal diarrhea . Linkage disequilibrium analyses have suggested the congenital chloride diarrhea gene (CLD) to lie within 0.37 cM from D7S496 in human chromosome 7q31 . To clone the CLD gene, we have constructed and refined a physical map based on a 2.7-Mb YAC contig around D7S496 and identified two candidate genes . The physical positions of 4 known genes (DRA, PRKAR2B, LAMB1, DLD), 7 polymorphic repeat markers, and 13 CpG islands were established . DRA (down-regulated in adenoma) is expressed in the gut and encodes a protein with sequence homology to anion transporters, whereas PRKAR2B encodes a regulatory subunit for protein kinase A . Both genes map within 450 kb from D7S496, making them functionally and positionally relevant candidates for CLD. Genome Res, 1996 Mar, 6(3), 187 - 94 The human CAS (cellular apoptosis susceptibility) gene mapping on chromosome 20q13 is amplified in BT474 breast cancer cells and part of aberrant chromosomes in breast and colon cancer cell lines; Brinkmann U et al.; The CAS (cellular apoptosis susceptibility) gene is the human homolog of the yeast chromosome segregation gene CSE1 . CAS may have a dual function in mammalian cells, one in apoptosis and another in cell proliferation . We have now mapped the CAS gene to chromosome 20q13 . This region is known to harbor amplifications that correlate with aggressive breast cancer . Southern hybridizations with a CAS cDNA fragment and fluorescent in situ hybridization (FISH) with a P1 clone containing the CAS gene show elevated copy numbers in one leukemia, three of four colon, and in three of seven breast cancer cell lines . Elevated CAS copy number in CEM leukemia and COLO201 colon cancer cells was attributable to additional copies of chromosome 20 . In SW480 and COLO205 colon cancer cells CAS is part of aberrant chromosomes containing large parts of 20q . In breast cancer cells CAS is also part of aberrant 20q chromosomes (MDA-MB-157 and UACC-812) or of additional 20q isochromosome in MDA-MB-134 . In MDA-MB361 and BT-474 breast cancer cells CAS is separated from other markers centromeric and telomeric of CAS on 20q . MDA-MB 361 contains one additional copy of CAS, separated from the centromeric 20q control probe . BT-474 cells have up to 12 additional CAS copies that we separated from nearby telomeric and centromeric probes on 20q and that are translocated to abnormal chromosomes. Genome Res, 1996 Mar, 6(3), 176 - 86 Construction of a consistent YAC contig for human chromosome region 3p14.1; Bardenheuer W et al.; Chromosomal deletions and translocations of human chromosome region 3p14 are observed in various human malignancies and suggest the existence of a tumor suppressor gene locus within this region . Tumors most frequently affected by these aberrations are small-cell lung cancer and renal-cell carcinoma . In continuation of our previously published YAC contig of chromosome region 3p14.2-p14.3, we report here on the construction of a YAC contig of at least 11 Mb that consisted of 171 YACs and covers the entire subregion 3p14.1 . This contig includes the t(3;8) breakpoint of a hereditary renal-cell carcinoma localized in 3p14.2 and extends into human chromosome region 3p12-p13 . It defines the order of 34 DNA probes in relation to reference markers D3S6 and D3S30 as well as the human protein tyrosine phosphatase-gamma gene . For 31 DNA probes we identified nonchimeric YACs by fluorescence in situ hybridization . The minimal tilling pathway consists of 16 yeast artificial chromosomes . As a prerequisite for identification of a putative tumor suppressor gene within this region, this contig renders human chromosome region 3p14.1 accessible to gene isolation. Plant J, 1996 Mar, 9(3), 369 - 80 Mutation of putative branchpoint consensus sequences in plant introns reduces splicing efficiency; Simpson CG et al.; Intron lariat formation between the 5' end of an intron and a branchpoint adenosine is a fundamental aspect of the first step in animal and yeast nuclear pre-mRNA splicing . Despite similarities in intron sequence requirements and the components of splicing, differences exist between the splicing of plant and vertebrate introns . The identification of AU-rich sequences as major functional elements in plant introns and the demonstration that a branchpoint consensus sequence was not required for splicing have led to the suggestion that the transition from AU-rich intron to GC-rich exon is a major potential signal by which plant pre-mRNA splice sites are recognized . The role of putative branchpoint sequences as an internal signal in plant intron recognition/definition has been re-examined . Single nucleotide mutations in putative branchpoint adenosines contained within CUNAN sequences in four different plant introns all significantly reduced splicing efficiency . These results provide the most direct evidence to date for preferred branchpoint sequences being required for the efficient splicing of at least some plant introns in addition to the important role played by AU sequences in dicot intron recognition . The observed patterns of 3' splice site selection in the introns studied are consistent with the scanning model described for animal intron 3' splice site selection . It is suggested that, despite the clear importance of AU sequences for plant intron splicing, the fundamental processes of splice site selection and splicing in plants are similar to those in animals. Jpn J Hum Genet, 1996 Mar, 41(1), 149 - 58 Physical map of a YAC contig containing the region of the human gene (HYRC) complementing hyper-radiosensitivity of the scid mouse mutation; Watanabe Y et al.; We previously mapped the putative human HYRC (the hyper-radiosensitivity of the scid mutation, complementing gene) to human chromosome 8q11.1 by fluorescence in situ hybridization (FISH) using Alu-based PCR products from a mouse-human scid radiation cell hybrid (RD15/5) as probes . From a cosmid library constructed from RD15/5, 57 cosmid clones containing human DNA inserts were isolated . 18 of which were mapped to 8q11 . Based on the sequences of plasmid subclones of the 18 cosmids, five novel sequence-tagged-sites (STSs) were made . By a screening of the CEPH-YAC library with these STSs, five yeast artificial chromosome (YAC) clones were isolated . All these YAC clones were confirmed not to be chimeric by FISH, but two of them showed deleted human insert DNAs . Using the other 3 non-deleted YACs, we constructed a physical map covering the HYRC region . We confirmed that the recently isolated gene (the DNA-PKcs gene) which is a strong candidate for HYRC is located within the present contig and spans less than 200 kb . This map will be useful for the analysis of the genomic structure of the DNA-PKcs gene and for isolation of other complementing genes in the HYRC region. J Clin Microbiol, 1996 Mar, 34(3), 615 - 21 Rapid identification of Candida species by DNA fingerprinting with PCR; Thanos M et al.; DNA polymorphisms in different species and strains of the genus Candida were assessed by amplifying genomic DNA with single nonspecific primers . This PCR method employed an arbitrary primer (the 10-mer AP3), a primer derived from the intergenic spacer regions (T3B), and the microsatellite primers (GTG)5 and (AC)10 . Distinctive and reproducible sets of amplification products were observed for 26 different Candida and 8 other fungal species . The numbers and sizes of the amplification products were characteristic for each species . All yeast species tested could be clearly distinguished by their amplification patterns . With all primers, PCR fingerprints also displayed intraspecies variability . However, PCR profiles obtained from different strains of the same species were far more similar than those derived from different Candida species . By comparing species-specific PCR fingerprints of clinical isolates with those of reference strains, clinical isolates could be identified to the species level even if they could not be identified by routine biochemical methods. Insect Biochem Mol Biol, 1996 Mar, 26(3), 235 - 47 Molecular cloning of an alpha-esterase gene cluster on chromosome 3r of Drosophila melanogaster; Russell RJ et al.; All or part of the alpha-esterase gene cluster in Drosophila melanogaster has been isolated by screening a YAC clone that spans cytological region 84D3-10 with consensus carboxyl/cholinesterase oligonucleotides . The cluster encompasses 11 putative esterase genes within 65 kb of genomic DNA and is one of the largest clusters of related protein-coding genes yet reported in Drosophila . The cluster must include the gene encoding the major alpha-esterase isozyme, EST9, which has previously been mapped to 84D3-5 . It probably also includes the genes encoding the EST23, MCE and ALI esterases that have previously been mapped to 84D3-E2 . The latter three are homologs of genes involved in organophosphate insecticide resistance in the sheep blowfly, Lucilia cuprina and the housefly, Musca domestica . Sequencing of one of the putative esterase genes in the Drosophila cluster, alpha E1, shows that it would encode features characteristic of an active carboxyl/cholinesterase, including the so-called catalytic triad, the nucleophilic elbow and oxyanion hole . It also shows that the closest relative of alpha E1 amongst previously published esterase sequences is ESTB1, which confers organophosphate resistance in Culex mosquitoes . We argue that we have cloned the D . melanogaster version of a major cluster of esterase genes which have variously mutated to confer organophosphate resistance in diverse Diptera. Hum Mol Genet, 1996 Mar, 5(3), 367 - 71 Large homozygous deletions of the 2q13 region are a major cause of juvenile nephronophthisis; Konrad M et al.; Juvenile nephronophthisis (NPH) is a genetically heterogeneous disorder representing the most frequent inherited cause of chronic renal failure in children . We recently assigned a gene (NPH1) to the 2q13 region which is responsible for approximately 85% of cases . Cloning this region in a yeast artificial chromosome contig revealed the presence of low copy repeats . Large-scale rearrangements were detected in 80% of the patients belonging to inbred or multiplex NPH1 families and in 65% of the sporadic cases . Surprisingly, these rearrangements seem to be, in most cases, large homozygous deletions of approximately 250 kb involving an 100 kb inverted duplication . This suggests a common genetic disease-causing mechanism, which could be responsible for the highest frequency of large rearrangements reported in an autosomal recessive trait . Our findings are also of major clinical interest, as they permit the diagnosis in the majority of sporadic cases without the need for kidney biopsy. Genomics, 1996 Mar 1, 32(2), 277 - 80 The chromosome localization and the HCF repeats of the human host cell factor gene (HCFC1) are conserved in the mouse homologue; Frattini A et al.; The gene encoding the human host cell factor (HCFC1) has recently been cloned and mapped to Xq28 . HCFC1 codes for a family of related polypeptides that apparently arise from posttranslational processing . Six extremely conserved 19-amino-acid (aa)long motifs, unique to HCFC1 and located in the middle of the protein, could play a role in this processing or could be instrumental to the physiological role of the protein . Aternatively, these repeats could have arisen from recent duplications and may not have any specific function . To resolve this issue, we cloned the homologous region from the mouse HCFC1 gene and demonstrated that the 19-aa motifs are extremely conserved in sequence, number, and genomic organization, while the "linker" region between the third and fourth repeat is not . This suggests an important function for these repeats . In addition, by RT-PCR analysis of human RNA and comparison to the human genomic sequence, an alternative transcript including a 44-aa in-frame insertion, deriving from the 3' end of intron 18, was found . The significance of this alternative transcript is unknown, since it was not detectable in the mouse . The mouse HCFC1 gene maps to a region syntenic to Xq28, and, as in human, is in close proximity to the Renin-binding protein gene, in a 100-kb region also including the Licam and Vasopressin receptor type 2 genes. Genomics, 1996 Mar 1, 32(2), 260 - 5 Localization of chi1-related helicase genes to human chromosome regions 12p11 and 12p13: similarity between parts of these genes and conserved human telomeric-associated DNA; Amann J et al.; The helicase enzymes are essential components of a number of multi-protein complexes, including those that regulate transcription, splicing, translation, and DNA repair . These enzymes assist in the unwinding of double-stranded DNA and RNA as an essential part of their function . The yeast Chl1 gene encodes a putative helicase that appears to be essential for normal chromosome transmission . Human cDNAs related to this yeast gene, hCHLR1 and hCHLR2, were recently isolated and shown to encode products that localize to the nucleus . Two corresponding genes have now been partially characterized and localized to human chromosome regions 12p11 and 12p13, indicating that this gene is contained within a duplicated region localized to 12p . In addition, a comparison of the hCHLR gene sequences with available databases indicates that a large portion of these genes, including exons encoding two functional domains of the carboxyl-terminal region of these proteins, has been duplicated as part of a larger human telomeric repeat sequence found on many human chromosomes . Our results suggest that duplication of a relatively large region of chromosome 12p containing this putative helicase gene has resulted in the creation of numerous pseudogenes as part of a subtelomeric repeat . The presence of these helicase pseudogenes, as well as pseudogenes for other genes such as the interleukin-9 receptor, within many subtelomeric regions support the possibility that the spread of this region is subject to exchange between different chromosomes and may have implications for elucidation of the mechanism of intra- and interchromosomal duplication events. Genomics, 1996 Mar 1, 32(2), 236 - 44 Structural analysis of the HLA-A/HLA-F subregion: precise localization of two new multigene families closely associated with the HLA class I sequences; Pichon L et al.; Positional cloning strategies for the hemochromatosis gene have previously concentrated on a target area restricted to a maximum genomic expanse of 400 kb around the HLA-A and HLA-F loci . Recently, the candidate region has been extended to 2-3 Mb on the distal side of the MHC . In this study, 10 coding sequences {hemochromatosis candidate genes (HCG) I to X} were isolated by cDNA selection using YACs covering the HLA-A/HLA-F subregion . Two of these (HCG II and HCG IV) belong to multigene families, as well as other sequences already described in this region, i.e., P5, pMC 6.7, and HLA class 1 . Fingerprinting of the four YACs overlapping the region was performed and allowed partial localization of the different multigene family sequences on each YAC without defining their exact positions . Fingerprinting on cosmids isolated from the ICRF chromosome 6-specific cosmid library allowed more precise localization of the redundant sequences in all of the multigene families and revealed their apparent organization in clusters . Further examination of these intertwined sequences demonstrated that this structural organization resulted from a succession of complex phenomena, including duplications and contractions . This study presents a precise description of the structural organization of the HLA-A/HLA-F region and a determination of the sequences involved in the megabase size polymorphism observed among the A3, A24, and A31 haplotypes. Genomics, 1996 Mar 1, 32(2), 191 - 9 Genomic organization of the human NSP gene, prototype of a novel gene family encoding reticulons; Roebroek AJ et al.; Recently, cDNA cloning and expression of three mRNA variants of the human NSP gene were described . This neuroendocrine-specific gene encodes three NSP protein isoforms with unique amino-terminal parts, but common carboxy-terminal parts . The proteins, with yet unknown function, are associated with the endoplasmic reticulum and therefore are named NSP reticulons . Potentially, these proteins are neuroendocrine markers of a novel category in human lung cancer diagnosis . Here, the genomic organization of this gene was studied by analysis of genomic clones isolated from lambda phage and YAC libraries . The NSP exons were found to be dispersed over a genomic region of about 275 kb . The present elucidation of the genomic organization of the NSP gene explains the generation of NSP mRNA variants encoding NSP protein isoforms . Multiple promoters rather than alternative splicing of internal exons seem to be involved in this diversity . Furthermore, comparison of NSP genomic and cDNA sequences with databank nucleotide sequences resulted in the discovery of other human members of this novel family of reticulons encoding genes. Drug Metab Dispos, 1996 Mar, 24(3), 303 - 6 A sensitive method for determination of cytochrome P4502D6 activity in vitro using bupranolol as substrate; Appanna G et al.; Previous studies have shown that bupranolol, a beta-adrenoceptor blocker, is a substrate of cytochrome P4502D6 (CYP2D6) . A sensitive in vitro assay was developed to quantify the formation of hydroxybupranolol using HPLC . A TLC method, using radiolabeled bupranolol, was also developed to test the reproducibility of the two methods . Both of them gave virtually identical results; however, the HPLC method was sensitive to 20 pmol and the TLC with radiolabeled substrate to <1 pmol of hydroxybupranolol . The KM value for bupranolol was lower than that reported for any other substrate of CYP2D6 . The KM value in microsomes of a typical human liver (L-1) was 0.272 +/- 0.02 (SE) mu M and the Vmax was 360 +/- 10 (SE) pmol/mg/min (0.83 +/- 0.02 pmol/pmol cytochrome P450/min) . The KM value for the CYP2D6 expressed in yeast was 0.076 +/- 0.003 (SE) mu M, and the Vmax was 43 +/- 1 (SE) pmol/mg/min (0.64 +/- 0.01 pmol/pmol cytochrome P450/min) . Quinidine competitively inhibited the formation of hydroxybupranolol, with Ki values of 5 nM in expressed CYP2D6 and 14.05 nM in human liver (L-1). Fundam Appl Toxicol, 1996 Mar, 30(1), 93 - 101 Failure of chloro-S-triazine-derived compounds to induce estrogen receptor-mediated responses in vivo and in vitro; Connor K et al.; The potential estrogenic activities of atrazine and simazine were investigated in vivo using the immature female Sprague-Dawley rat uterus and in vitro using the estrogen-responsive MCF-7 human breast cancer cell line and the estrogen-dependent recombinant yeast strain PL3 . Animals that were dosed with 50, 150, or 300 mg/kg of atrazine or simazine alone for 3 consecutive days did not exhibit any significant increases in uterine wet weight while decreases in cytosolic progesterone receptor (PR) binding levels and uterine peroxidase activity were observed . 17 beta-estradiol (E2)-induced increases in uterine wet weight were not significantly affected by cotreatment with either chemical; however, some dose-independent decreases in E2-induced cytosolic PR binding and uterine peroxidase activity were observed . In vitro, atrazine and simazine did not affect basal or E2-induced MCF-7 cell proliferation or the formation of nuclear PR-DNA complexes as determined by gel electrophoretic mobility shift assays . In addition, these chloro-S-triazines did not display agonist activity or antagonize E2-induced luciferase activity in MCF-7 cells transiently transfected with a Gal4-human estrogen receptor chimera (Gal4-HEGO) and a Gal4-regulated luciferase reporter gene (17m5-G-Luc) . Moreover, the estrogen-dependent PL3 yeast strain was not capable of growth on minimal media supplemented with atrazine or simazine in place of E2 . Collectively, these results indicate that the reported estrogenic and antiestrogenic effects elicited by these chemicals are not mediated by the estrogen receptor. Curr Biol, 1996 Mar 1, 6(3), 276 - 8 Rehearsing the ABCs . Protein translocation; Cleves AE et al.; Recent evidence that vacuolar enzymes in yeast can be delivered directly from the cytosol, rather than via the secretory pathway, alerts us to the increasing evidence for 'non-classical' forms of protein translocation that may involve ABC transporters. Hum Genet, 1996 Mar, 97(3), 399 - 403 Integration of four genes, a pseudogene, thirty-one STSs, and a highly polymorphic STRP into the 7-10 Mb YAC contig of 5q34-q35; Kostrzewa M et al.; Thirty-one sequence tagged sites and a highly polymorphic short tandem repeat polymorphism have been isolated from 5q34-q35 and integrated into the yeast artificial chromosome (YAC) contig of 5q34-q35 . In addition, four genes (MSX2, CSX, DRD1, and CL100) and a pseudogene (GLUT6) were localized on this map . The high density of new markers in the region allowed further refinement of the YAC contig of distal 5q . This is a prerequisite for the conversion of this YAC into a cosmid contig. Hum Genet, 1996 Mar, 97(3), 345 - 52 The gene for X-linked hypophosphataemic rickets maps to a 200-300kb region in Xp22.1, and is located on a single YAC containing a putative vitamin D response element (VDRE); Rowe PS et al.; The location of the HYP gene, which determines X-linked hypophosphataemic rickets, has been refined considerably by linkage analysis, and three new microsatellite primers isolated, Cap32 (DXS7473), Cap29 (DXS7474) and 7v2 (DXS7475) . The locations of four other markers have also been determined (DXS1226, AFMa176zb1, AFMa152wc5, and AFM346azc1) . Markers Cap29 and Cap32 are the closest distal markers to the gene with zetamax=11.93, thetamax= 0.018 and zetamax=12.03, thetamax = 0.015 respectively . Both Cap29 and Cap32 are proximal to DXS365 and AFMa176zb1, as deduced by screening non-chimaeric yeast artificial chromosomes (YACs) from a contig spanning the HYP gene . A single crossover places AFMa176zbl distal to the disease gene . There are no recombinations between 7v2 and HYP (zetamax=12.9, thetamax=0.0), or between 7v2 and adjacent markers Cap32, Cap29, AFMa176zb1, DXS1683 and DXS365 . However screening of YAC clones encompassing the HYP gene and also P1 clones localises 7v2 distal to Cap29 and Cap32, and proximal to DXS443 . Marker DXS1226 is placed outside the region containing the gene, and is located proximal to DXS274 as confirmed by a crossover for this marker and DXS41 against HYP and its presence on YAC 83B05 . Genetic mapping of CEPH pedigrees, and screening of YACs places AFMa152wc5 and AFMa346zcl between DXS1683 and DXS1052 . The following gene marker map presents the best order for the HYP region: Xptel-DXS43-DXS999-DXS443-(DXS365/DXS74 75/AFMa176zb1)-(DXS7474/DXS7473)-HYP- DXS1683-(AFMa152wc5/AFMa346zc1)-DXS1052-DXS 274 -(DXS41/DXS1226)-Xcen . The distance between the cluster of distal flanking markers Cap29 (DXS7474), Cap32 (DXS7473), and DXS1683 is approximately 300 kb, as deduced from physical map data from a YAC contig spanning the gene . Thus the gene for HYP is contained within a single YAC (900AO472) . Of further interest, is the location of a putative vitamin D response element (VDRE) on this YAC. Indian J Exp Biol, 1996 Mar, 34(3), 275 - 6 Antipyretic activity of Nelumbo nucifera rhizome extract; Mukherjee PK et al.; Antipyretic activity of methanolic extract of rhizome of N . nucifera was studied on normal body temperature and yeast induced pyrexia in rats . Yeast suspension (10 ml/kg, s.c.) increased rectal temperature after 19 hr of administration . The extract, in doses of 200, 300 or 400 mg/kg (po) produced significant dose dependent lowering of normal body temperature and yeast provoked elevation of body temperature in rats . The effect produced was comparable with the standard antipyretic drug, paracetamol (150 mg/kg, i.p.). Indian J Exp Biol, 1996 Mar, 34(3), 272 - 3 Laboratory evaluation of toxic baits for mosquito (Aedes aegypti) larvae; Vartak PH et al.; Toxic bait discs were prepared by incorporating the larvicide Abate (Temephos) technical, with yeast and dog biscuit (1:1), the latter forming the attractant source . These were assessed in 1, 10 and 25 litre aquaria and 100 litre drums against Aedes aegypti larvae . Floating as well as sinking bait discs were found equally effective in controlling free moving larvae . Larvae confined in artificially suspended cages were not affected . The baits were found effective for 5 to 6 weeks . Toxic disc bait preparation methodology and evaluation protocols are discussed in this communication. Anal Chem, 1996 Mar 1, 68(5), 850 - 8 Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels; Shevchenko A et al.; Proteins from silver-stained gels can be digested enzymatically and the resulting peptide analyzed and sequenced by mass spectrometry . Standard proteins yield the same peptide maps when extracted from Coomassie- and silver-stained gels, as judged by electrospray and MALDI mass spectrometry . The low nanogram range can be reached by the protocols described here, and the method is robust . A silver-stained one-dimensional gel of a fraction from yeast proteins was analyzed by nano-electrospray tandem mass spectrometry . In the sequencing, more than 1000 amino acids were covered, resulting in no evidence of chemical modifications due to the silver staining procedure . Silver staining allows a substantial shortening of sample preparation time and may, therefore, be preferable over Coomassie staining . This work removes a major obstacle to the low-level sequence analysis of proteins separated on polyacrylamide gels. J Neurochem, 1996 Mar, 66(3), 889 - 97 The Sec1 family: a novel family of proteins involved in synaptic transmission and general secretion; Halachmi N et al.; The Sec1 family, a novel family of proteins involved in synaptic transmission and general secretion, is described . To date, 14 members of this family have been identified: four yeast proteins, Sec1, Sly1, Slp1/Vps33, and Vps45/Stt10; three nematode proteins, Unc-18 and the homologues of Sly1 and Slp1; the Drosophila Rop; and six mammalian proteins, the rat Munc-18/n-Sec1/rbSec1A and rbSec1B, the mouse Munc-18b/muSec1 and Munc-18c, and the bovine Munc-18 and mSec1 . The mammalian proteins share 44-63% sequence identity with the nematode Unc-18 and Drosophila Rop proteins and 20-29% with the yeast proteins and their nematode homologues . The Sec1 proteins are mostly hydrophilic and lack a transmembrane domain . Nevertheless, Sec1 proteins are found as membrane-bound proteins . Some of them are also found as soluble, cytoplasmic proteins . Binding of the rat brain Sec1 to the presynaptic membrane may be due to strong interaction with syntaxin, an integral component of this membrane . The rat brain Sec1 is also bound to Cdk5, a neural cyclin-dependent kinase . The Sec1 proteins play a positive role in exocytosis . Loss of function mutations in SEC1, SLY1, or SLP1 result in blocking of protein transport between distinct yeast sub-cellular compartments . Inactivation of unc-18 and rop results in inhibition of neurotransmitter release and, in the case of rop, inhibition of general secretion as well . In addition, studies of Rop and n-Sec1 indicate that they also play a negative role in synaptic transmission, mediated by their interaction with syntaxin . A working model addressing the dual regulative role of the Sec1 proteins in secretion is presented. Mycoses, 1996 Mar-Apr, 39(3-4), 125 - 8 Sporothrix schenckii isolated from a cat in Japan; Nakamura Y et al.; A yeast-like fungus isolated from a granulomatous and ulcerative skin lesion in a mongrel cat was mycologically examined . The isolate was identified as Sporothrix schenckii from the morphological findings and its pathogenicity in a mouse, confirming the first case of feline sporotrichosis in Japan . Fortunately, no transmission to humans occurred in this case, however the risk of humans contracting Sporothrix schenckii infection increases with the increase in the number of animals with sporotrichosis . Consequently when handling such animals protective clothing such as gloves should be worn to reduce the risk of transmission of the fungus through open wounds. Mycoses, 1996 Mar-Apr, 39(3-4), 85 - 93 Induction and detection of cell-mediated reactions with different Blastomyces dermatitidis antigenic preparations; Abuodeh RO et al.; Blastomyces dermatitidis yeast-phase antigens (killed cell, KWY; lysate, Lys; and filtrate, Fil) from canine isolate T-58 were compared with respect to the induction and detection of cellular immune responses in mice . The antigens exhibited good sensitivity and specificity when used to detect a delayed-type hypersensitivity (DTH) response in mice previously immunized with T-58 or Histoplasma capsulatum antigens . Greater reactivity was observed with the KWY and Lys antigens as DTH-inducing agents (immunogens) than with the Fil antigen . The antigens were also compared with regard to the induction and detection of a lymphoproliferative reaction using splenocytes from normal and sensitized mice, and optimal reactivity was demonstrated with the KWY and Fil antigen preparations both as immunogens (absorbance values 0.22-1.60 and 0.20-0.90, respectively) and as in vitro testing antigens (absorbance values 0.60-1.60 and 0.35-1.00 respectively) (P < 0.01) . Peritoneal macrophages from mice sensitized with Fil and KWY antigens showed the greatest in vitro replication inhibition (RI) of B . dermatitidis yeast cells (RI values of 53% and 50% respectively) (P < 0.05) . When mitogenic or antigenic lymphocytic supernatants were compared with respect to their ability to enhance the phagocytic activity of unelicited macrophages from normal mice to kill yeast cells, the T-cell mitogens (concanavalin A and phytohaemagglutinin) optimally activated the naive macrophages (45% and 44% RI respectively) compared with the B-cell mitogen (LPS) (23% RI) (P < 0.05) . Similar results were obtained with the lymphocytic supernatants from mice immunized with KWY cells when activated using KWY or Fil antigens (46% and 40% RI respectively) (P < 0.05). Neurobiol Aging, 1996 Mar-Apr, 17(2), 173 - 82 Rational design of an animal model for Alzheimer's disease: introduction of multiple human genomic transgenes to reproduce AD pathology in a rodent; Loring JF et al.; A major obstacle to understanding the pathogenesis of Alzheimer's disease is the lack of easily studied animal models . Our approach is to apply transgenic methods to humanize mice and rats, employing methods that introduce large genomic transgenes, because this improves the level of transgene protein expression and the tissue specificity of expression . Our plan is to reproduce AD pathology in rodents by making them transgenic for several human proteins involved in AD . This report describes transgenic animal lines that we have produced, and summarizes our current approach and future plans . Two human genes known to be involved in AD pathology are the amyloid precursor protein (APP) and the E4 isoform of apolipoprotein E (apoE4) . So far, we have produced and analyzed a transgenic line carrying the entire human APP gene cloned in a yeast artificial chromosome . We have also produced but not yet analyzed a mouse carrying the human apoE4 gene . Work is in progress to produce a transgenic line carrying a disease-causing mutation in the human APP gene . As we produce these animals, we are breeding them together, and also breeding them with a mouse line that lacks endogenous apoE, to produce an animal model carrying several human proteins whose interaction is believed to be instrumental in development of AD pathology . These transgenic animals will be useful for dissecting the biochemical and physiological steps leading to AD, and for development of therapies for disease intervention. Braz J Med Biol Res, 1996 Mar, 29(3), 309 - 18 Myosin-V: a class of unconventional molecular motors; Larson RE; In this review we focus on the biochemical and structural properties of the myosin-V class of unconventional myosins as an example of the diversity of molecular motors within the myosin superfamily . A member of this class was first identified as a novel calmodulin-binding protein in mammalian brain (Larson RE, Pitta DE and Ferro JA (1988) . Brazilian Journal of Medical and Biological Research, 21: 213-217) . To date, the myosin-V class is represented by two molecules from yeast, one from nematodes, several from vertebrates (chickens, rats, mice and humans) and possibly one from plants . The domain structure of these myosins features a highly conserved head containing the ATP-hydrolysis and actin-binding sites, an extended neck composed of six tandem IQ-motifs which are sites for calmodulin binding and a large tail which has coiled-coil segments intercalated with globular regions of as yet unknown function . Biochemical studies on purified myosin-V from vertebrate brains and the description of myosin-V mutants in yeast and mice have made myosin-V one of the best characterized, unconventional myosin classes at the present time, surpassed only by the well-studied myosin-I class. Baillieres Clin Gastroenterol, 1996 Mar, 10(1), 113 - 34 Trefoil peptides; Poulsom R; There is a growing body of evidence supporting the hypothesis that members of the trefoil peptide family are involved actively in maintaining the integrity of the gastrointestinal mucosa and facilitating its repair . To date, three trefoil peptides are known in man: pS2, ITF and SP . Each is a secretory peptide expressed in specific compartments throughout the gut, in patterns that appear generally to be conserved between mammalian species . Ulceration, whether due to common pathological processes or experimentally induced, results in altered local expression of trefoil peptides . In diverse chronic ulcerative conditions in man, glandular structures develop within the mucosa, derived from the UACL . These UACL glands express three trefoil peptides, EGF and lysozyme, all potentially able to contribute to the healing process . In fact local goblet and endocrine cell types may also be recruited to secrete pS2 into the local environment . In experimental ulcers, in rate stomach or intestinal resection margins, there is also accentuation of trefoil peptide expression at the margins and in the poorly differentiated mucous cells extending out presumably in attempts to restore epithelial integrity . Several trefoil peptides have been expressed as 'recombinant' proteins in bacterial, baculoviral or yeast systems, and these procedures have allowed some of the biological properties of these peptides to be determined . In vitro, rITF, hITF and hSP are motogens, able to promote migration of epithelial cells . In vivo, rITF and hSP are able to prevent much of the gastric damage effect by a single dose of indomethacin, when given systemically . There is synergy between EGF and rITF both in vitro and in vivo, which may allow the development of new peptide therapies for ulceration that will maximize repair and minimize cell proliferation. Genes Chromosomes Cancer, 1996 Mar, 15(3), 191 - 4 Identification of a YAC spanning the translocation breakpoint t(8;22) associated with acute monocytic leukemia; Soenen V et al.; Using a series of yeast artificial chromosomes (YAC) from the Bp11-12 chromosome region, we have analyzed a t(8;22) translocation present in two patients suffering from acute leukemia by using fluorescence in situ hybridization (FISH) . We have identified a YAC that spans the breakpoint in both cases. Dis Markers, 1996 Mar, 12(4), 241 - 6 Establishment of sequence-tagged sites on 15q11-q13 by Alu-vector PCR cloning of YAC-generated fragments; Kim WS et al.; Angelman syndrome (AS) is caused by the loss of function of undefined gene(s) on human chromosome 15 . The majority of subjects have deletions involving maternally-derived chromosome 15q11-q13, and the shortest region of deletion overlap (SRO) has been localized to the region between D15S10 and D15S113 . In this study, yeast artificial chromosomes (YACs), 6G-D4, 9H-D2 and 37D-F9, mapping within the AS SRO, were isolated from the ICI YAC library . Alu-vector PCR products were amplified from the YACs and from YACs A229A2 and A33F10 which had been obtained from the St . Louis YAC library . The PCR products were cloned and sequenced, and three new sequence-tagged sites were generated within the AS SRO, facilitating the characterization of gene(s) involved in the Angelman syndrome. Cancer Res, 1996 Mar 1, 56(5), 978 - 83 Potential gastrointestinal tumor suppressor locus at the 3p14.2 FRA3B site identified by homozygous deletions in tumor cell lines; Kastury K et al.; A number of DNA fragments, identified by representational difference analysis, which were homozygously deleted in various cancer cell lines were previously mapped to human chromosomal arms . One of these, BE758-6, which was homozygously deleted in a number of colon carcinoma cell lines, had been mapped to chromosome region 3p . We have further localized the probe to 3p14.2, approximately 350kbp telomeric to the 3p14.2 break of the t(3;8) hereditary renal cell carcinoma chromosome translocation, within or near the 3p14.2 FRA3B, the most common human fragile site . We determined the sizes of the homozygous deletions in a number of cancer cell lines after isolation of a yeast artificial chromosome contig and development of STS markers which fall between D3S1234 and D2S1481, which flank the deletions . Homozygous deletions were observed and sized not only in the cell lines originally reported but also in a number of nasopharyngeal carcinoma cell lines and a gastric carcinoma cell line . About 50% of uncultured stomach and colon carcinomas were then shown to lose heterozygosity for alleles in the same region, with a common region of loss between the D3S1234 and D3S1481 markers . Thus, it is likely that the homozygous deletion observed in these cancer cell lines harbors an important tumor suppressor gene for several tumor types. Plant Mol Biol, 1996 Mar, 30(5), 935 - 46 Cloning and characterization of cDNA encoding farnesyl diphosphate synthase from rubber tree (Hevea brasiliensis); Adiwilaga K et al.; Commercially used natural rubber (cis-1,4-polyisoprene) is a secondary metabolite of the rubber tree (Hevea brasiliensis) . Previous studies have shown the involvement of a prenyl transferase in the final steps of natural rubber biosynthesis which includes polymerization of isopentenyl pyrophosphate into rubber . Using synthetic oligonucleotides corresponding to the partial amino acid sequences of this protein as probes to screen a laticifer-specific cDNA library, we have isolated a full-length cDNA which encodes a 47 kDa protein with strong homology to farnesyl diphosphate synthases from many species . The catalytic activity of this protein was confirmed by complementing the deletion yeast mutant . In Hevea, this gene is expressed in latex producing cells and in the epidermal region of the rubber plant suggesting a dual role for the protein in the biosyntheses of rubber and other isoprenoids . Although the expression level of this gene is not significantly affected by hormone treatment (e.g . ethylene), regeneration of latex due to tapping increases its expression level. Plant Mol Biol, 1996 Mar, 30(5), 1051 - 8 Isolation by PCR of a cDNA clone from pea petals with similarity to petunia and wheat zinc finger proteins; Michael AJ et al.; The C2H2 TFIIIA/Kruppel class of zinc finger proteins are an important group of regulatory nucleic acid binding factors and have been extensively studied in humans, Drosophila and yeast . We have employed 3' RACE PCR, using a highly degenerate oligonucleotide primer, for the facile isolation of a C2H2 zinc finger protein cDNA (Pszf1) from pea petals . The Pszf1 cDNA open reading frame potentially encodes a protein with two widely separated zinc fingers similar to zinc finger proteins from petunia and wheat . This class of two-fingered zinc finger proteins, possessing a wide and variable linker sequence, appears to be unique to plants . Three regions outside the zinc finger domains are also conserved between the members of the plant zinc finger protein family and one of these regions is a candidate nuclear localisation signal . The Pszf1 amino acid sequence is most similar to that of the petunia Epf1 protein, they possess an interfinger linker sequence of approximately the same length and they have a similar expression pattern with maximal transcript accumulation in mature petals, suggesting that Pszf1 may be the pea homologue of the petunia Epf1 zinc finger gene. Development, 1996 Mar, 122(3), 961 - 9 The Drosophila tissue polarity gene inturned acts cell autonomously and encodes a novel protein; Park WJ et al.; Mutations in the inturned (in) gene result in abnormal wing hair polarity and in many wing cells forming two or more hairs instead of the normal single hair . We have generated genetic mosaics in a number of different experiments and find that the in gene is required in all regions of the wing and that it functions in a cell autonomous fashion . We report the molecular cloning of the in gene, the molecular mapping of in mutations and the isolation and sequencing of an in cDNA clone . The in gene encodes a novel protein whose sequence suggests it will be membrane bound . The ability of an in cDNA, the expression of which is driven by the basal activity of the hsp70 promoter to rescue an in mutation suggests that patterned expression of in is unlikely to play a role in the function of the gene. Development, 1996 Mar, 122(3), 747 - 51 A test for cell autonomy, based on di-cistronic messenger translation; Hart K et al.; We have devised a test for cell autonomy of a gene that is switched on ectopically in a clone of cells, allowing us to ask whether the wild-type activity of this gene can influence neighbouring cells . To switch on the test gene, we used the yeast FRT system, and marked the FRT-generated cell clone by co-expressing beta-galactosidase . Co-expression is achieved by a stretch of 5' untranslated mRNA from the homeotic gene Ultrabithorax (Ubx), which is inserted between the two coding sequences . We show that this Ubx sequence mediates efficient and reliable di-cistronic mRNA translation in wing imaginal discs of Drosophila . Applying our test to Ubx, we find that ectopic Ubx in wing discs strictly coincides with beta-galactosidase expression . Consequently, wing cells are transformed into cells that appear to be intermediates between wing and haltere cells, contesting the view that homeotic genes act as binary switches. Mol Cell Biol, 1996 Mar, 16(3), 990 - 7 Finely tuned regulation of cytoplasmic retention of Xenopus nuclear factor 7 by phosphorylation of individual threonine residues; Shou W et al.; Xenopus nuclear factor 7 (xnf7) is a maternal gene product that functi ons in dorsal/ventral patterning of the embryo . The xnf7 protein is stored in the oocyte nucleus germinal vesicle in a hypophosphorylated state . At oocyte maturation, xnf7 is hyperphosphorylated and released into the cytoplasm, where it is anchored until the midblastula stage, where it is dephosphorylated and enters the nucleus . We demonstrated that cytoplasmic anchoring of xnf7 was regulated by changes in the phosphorylation status of four threonines within two sites, site 1 (Thr-103) and site 2 (Thr-209, Thr-212, and Thr-218), which function in an additive manner . A mutant form of xnf7 (xnf7thr-glu) in which the threonines at sites 1 and 2 were mutated to glutamic acids to mimic a permanent state of phosphorylation was retained in the cytoplasm in oocytes and embryos through the gastrula stage . The cytoplasmic form of xnf7 was detected in a large 670-kDa protein complex probably consisting of xnf7 and several other unknown protein components . Anchoring of xnf7 was not dependent on association with either microtubule or microfilament components of the cytoskeleton, since treatment with cytochalasin B and nocodazole did not affect cytoplasmic retention . Both wild-type xnf7 and xnf7thr-glu form dimers in the yeast two-hybrid system; however, homodimerization was not required for cytoplasmic retention . We suggest that the cytoplasmic retention of xnf7 depends on the phosphorylation state of the protein whereas the cytoplasmic anchoring machinery appears to be constitutively present in oocytes and throughout development until the gastrula stage. Mol Cell Biol, 1996 Mar, 16(3), 968 - 76 Mapping of a replication origin within the promoter region of two unlinked, abundantly transcribed actin genes of Physarum polycephalum; Benard M et al.; We analyzed the replication of two unlinked actin genes, ardB and ardC , which are abundantly transcribed in the naturally synchronous plasmodium of the slime mold Physarum polycephalum . Detection and size measurements of single-stranded nascent replication intermediates (RIs) demonstrate that these two genes are concomitantly replicated at the onset of the 3-h S phase and tightly linked to replication origins . Appearance of RIs on neutral-neutral two-dimensional gels at specific time points in early S phase and analysis of their structure confirmed these results and further established that, in both cases, an efficient, site-specific, bidirectional origin of replication is localized within the promoter region of the gene . We also determined similar elongation rates for the divergent replication forks of the ardC gene replicon . Finally, taking advantage of a restriction fragment length polymorphism, we studied allelic replicons and demonstrate similar localizations and a simultaneous firing of allelic replication origins . Computer search revealed a low level of homology between the promoters of ardB and ardC and, most notably, the absence of DNA sequences similar to the yeast autonomously replicating sequence consensus sequence in these Physarum origin regions . Our results with the ardB and ardC actin genes support the model of early replicating origins located within the promoter regions of abundantly transcribed genes in P . polycephalum. Mol Cell Biol, 1996 Mar, 16(3), 932 - 42 Drosophila homologs of the proto-oncogene product PEBP2/CBF beta regulate the DNA-binding properties of Runt; Golling G et al.; The Drosophila runt gene is the founding member of the Runt domain family of transcriptional regulators . Mammalian Runt domain genes encode the alpha subunit of the heterometric DNA-binding factor PEBP2/CBF . The unrelated PEBP2/CBF beta protein interacts with the Runt domain to increase its affinity for DNA . The conserved ability of the Drosophila Runt protein to respond to the stimulating effect of mammalian PEBP2/CBF beta indicated that flies were likely to have a homologous beta protein . Using the yeast two-hybrid system to isolate cDNAs for Runt-interacting proteins, we identified two Drosophila genes, referred to as Brother and Big-brother, that have substantial sequence homology with PEBP2/CBF beta . Yeast two-hybrid experiments as well as in vitro DNA-binding studies confirmed the functional homology of the Brother, Big-brother, and PEBP2/CBF beta proteins and demonstrated that the conserved regions of the Runt and Brother proteins are required for their heterodimeric interaction . The DNA-bending properties of Runt domain proteins in the presence and absence of their partners were also examined . Our results show that Runt domain proteins bend DNA and that this bending is influenced by Brother protein family members, supporting the idea that heterodimerization is associated with a conformational change in the Runt domain . Analysis of expression patterns in Drosophila embryos revealed that Brother and Big-brother are likely to interact with runt in vivo and further suggested that the activity of these proteins is not restricted to their interaction with Runt. J Biol Chem, 1996 Mar 1, 271(9), 5258 - 64 Physical interaction between the mitogen-responsive serum response factor and myogenic basic-helix-loop-helix proteins; Groisman R et al.; Terminal differentiation of muscle cells results in opposite effects on gene promoters: muscle-specific promoters, which are repressed during active proliferation of myoblasts, are turned on, whereas at least some proliferation-associated promoters, such as c-fos, which are active during cell division, are turned off . MyoD and myogenin, transcription factors from the basic-helix-loop-helix (bHLH) family, are involved in both processes, up-regulating muscle genes and down-regulating c-fos . On the other hand, the serum response factor (SRF) is involved in the activation of muscle-specific genes, such as c-fos, as well as in the up-regulation of a subset of genes that are responsive to mitogens . Upon terminal differentiation, the activity of these various transcription factors could be modulated by the formation of distinct protein-protein complexes . Here, we have investigated the hypothesis that the function of SRF and/or MyoD and myogenin could be modulated by a physical association between these transcription factors . We show that myogenin from differentiating myoblasts specifically binds to SRF . In vitro analysis, using the glutathione S-transferase pull-down assay, indicates that SRF-myogenin interactions occur only with myogenin-E12 heterodimers and not with isolated myogenin . A physical interaction between myogenin, E12, and SRF could also be demonstrated in vivo using a triple-hybrid approach in yeast . Glutathione S-transferase pull-down analysis of various mutants of the proteins demonstrated that the bHLH domain of myogenin and that of E12 were necessary and sufficient for the interaction to be observed . Specific binding to SRF was also seen with MyoD . In contrast, Id, a natural inhibitor of myogenic bHLH proteins, did not bind SRF in any of the situations tested . These data suggest that SRF, on one hand, and myogenic bHLH, on the other, could modulate each other's activity through the formation of a heterotrimeric complex. J Clin Psychiatry, 1996 Mar, 57(3), 99 - 104 The making of a user friendly MAOI diet; Gardner DM et al.; BACKGROUND: Many monoamine oxidase inhibitor (MAOI) diets are considered to be excessively restrictive and founded on poor scientific evidence . We present a safe and practical MAOI diet based on the related clinical and analytic data . METHOD: We used a critical review of the literature and our own tyramine assay results to categorize foods to be restricted absolutely, taken in moderation only, or unrestricted . RESULTS: We recommend that users avoid aged cheese; aged or cured meats (e.g., air-dried sausage); any potentially spoiled meat, poultry, or fish; broad (fava) bean pods; Marmite concentrated yeast extract; sauerkraut; soy sauce and soy bean condiments; and tap beer . Wine and domestic bottled or canned beer are considered safe when consumed in moderation . Other foods not mentioned are considered unrestricted . CONCLUSION: The concerns about perpetuating an overly restrictive MAOI diet include the avoidance by prescribers of a potentially useful treatment option, excessive limitations on lifestyle for patients, and increased risk to patients secondary to noncompliance with the diet . We propose an MAOI diet that has a solid scientific and clinical basis and that is, above all, practical. Exp Parasitol, 1996 Mar, 82(2), 87 - 96 Leishmania chagasi: a gene encoding a protein kinase with a catalytic domain structurally related to MAP kinase kinase; Li S et al.; The gene for a serine/threonine protein kinase was isolated from Leishmania chagasi . The deduced amino acid sequence encoded by this Leishmania protein kinase (LPK-1) gene possesses all of the 11 conserved subdomains found in most other serine/threonine protein kinase catalytic domains . Sequence alignment with other known protein kinases demonstrates that the 42-kDa LPK-1 is a member of the mitogen-activated protein kinase kinase family involved in the signal-transduction pathways in yeast and mammalian cells . The single copy gene for LPK-1 specifies a 3.1-kb mRNA that is expressed in both logarithmic and stationary phase promastigotes with a twofold higher steady-state level in the infective stationary phase promastigotes. Br J Haematol, 1996 Mar, 92(3), 673 - 80 Secondary acute leukaemias with 11q23 rearrangement: clinical, cytogenetic, FISH and FICTION studies; Zhang Y et al.; Three patients with secondary acute leukaemia after treatment with topoisomerase II inhibitor agents are described . Two patients had acute myeloid leukaemia (AML) . FAB M5a, one had pro-B-acute lymphoblastic leukaemia (ALL) . The interval between initiation of chemotherapy and the onset of secondary acute leukaemia was 19-20 months . 11q23 rearrangements were detected in all cases . They were due to translocations t(11;19) (q23;p13.3), t(11;16)(q23;p13) and t(4;11)(q21;q23), respectively . Fluorescence in situ hybridization (FISH) with Yeast Artificial Chromosome (YAC) probe 13HH4 spanning the ALL-1 gene on 11q23 confirmed that in each case the ALL-1 gene had been disrupted by the translocations . The study underlined the relationship between the development of secondary acute leukaemias with 11q23 rearrangement and previous chemotherapy with topisomerase II inhibitor agents . So far, however, only six adult patients with secondary ALL with t(4;11) after treatment with topoisomerase II inhibitor agents have been reported . All with t(4;11) mostly occurs in infants or young children . Our patient received epirubicin continuously for >19 months . This indicates that both myeloid and lymphoid leukaemias with involvement of the ALL-1 gene can be induced by exogenous agents, especially topoisomerase II inhibitors . Thus they may have a common biological background . This hypothesis was substantiated by means of combined immunophenotyping and FISH (FICTION) . In the case of AML M5a with t(11;19), the tumour cells with ALL-1 rearrangement expressed CD34 . Moreover, the pro-B-ALL with t(4;11) was CD34 positive . These findings suggest that the cell of origin of secondary AML and ALL with 11q23 rearrangement is an immature haemopoietic progenitor cell. RNA, 1996 Mar, 2(3), 213 - 25 The canonical GU dinucleotide at the 5' splice site is recognized by p220 of the U5 snRNP within the spliceosome; Reyes JL et al.; Specific recognition of the 5' splice site (5'SS) by the spliceosome components was studied using a simple in vitro system in which a short 5'SS RNA oligonucleotide specifically induces the assembly of snRNP particles into spliceosome-like complexes and actively participates in a trans-splicing reaction . Short-range cross-liking demonstrates that a U5 snRNP protein component, p220 (the human analogue of the yeast Prp8) specifically interacts with the invariant GU dinucleotide at the 5' end of the intron . The GU:p220 interaction can be detected in the functional splicing complex B . Although p220 has been known to contact several nucleotides around the 5' splice junction, the p220:GU dinucleotide interaction described here is remarkably specific . Consistent with the high conservation of the GU, even minor modifications of this element affect recognition of the 5'SS RNA by p220 . Substitution of uridine at the GU with base analogues containing a large methyl or iodo group, but not a smaller flouro group at base position 5, interferes with association of 5'SS RNA with snRNP complexes and their functional participation in splicing. RNA, 1996 Mar, 2(3), 201 - 12 A central pseudoknotted three-way junction imposes tRNA-like mimicry and the orientation of three 5' upstream pseudoknots in the 3' terminus of tobacco mosaic virus RNA; Felden B et al.; A three-dimensional model of the histidylable 3'-terminal tRNA-like domain of tobacco mosaic virus RNA is proposed on the basis of a comparative structural analysis, chemical and enzymatic probing, combined with graphical modeling of three RNA constructs of increasing size (38, 108, and 182 nt) derived from the 3'-terminal viral RNA sequence . The comparison between the probing patterns of the three RNAs allowed the determination of the relative orientation of these structural domains in the full-length viral tRNA-like structure . Modeling data indicate that only one of the two possible isomers of the three-way junction located at a central position of the tRNA-like domain is in agreement with structural data . Interestingly, this isomer gives rise to a molecule bearing a structural mimicry with the L-shape of canonical tRNAs . A pseudoknotted acceptor branch containing a T-like loop is located perpendicularly to an anticodon-like branch . Moreover, a single-stranded RNA stretch belonging to the pseudoknotted central core mimics a D-like loop and it is proposed that it interacts via two conserved guanosines with nucleotides of the T-like loop as found in canonical tRNAs . This model is valid for the 3' noncoding regions of tobamoviral RNAs as well as for the tRNA-like domain of the satellite tobacco mosaic virus RNA . All three molecules are substrates for yeast HisRS; however, whereas the complete viral genome is required for optimal histidylation capacities, both charging levels and affinity constants are decreased for the three RNA transcripts, suggesting that additional contacts located outside the tRNA-like domain are needed for an optimal aminoacylation process. Genes Dev, 1996 Mar 1, 10(5), 528 - 40 CBP as a transcriptional coactivator of c-Myb; Dai P et al.; CBP (CREB-binding protein) is a transcriptional coactivator of CREB (cAMP response element-binding) protein, which is directly phosphorylated by PKA (cAMP-dependent protein kinase A) . CBP interacts with the activated phosphorylated form of CREB but not with the nonphosphorylated form . We report here that CBP is also a coactivator of the c-myb proto-oncogene product (c-Myb), which is a sequence-specific transcriptional activator . CBP directly binds to the region containing the transcriptional activation domain of c-Myb in a phosphorylation-independent manner in vitro . The domain of CBP that touches c-Myb is also required for binding to CREB . A c-Myb/CBP complex in vivo was demonstrated by a yeast two-hybrid assay . CBP stimulates the c-Myb-dependent transcriptional activation . Conversely, the expression of antisense RNA of CBP represses c-Myb-induced transcriptional activation . In addition, adenovirus EIA, which binds to CBP, inhibits c-Myb-induced transcriptional activation . Our data thus identify CBP as a coactivator of c-Myb . These results suggest that CBP functions as a coactivator for more transcriptional activators than were thought previously. Curr Genet, 1996 Mar, 29(4), 335 - 43 Double-strand break-induced mitotic gene conversion: examination of tract polarity and products of multiple recombinational repair events; Weng YS et al.; Double-strand break (DSB)-induced gene conversion in yeast was studied in crosses between ura3 heteroalleles carrying phenotypically silent markers at approximately 100-bp intervals, which allow high-resolution analyses of tract structures . DSBs were introduced in vivo by HO nuclease at sites within shared homology and were repaired using information donated by unbroken alleles . Previous studies with these types of crosses showed that most tracts of Ura+ products are continuous, unidirectional, and extend away from frameshift mutations in donor alleles . Here we demonstrate that biased tract directionality is a consequence of selection pressure against Ura- products that results when frameshift mutations in donor alleles are transferred to recipient alleles . We also performed crosses in which frameshift mutations in recipient and donor alleles were arranged such that events initiated at DSBs could not convert broken alleles to Ura+ via a single gap repair event or a single long-tract mismatch repair event in heteroduplex DNA . This constraint led to low recombination frequencies relative to unconstrained crosses, and inhibited preferential conversion of broken alleles . Physical analysis of 51 DSB-induced products arising from multiple recombinational repair events suggested that hDNA formation is generally limiting, but that some hDNA regions may extend more than 600 bp . Among these products, markers separated by 20 bp were independently repaired about 40% of the time. Biochim Biophys Acta, 1996 Mar 1, 1305(3), 105 - 8 A novel cDNA encoding a human homologue of ribosomal protein L29; Law PT et al.; During the large scale partial sequencing of human heart cDNA clones, a novel clone which is very similar to the rat ribosomal protein L29 in both DNA and amino acid sequences was found . The cDNA encodes a protein with a deduced molecular weight of 17751 (159 aa) . It shows 80.4% homology to protein L29 from the large ribosomal subunit of rat and is related to yeast YL43 . The putative protein was named human ribosomal protein L29 (hRPL29) . hRPL29 has a large excess of basic residues over acidic ones . The large amount of charged residues makes the protein very hydrophilic and the protein has a deduced pI of 12.16 . Internal repeats have been characterised in many ribosomal proteins and a tandem repeat of KAKAKAKA was found to be unique to hRPL29 . Analysis of gene organisation by Southern blotting shows that of the approximate 10 copies of hrpL29, all but one are pseudogenes . Northern analysis indicated that the mRNA that encodes human L29 is approx . 800 base pairs in length . An intron of hrpL29 has also been cloned and sequenced by polymerase chain reaction using human genomic DNA as the template. Diabetes, 1996 Mar, 45(3), 291 - 4 An approach for identifying simple sequence repeat DNA polymorphisms near cloned cDNAs and genes . Linkage studies of the islet amyloid polypeptide/amylin and liver glycogen synthase genes and NIDDM; Gambino V et al.; Genetic factors contribute to the development of NIDDM, and genes involved in regulating pancreatic beta-cell function and insulin's effects on glucose metabolism are good candidates for being NIDDM susceptibility loci . However, testing candidate genes for linkage to NIDDM depends on the identification of highly informative DNA polymorphisms in or near the candidate locus . Here we describe an approach for identifying highly polymorphic markers near candidate genes that utilizes the emerging physical map of the human genome . A sequence-tagged site from the candidate gene is used to screen the Centre d'Etude du Polymorphisme Humain megabase-insert yeast artificial chromosome library, which contains information on the physical localization of >3,000 genetically mapped simple sequence repeat DNA polymorphisms . Thus, identification of a yeast artificial chromosome containing the candidate locus will in many instances also identify a physically linked simple sequence repeat DNA polymorphism that can be used as a marker for the candidate gene in linkage studies . We have used this approach to identify a marker for the islet amyloid polypeptide gene on chromosome 12 . The physical mapping of this gene to a yeast artificial chromosome showed that it was in the same yeast artificial chromosome as the gene encoding liver glycogen synthase, another possible NIDDM susceptibility gene . Affected sib pair studies using a simple sequence repeat DNA polymorphism physically linked to the islet amyloid polypeptide and liver glycogen synthase genes showed no evidence for linkage with NIDDM, indicating that they are not major genes contributing to NIDDM susceptibility. DNA Res, 1996 Feb 29, 3(1), 17 - 24 Prediction of the coding sequences of unidentified human genes . V . The coding sequences of 40 new genes (KIAA0161-KIAA0200) deduced by analysis of cDNA clones from human cell line KG-1; Nagase T et al.; As part of our continuing efforts to accumulate information on the coding region of unidentified human genes, we newly determined the sequences of 40 cDNA clones of human cell line KG-1 which correspond to relatively long and nearly full-length transcripts, and predicted the coding sequences of the corresponding genes, named KIAA0161 to 0200 . The average size of the cDNA clones analyzed was approximately 5.0 kb . A computer search of the sequences in public databases indicated that the sequences of 20 genes were unrelated to any reported genes, while the remaining 20 genes carried sequences which show some similarities to known genes . Among the genes in the latter category, KIAA0167 contained a Zn-finger motif with significant structural similarity to that of the yeast transcription factor GCS1, and KIAA0189 was classified into the RhoGAP gene family . Stretches of typical CAG (Gln) repeats, which were often correlated with genetic disorders, were found in KIAA0181 and KIAA0192 . Another novel repeat composed of alternating Arg and Glu was identified in KIAA0182 . Northern hybridization analysis demonstrated that 10 genes are expressed in a cell- or tissue-specific manner. Mutat Res, 1996 Feb 29, 359(2), 95 - 102 'Non-genotoxic' carcinogens evaluated using the white-ivory assay of Drosophila melanogaster; Consuegra S et al.; Seven carcinogenic compounds (urethane, ethionine, auramine O, safrole, amitrole, acetamide and thioacetamide) were tested using the white-ivory (Wi) assay of Drosophila melanogaster . These compounds were chosen because they were considered as Ames-test negative but produced positive results in the yeast DEL assay, which estimates the introduction of intrachromosomal recombination . Only one compound, urethane, produced positive results in the Wi assay, while the remaining were classified as negative . These results indicate that, in contrast with which has been postulated in yeast, these carcinogens do not induce any event associated to intrachromosomal recombination in D . melanogaster. FEBS Lett, 1996 Feb 26, 381(1-2), 123 - 6 The sequence of a small subunit of cytochrome c oxidase from Crithidia fasciculata which is homologous to mammalian subunit IV; Speijer D et al.; The sequence of subunit 8 of cytochrome c oxidase from Crithidia fasciculata was determined by sequencing cDNA and N-terminus of the mature protein (Mr = 15.7 kDA) . The (inferred) protein is homologous to mammalian cox IV and the corresponding cox subunits from yeast, Neurospora crassa and Dictyostelium discoideum, which is reflected in a very similar hydropathy profile . Elements that are conserved in the C . fasciculata sequence include (i) an N-terminal (D/E)-(K/R)-X-K-(X2)-W-(X2)-(I/L) motif, (ii) a putative membrane-spanning region in the middle portion of the protein, and (iii) a C-terminal W-(X13)-(N/D)-P motif . The C . fasciculata protein is synthesized with a cleavable presequence. J Biol Chem, 1996 Feb 23, 271(8), 4539 - 44 Phosphorylation of plant eukaryotic initiation factor-2 by the plant-encoded double-stranded RNA-dependent protein kinase, pPKR, and inhibition of protein synthesis in vitro; Langland JO et al.; Regulation of protein synthesis by eukaryotic initiation factor-2alpha (eIF-2alpha) phosphorylation is a highly conserved phenomenon in eukaryotes that occurs in response to various stress conditions . Protein kinases capable of phosphorylating eIF-2alpha have been characterized from mammals and yeast . However, the phenomenon of eIF2-alpha-mediated regulation of protein synthesis and the presence of an eIF-2alpha kinase has not been demonstrated in higher plants . We show that plant eIF-2alpha (peIF-2alpha) and mammalian eIF-2alpha (meIF-2alpha) are phosphorylated similarly by both the double-stranded RNA-binding kinase, pPKR, present in plant ribosome salt wash fractions and the meIF-2alpha kinase, PKR . By several criteria, phosphorylation of peIF-2alpha is directly correlated with pPKR protein and autophosphorylation levels . Significantly, pPKR is capable of specifically phosphorylating Ser51 in a synthetic eIF-2alpha peptide, a key characteristic of the eIF-2alpha kinase family . Taken together, these data support the concept that pPKR is a member of the eIF-2alpha kinase family . In addition, the inhibition of brome mosaic virus RNA in vitro translation in wheat germ lysates by the addition of double-stranded RNA, phosphorylated peIF-2alpha, meIF-2alpha, or activated human PKR suggests that plant protein synthesis may be regulated via phosphorylation of eIF-2alpha. J Biol Chem, 1996 Feb 23, 271(8), 3967 - 70 IL-1Rrp is a novel receptor-like molecule similar to the type I interleukin-1 receptor and its homologues T1/ST2 and IL-1R AcP; Parnet P et al.; A novel member of the interleukin-1 receptor family has been cloned by polymerase chain reaction using degenerate oligonucleotide primers derived from regions of sequence conservation, using as template a yeast artificial chromosome known to contain both interleukin-1 (IL-1) receptors and T1/ST2 . The new receptor, called IL-1 receptor-related protein or IL-1Rrp, fails to bind any of the known IL-1 ligands . A chimeric receptor, in which the IL-1Rrp cytoplasmic domain is fused to the extracellular and transmembrane regions of the IL-1 receptor, responds to IL-1 following transfection into COS cells by activation of NFkappaB and induction of IL-8 promoter function. Biochem Pharmacol, 1996 Feb 23, 51(4), 503 - 15 Development of an in situ toxicity assay system using recombinant baculoviruses; Grant DF et al.; A new method for experimentally analyzing the role of enzymes involved in metabolizing mutagenic, carcinogenic, or cytotoxic chemicals is described . Spodoptera fugiperda (SF-21) cells infected with recombinant baculoviruses are used for high level expression of one or more cloned enzymes . The ability of these enzymes to prevent or enhance the toxicity of drugs and xenobiotics is then measured in situ . Initial parameters for the system were developed and optimized using baculoviruses engineered for expression of the mouse soluble epoxide hydrolase (msEH, EC 3.3.2.3) or the rat cytochrome P4501A1 . SF-21 cells expressing msEH were resistant to trans-stilbene oxide toxicity as well as several other toxic epoxides including: cis-stilbene oxide, 1,2,7,8-diepoxyoctane, allylbenzene oxide, and estragole oxide . The msEH markedly reduced DNA and protein adduct formation in SF-21 cells exposed to {3H}allylbenzene oxide or {3H}estragole oxide . On the other hand, 9,10-epoxyoctadecanoic acid and methyl 9,10-epoxyoctadecanoate were toxic only to cells expressing sEH, suggesting that the corresponding fatty acid diols were cytotoxic . This was confirmed by showing that chemically synthesized diols of these fatty acid epoxides were toxic to control SF-21 cells at the same concentration as were the epoxides to cells expressing sEH . A recombinant baculovirus containing a chimeric cDNA formed between the rat P4501A1 and the yeast NADPH-P450 reductase was also constructed and expressed in this system . A model compound, naphthalene, was toxic to SF-21 infected with the rat P4501A1/reductase chimeric co-infecting SF-21 cells with either a human or a rat microsomal EH virus along with P4501A1/reductase virus . These results demonstrate the usefulness of this new system for experimentally analyzing the role of enzymes hypothesized to metabolize endogenous and exogenous chemicals of human health concern. Science, 1996 Feb 23, 271(5252), 1120 - 2 The p21(RAS) farnesyltransferase alpha subunit in TGF-beta and activin signaling; Wang T et al.; The alpha subunit of p21(RAS) farnesyltransferase (FNTA), which is also shared by geranylgeranyltransferase, was isolated as a specific cytoplasmic interactor of the transforming growth factor-beta (TGF-beta) and activin type I receptors with the use of the yeast two-hybrid system . FNTA interacts specifically with ligand-free TGF-beta type l receptor but is phosphorylated and released upon ligand binding . Furthermore, the release is dependent on the kinase activity of the TGF-beta type II receptor . Thus, the growth inhibitory and differentiative pathways activated by TGF-beta and activin involve novel mechanisms of serine-threonine receptor phosphorylation-dependent release of cytoplasmic interactors and regulation of the activation of small G proteins, such as p21(RAS). FEBS Lett, 1996 Feb 19, 380(3), 229 - 32 Functional expression of the plant K+ channel KAT1 in insect cells; Marten I et al.; Following the biophysical analysis of plant K+ channels in their natural environment, three members from the green branch of the evolutionary tree of life KAT1, AKT1, and KST1 have recently been identified on the molecular level . Among them, we focussed on the expression and characterization of the Arabidopsis thaliana K+ channel KAT1 in the insect cell line Sf9 . The infection of Sf9 cells with KAT1-recombinant baculovirus resulted in functional expression of KAT1 channels, which was monitored by inward-rectifying, K+-selective (impermeable to Na+ and even NH4+) ionic conductance in whole-cell patch-clamp recordings . A voltage threshold as low as -60 to -80mV for voltage activation compared to other plant inward rectifiers in vivo, and to in vitro expression of KAT1 in Xenopus oocytes or yeast, may be indicative for channel modulation by the expression system . A rise in cytoplasmic Ca2+ concentration (up to 1 mM), a regulator of the inward rectifier in Vicia faba guard cells, did not modify the voltage dependence of KAT1 in Sf9 cells . The access to channel function on one side and channel protein on the other make Sf9 cells a suitable heterologous system for studies on the biophysical properties, post-traditional modification and assembly of a green inward rectifier. J Biol Chem, 1996 Feb 16, 271(7), 3727 - 36 cDNA cloning and characterization of murine transcriptional enhancer factor-1-related protein 1, a transcription factor that binds to the M-CAT motif; Yockey CE et al.; The M-CAT motif is a cis-regulatory DNA sequence that is essential for muscle-specific transcription of several genes . Previously, we had shown that both muscle-specific (A1) and ubiquitous (A2) factors bind to an essential M-CAT motif in the myosin heavy chain beta gene and that the ubiquitous factor is transcriptional enhancer factor (TEF)-1 . Here we report the isolation of mouse cDNAs encoding two forms (a and b) of a TEF-1-related protein, TEFR1 . The TEFR1a cDNA encodes a 427-amino acid protein . The coding region of TEFR1b is identical to 1a in both nucleotide and predicted amino acid sequence except for the absence of 43 amino acids downstream of the TEA DNA-binding domain . Three TEFR1 transcripts (approximately 7, approximately 3.5, and approximately 2 kilobase pairs) are enriched in differentiated skeletal muscle (myotubes) relative to undifferentiated skeletal muscle (myoblasts) and non-muscle cells in culture . In situ hybridization analysis indicated that TEFR1 transcripts are enriched in the skeletal muscle lineage during mouse embryogenesis . Transient expression of fusion proteins of TEFR1 and the yeast GAL4 DNA-binding domain in cell lines activated the expression of chloramphenicol acetyltransferase (CAT) reporter constructs containing GAL4 binding sites, indicating that TEFR1 contains an activation domain . An anti-TEFR1 polyclonal antibody supershifted the muscle-specific M-CAT.A1 factor complex in gel mobility shift assays, suggesting that TEFR1 is a major component of this complex . Our results suggest that TEFR1 might play a role in the embryonic development of skeletal muscle in the mouse. J Mol Biol, 1996 Feb 16, 256(1), 89 - 107 Characterization of actin and poly-L-proline binding sites of Acanthamoeba profilin with monoclonal antibodies and by mutagenesis; Kaiser DA et al.; We characterized several deletion and substitution mutations of Acanthamoeba profilin and nine monoclonal antibodies to Acanthamoeba profilin . The results provide two independent lines of evidence about the binding sites for actin and poly-L-proline on the profilin molecule . This new evidence is consistent with the main conclusions about these binding sites from previous structural and mutagenic studies . Mutagenesis also revealed that the native structure of profilin is very sensitive to substitutions and deletions at the C terminus . For example, profilin with a deletion of the eight C-terminal residues has many of the physical properties of a molten globule, yet remarkably still binds to actin . This instability may account for the lack of function of similar mutants in yeast. J Mol Biol, 1996 Feb 16, 256(1), 187 - 200 Fast and one-step folding of closely and distantly related homologous proteins of a four-helix bundle family; Kragelund BB et al.; Bovine acyl-coenzyme A binding protein is a four-helix bundle protein belonging to a group of homologous eukaryote proteins that binds medium and long-chain acyl-coenzyme A esters with a very high affinity . The three-dimensional structure of both the free and the ligated protein together with the folding kinetics have been described in detail for the bovine protein and with four new sequences reported here, a total of 16 closely related sequences ranging from yeasts and plants to human are known . The kinetics of folding and unfolding in different concentrations of guanidine hydrochloride together with equilibrium unfolding have been measured for bovine, rat and yeast acyl-coenzyme A binding protein . The bovine and rat sequences are closely related whereas the yeast is more distantly related to these . In addition to the three natural variants, kinetics of a bovine mutant protein, Tyr31 --> Asn, have been studied . Both the folding and unfolding rates in water of the yeast protein are 15 times faster than those of bovine . The folding rates in water of the two mammalian forms, rat and bovine, are similar, though still significantly different . A faster unfolding rate both for rat and the bovine mutant protein results from a lower stability of the native states of these . These hydrophobic regions, mini cores, have been identified in the three-dimensional structure of the bovine protein and found to be formed primarily by residues that have been conserved throughout the entire eukaryote evolution from yeasts to both plants and mammals as seen in the sample of 16 sequences . The conserved residues are found to stabilize helix-helix interactions and serve specific functional purposes for ligand binding . The fast one-step folding mechanism of ACBP has been shown to be a feature that seems to be maintained throughout evolution despite numerous differences in sequence and even dramatic differences in folding kinetics and protein stability . The protein study raises the question to what extent does the conserved hydrophobic residues provide a scaffold for an efficient one-step folding mechanism. Genomics, 1996 Feb 15, 32(1), 97 - 103 High-resolution mapping of a 130-kb core region of the MYCN amplicon in neuroblastomas; Reiter JL et al.; The MYCN proto-oncogene is amplified in 25% of neuroblastomas, and amplification is strongly correlated with advanced disease stage and with rapid tumor progression . We have generated a high-resolution restriction map of nearly 500 kb spanning the MYCN locus by subcloning yeast artificial chromosomes into cosmids . Cosmids plus additional amplified probes were hybridized to DNA from 33 MYCN amplified neuroblastomas, and we determined that the amplicons range from 350 kb to over 1 Mb . Deletions and rearrangements of the MYCN amplicon occurred less frequently in primary tumors than in cell lines . We have defined a 130-kb region that was amplified in 32 of 33 tumors . One additional tumor deleted 65 kb of this region from its amplicon, while amplifying a large amount of flanking DNA . The only CpG island found in this region was within the MYCN gene . In conclusion, our data demonstrate that, despite the large size of most amplicons, the core domain that is consistently amplified in neuroblastomas probably contains little more than the MYCN gene. Genomics, 1996 Feb 15, 32(1), 29 - 38 Integrated map of the chromosome 8p12-p21 region, a region involved in human cancers and Werner syndrome; Imbert A et al.; Detailed physical maps of the human genome are important resources for the identification and isolation of disease genes and for studying the structure and function of the genome . To improve the definition of the 8p12-p21 chromosomal region, an integrated physical and genetic map was constructed extending from the genes . NEFL to FGFR1 . The map comprises a series of contigs (the larger of these being around 9 Mb) of yeast artificial chromosomes (YACs) spanning the proximal region of deletion involved in a broad range of human cancers, including breast carcinomas, and in the Werner syndrome . In addition, losses of heterozygosity at 8p markers and linkage analysis of breast cancer families were also detailed . Finally, several genes potentially involved in 8p-associated diseases, namely GTF2E2, PPP2CB, and HGL, were precisely mapped within the YAC contigs . The reported map and contigs of YACs should facilitate the search for putative genes involved in sporadic and familial breast cancer as well as in the Werner syndrome. Genomics, 1996 Feb 15, 32(1), 117 - 20 An integrated physical map covering 25 cM of human chromosome 8; Chen W et al.; We have developed an integrated physical map for human chromosome 8q23-q24.1 based on STS content analysis of somatic cell hybrids and YAC contigs . Among the 63 STSs used are markers from multiple genetic maps and several previously unmapped polymorphisms, thus integrating genetic markers from different sources into comprehensive, ordered sets based on their positions in the physical map . These maps will facilitate the isolation of genes causing human inherited diseases that map to these chromosomal regions. Genomics, 1996 Feb 15, 32(1), 1 - 14 Integration of the cytogenetic, genetic, and physical maps of the human genome by FISH mapping of CEPH YAC clones; Bray-Ward P et al.; The physical locations on human metaphase chromosomes of over 950 yeast artificial chromosome (YAC) clones from the CEPH library have been determined by fluorescence in situ hybridization and described as fractional chromosome length relative to the terminus of the short arm . Collectively, these clones contain approximately 1 billion basepairs of human DNA, about one-third of the human genome . In addition, the locations of 337 of these clones were established in terms of cytogenetic bands for chromosomes 1-18, 20, and X . Since most clones are positive for one or more of the Genethon polymorphic STS markers with defined genetic linkage distances corresponding to their physical locations, these data facilitate the integration of the cytogenetic, genetic, and physical maps of the human genome . Use of these mapping data in conjunction with public database information on CEPH YACs greatly facilitates the identification of YACs or polymorphic markers at specific locations in the genome. Microsc Res Tech, 1996 Feb 15, 33(3), 262 - 5 Slow-speed freezing of chemically unfixed biological tissues and long-term storage of frozen samples for cryoscanning electron microscopy; Adler K et al.; We describe a procedure in which plant tissue as well as a yeast culture on agar are frozen with slow cooling rates for observation of surface structures in a cryoscanning electron microscope . A system is also presented for long-term storage of frozen specimens under liquid nitrogen, in which the material is maintained for direct observation . Some small tools are described, which are essential for making preparations using slow-speed freezing and for the storage of prepared samples . Three examples of preparations with different complications are given: the "sculptures" on the surface of a leaf of Allium schoenoprasum, an early stage of flower development of Allium cernuum, and a part of an agar-grown colony of Arxula adeninivorans . In our experience, it is possible to store fully hydrated samples under the described conditions for more than a year without damaging the fine structures. EMBO J, 1996 Feb 15, 15(4), 900 - 9 Overexpression of poly(A) binding protein prevents maturation-specific deadenylation and translational inactivation in Xenopus oocytes; Wormington M et al.; The translational regulation of maternal mRNAs is the primary mechanism by which stage-specific programs of protein synthesis are executed during early development . Translation of a variety of maternal mRNAs requires either the maintenance or cytoplasmic elongation of a 3' poly(A) tail . Conversely, deadenylation results in translational inactivation . Although its precise function remains to be elucidated, the highly conserved poly(A) binding protein I (PABP) mediates poly(A)-dependent events in translation initiation and mRNA stability . Xenopus oocytes contain less than one PABP per poly(A) binding site suggesting that the translation of maternal mRNAs could be either limited by or independent of PABP . In this report, we have analyzed the effects of overexpressing PABP on the regulation of mRNAs during Xenopus oocyte maturation . Increased levels of PABP prevent the maturation-specific deadenylation and translational inactivation of maternal mRNAS that lack cytoplasmic polyadenylation elements . Overexpression of PABP does not interfere with maturation-specific polyadenylation, but reduces the recruitment of some mRNAs onto polysomes . Deletion of the C-terminal basic region and a single RNP motif from PABP significantly reduces both its binding to polyadenylated RNA in vivo and its ability to prevent deadenylation . In contrast to a yeast PABP-dependent poly(A) nuclease, PABP inhibits Xenopus oocyte deadenylase in vitro . These results indicate that maturation-specific deadenylation in Xenopus oocytes is facilitated by a low level of PABP consistent with a primary function for PABP to confer poly(A) stability. EMBO J, 1996 Feb 15, 15(4), 810 - 6 Ral-GTPases mediate a distinct downstream signaling pathway from Ras that facilitates cellular transformation; Urano T et al.; Ral proteins (RalA and RalB) comprise a distinct family of Ras-related GTPases (Feig and Emke