Yeasts

J Biochem (Tokyo), 1998 Jul, 124(1), 28 - 34
Stable isotope-edited NMR analysis of Ascaris suum mitochondrial tRNAMet having a TV-replacement loop; Ohtsuki T et al.; Most nematode mitochondrial (mt) tRNAs have a TV-replacement loop (TV loop) which replaces the normal T arm and the variable loop in standard tRNAs with a less-structured loop . The tertiary structure of such tRNAs has been discussed theoretically with reference to the crystal structure of yeast tRNAPhe {Wolstenholme et al . (1994) Nucleic Acids Res . 22, 4300-4306} and examined experimentally by chemical and enzymatic probing {Watanabe et al . (1994) J . Biol . Chem . 269, 22902-22906} . The results suggest that most regions of the tRNA other than the TV loop are folded in a similar manner to yeast tRNAPhe . To confirm this notion more clearly, the tertiary structure of Ascaris suum mt tRNAMet was analyzed by NMR using various synthetic tRNAs site-specifically labeled with stable isotopes, which were prepared by a combination of chemical synthesis and enzymatic ligation . Tertiary interactions involving G(L2), G(L3), U(L4), and U8 were observed in the NMR spectra of the labeled tRNAs, but those relating to G(L5) were not . On the basis of these results, a possible tertiary structural model of nematode mitochondrial tRNAMet was constructed.

J Med Genet, 1998 Jun, 35(6), 491 - 6
Duplication of 8p23.1: a cytogenetic anomaly with no established clinical significance; Barber JC et al.; We present seven families with a cytogenetic duplication of the short arm of chromosome 8 at band 8p23.1 . The duplication has been transmitted from parents to offspring in four of the seven families . In three families, the source of the extra material and its euchromatic origin were established using FISH with a YAC which was mapped to 8p23.1 and a whole chromosome paint for chromosome 8 . FISH signals from this YAC were significantly larger on the duplicated chromosome compared with the normal chromosome in all six family members tested . Comparative genomic hybridisation (CGH) on a representative subject was consistent with these results . The families were ascertained for a variety of mostly incidental reasons including prenatal diagnosis for advanced maternal age . The transmission of this duplication by multiple phenotypically normal family members with no history of reproductive loss suggests the existence of a novel class of 8p23.1 duplications, which can be regarded as euchromatic variants or duplications with no phenotypic effect.

J Biol Chem, 1998 Jul 3, 273(27), 16985 - 92
Interaction of BAG-1 with retinoic acid receptor and its inhibition of retinoic acid-induced apoptosis in cancer cells; Liu R et al.; BAG-1 (also known as RAP46) is an anti-apoptotic protein, which has been shown previously to interact with a number of nuclear hormone receptors, including receptors for glucocorticoid, estrogen, and thyroid hormone . We show here that BAG-1 also interacts with retinoic acid receptor (RAR) . Gel retardation assays demonstrated that in vitro translated BAG-1 protein could effectively inhibit the binding of RAR but not retinoid X receptor (RXR) to a number of retinoic acid (RA) response elements (RAREs) . A glutathione S-transferase-BAG-1 fusion protein also specifically bound RAR but not RXR . Interaction of BAG-1 and RAR could also be demonstrated by yeast two-hybrid assays . In transient transfection assays, co-transfection of BAG-1 expression plasmid inhibited the transactivation activity of RAR/RXR heterodimers but not RXR/RXR homodimers . When stably expressed in breast cancer cell lines, BAG-1 inhibited binding of RAR/RXR heterodimer to a number of RAREs and suppressed RA-induced growth inhibition and apoptosis . In addition, RA-induced suppression of Bcl-2 expression was abrogated by overexpression of BAG-1 . These results demonstrate that BAG-1 can regulate retinoid activities through its interaction with RAR and suggest that elevated levels of BAG-1 protein could potentially contribute to retinoid resistance in cancer cells.

J Biol Chem, 1998 Jul 3, 273(27), 16651 - 4
Steroid receptor coactivator-1 coactivates activating protein-1-mediated transactivations through interaction with the c-Jun and c-Fos subunits; Lee SK et al.; Steroid receptor coactivator-1 (SRC-1) specifically bound to the transcription factor AP-1 subunits c-Jun and c-Fos, as demonstrated by the yeast two-hybrid tests and glutathione S-transferase pull down assays . The c-Jun and c-Fos binding sites were localized to the C-terminal subregion of SRC-1 (amino acids 1101-1441) that encompasses the previously described histone acetyltransferase and receptor-binding domains . In mammalian cells, SRC-1, similar to the previous results with CBP-p300 (Arias, J., Alberts, A . S., Brindle, P., Claret, F . X., Smeal, T., Karin, M., Feramisco, J., and Montminy, M . (1994) Nature 370, 226-229; Bannister, A . J., and Kouzarides, T . (1995) EMBO J . 14, 4758-4762), potentiated the AP-1-mediated transactivations in a dose-dependent manner and derepressed the mutual inhibitions between nuclear receptors and AP-1 . Furthermore, coexpression of p300 further enhanced this SRC-1-potentiated level of transactivations . Thus, we concluded that at least two distinct coactivator molecules may cooperate to regulate AP-1-dependent transactivations and mediate transrepression between AP-1 and nuclear receptors in vivo.

Biochem Biophys Res Commun, 1998 Jun 18, 247(2), 524 - 9
Interleukin-2 induces N-glycosylation in T-cells: characterization of human lymphocyte oligosaccharyltransferase; Kumar V et al.; We have investigated the enzyme mediating N-glycosylation in "resting" and activated lymphocytes . Normal peripheral blood lymphocytes (PBLs) were found to have low activity for glycosylation of a synthetic glycan acceptor peptide . N-glycosylation activity increased 10-fold after mitogen activation of PBLs . N-glycosylation activity remained elevated during long-term culture and expansion of human lymphocytes when growth was supported by interleukin-2 . To our knowledge, this is the first biochemical evidence for induction of endoplasmic reticulum functions during T-cell activation . The enzyme mediating N-glycosylation in lymphocytes was localized predominantly but not entirely to a microsomal organelle by subcellular fractionation . After solubilization and 85-fold purification from salt-washed microsomes, the enzyme preparation contained four predominant proteins . N-terminal sequence analysis identified the proteins as ribophorin I, ribophorin II (doublet), and a 50-kDa homologue of Wbp1, a yeast protein essential for N-glycosylation .

J Mol Biol, 1998 Jun 26, 279(5), 1187 - 91
Conformational constraints for protein self-cleavage in the proteasome; Ditzel L et al.; The proteasome is the central enzyme of protein degradation in the cytosol and the nucleus . It is involved in the removal of abnormal, misfolded or incorrectly assembled proteins, in the processing or degradation of transcriptional regulators in stress response, in degradation of cyclins in cell-cycle control, in the destruction of transcription factors or metabolic enzymes in cell differentiation and metabolic response, and in MHC class I mediated cellular immune response . By the analysis of the crystal and molecular structures of the 20 S proteasomes from the archaeon Thermoplasma acidophilum and from yeast it was shown that the beta-type subunits in which the proteolytic activities reside are members of the N-terminal nucleophile (Ntn) protein family . They are synthesized as proproteins and become active by autoprocessing at a Gly-1-Thr1 bond . The Thr1Ala mutant of subunit beta1/Pre3 of the 20 S proteasome from yeast is unable to autolyse . Its crystal and molecular structure at 2.2 A resolution described here shows that the pro-segment adopts a well-defined gamma-turn conformation at Gly-1 and provides a first view at an autolysis site in Ntn hydrolases . The Gly-1 carbonyl oxygen displays two hydrogen bonds . The modelled Thr1 side-chain is located above the gamma-turn bulge such that addition of its nucleophilic hydroxyl group to the electrophilic Gly-1 carbonyl carbon atom may proceed by very small motions . The pro-segment binding site and the catalytic site provide a rigid structural framework and appropriate hydrogen bond donors for this reaction . The same structure also supports addition of the Thr1 hydroxyl group to the carbonyl carbon atom of Leu-2 as a model for the first step in substrate hydrolysis by the proteasome .

Curr Opin Cell Biol, 1998 Jun, 10(3), 346 - 53
Chromatin-remodeling factors: machines that regulate?
Varga-Weisz PD, Becker PB.
Chromatin has shifted into the focus of attention as a key to understanding the regulation of nuclear processes such as transcription . Protein machines have been described that use the energy of ATP to render chromatin dynamic and hence active, but which may also be involved in chromatin assembly . The discovery of three different Drosophila nucleosome remodeling complexes that contain imitation switch (ISWI), an ATPase with a high degree of sequence conservation from yeast to human, points to a central function of this ATPase in chromatin dynamics.

Shi Yan Sheng Wu Xue Bao, 1996 Sep, 29(3), 247 - 53
{Study on the transferring of YACs to new host by means of KAR cross}; Yang ZY et al.; The donor yeast strain YAC23 containing a 340 kb human genomic DNA fragment in YAC (Yeast Artificial Chromosome) was mated with recipient strain YLB504 by means of improved kar cross and the condidate YACductants were assayed by PCR and the amplification band of 404 bp indicated that they were the same as those of recipient strains (i.e . MAT alpha) . Further analysis of the electrokaryotype of the candidate YACductants by PFGE (Pulsed Field Gel Electrophoresis) confirmed that the YACs had succeeded in entering into the recipient cells . The YACs had thus completed the process of transferring from one host to the other.

Biochim Biophys Acta, 1998 Jul 1, 1407(1), 84 - 91
Assembly of a high-resolution map of the Acadian Usher syndrome region and localization of the nuclear EF-hand acidic gene; DeAngelis MM et al.; Usher syndrome type 1C (USH1C) occurs in a small population of Acadian descendants from southwestern Louisiana . Linkage and linkage disequilibrium analyses localize USH1C to chromosome 11p between markers D11S1397 and D11S1888, an interval of less than 680 kb . Here, we refine the USH1C linkage to a region less than 400 kb, between genetic markers D11S1397 and D11S1890 . Using 17 genetic markers from this interval, we have isolated a contiguous set of 60 bacterial artificial chromosomes (BACs) that span the USH1C critical region . Exon trapping of BAC clones from this region resulted in the recovery of an exon of the nuclear EF-hand acidic (NEFA) gene . However, DNA sequence analysis of the NEFA cDNA from lymphocytes of affected individuals provided no evidence of mutation, making structural mutations in the NEFA protein unlikely as the cellular cause of Acadian Usher syndrome.

Mutat Res, 1998 Jun 5, 401(1-2), 27 - 32
Caffeine does not potentiate gamma-radiation induced DNA damage in ataxia telangiectasia lymphoblastoid cells; Bebb DG et al.; Ataxia telangiectasia (AT) cells display a profound sensitivity to ionizing radiation, exhibiting more frequent chromosomal breaks, increased micronuclei formation and abnormal DNA repair kinetics following exposure . Despite the recent cloning of the ATM gene there remains a need for a simple and rapid means of discriminating AT heterozygotes from normal individuals . Caffeine (1,3,7-trimethyl xanthine), known to inhibit the repair of double-strand DNA breaks following ionizing radiation, increases the frequency of radiation induced chromosomal breaks in normal cells . Here we report that caffeine potentiates the induction of chromosomal breaks in G2 arrested AT heterozygote and normal lymphoblastoid cells, but not in homozygous AT lymphoblastoid cells . This observation parallels the findings reported by others that caffeine fails to potentiate the effect of ionizing radiation in radiation-sensitive yeast strains and radiation sensitive CHO cells . It also suggests that caffeine may somehow mimic the effect of the ATM gene product in normal cells . We also report that caffeine is unlikely to be useful in helping to discriminate AT heterozygotes from normal individuals .

Dev Genes Evol, 1998 May, 208(3), 128 - 34
Reduced expression of pax-3 is associated with overexpression of cdc46 in the mouse embryo; Hill AL et al.; CDC46/MCM5 encodes a protein that is highly conserved among yeast, plants, and animals . It is found in a complex which exhibits DNA replication licensing activity, which is proposed to regulate the synthesis of DNA once and only once per cell cycle . In yeast, loss of function mutations of CDC46/MCM5 decrease DNA synthesis . Very little is known about the regulation of CDC46/MCM5 in any species . We report here that, in the mouse embryo, expression of cdc46 is increased in unfused portions of the neural tube when the gene encoding the transcription factor, Pax-3, is either nonfunctional or underexpressed . These results were observed both in embryos of diabetic mice, which we have previously shown express significantly reduced levels of Pax-3 mRNA, and in Splotch embryos, which carry loss of function Pax-3 alleles . This indicates that expression of cdc46 is negatively regulated as part of a Pax-3-dependent pathway . Since cdc46 appears to regulate DNA synthesis and cell cycle progression, it is possible that its overexpression is involved in defective embryonic development that is associated with loss of Pax-3 function.

Biochem J, 1998 Jun 1, 332 ( Pt 2), 281 - 92
The extended protein kinase C superfamily; Mellor H et al.; Members of the mammalian protein kinase C (PKC) superfamily play key regulatory roles in a multitude of cellular processes, ranging from control of fundamental cell autonomous activities, such as proliferation, to more organismal functions, such as memory . However, understanding of mammalian PKC signalling systems is complicated by the large number of family members . Significant progress has been made through studies based on comparative analysis, which have defined a number of regulatory elements in PKCs which confer specific location and activation signals to each isotype . Further studies on simple organisms have shown that PKC signalling paradigms are conserved through evolution from yeast to humans, underscoring the importance of this family in cellular signalling and giving novel insights into PKC function in complex mammalian systems.

Electrophoresis, 1998 May, 19(6), 939 - 45
Identification of gel-separated proteins by liquid chromatography-electrospray tandem mass spectrometry: comparison of methods and their limitations; Haynes PA et al.; We have compared several different experimental systems currently in use in our laboratory for protein identification by high performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) . The efficiency of peptide recovery from trypsin-digested gel bands or electroblotted membrane slices was examined using 35S-labeled yeast proteins, and was found to be in excess of 80% . A dilution series of two standard proteins, bovine serum albumin (BSA) and carbonic anhydrase (CA), was analyzed by HPLC-ESI-MS/MS to determine what amount of protein could be loaded onto a gel and successfully identified, a measure we refer to as the practical detection limit . We were able to identify both standards at the 500 ng level in samples prepared from gel slices, using either a regular spray or a flow-split microspray HPLC-MS interface system . In samples prepared from membrane pieces, carbonic anhydrase was also identified at the 500 ng level, while bovine serum albumin could only be identified in samples of more than 1000 ng . In general, protein identification was slightly better in samples prepared from gels rather than membranes . A dilution series of lesser amounts of the same standard proteins was also analyzed using a gradient capillary LC system and we were able to successfully identify 50 ng of carbonic anhydrase and 100 ng of BSA.

Exp Cell Res, 1998 Jun 15, 241(2), 363 - 72
PKN interacts with a paraneoplastic cerebellar degeneration-associated antigen, which is a potential transcription factor; Takanaga H et al.; PKN is a fatty acid-activated serine/threonine protein kinase, having a catalytic domain homologous to protein kinase C family . PKN has been recently reported to interact with a small GTP-binding protein Rho and cytoskeletal proteins such as neurofilament and alpha-actinin . To identify the new components of the PKN-signaling pathway, the yeast two-hybrid system was employed . Using the amino-terminal regulatory domain of PKN as a bait, cDNA encoding a neural antigen PCD17, which is recognized by characteristic antibodies of patients with paraneoplastic cerebellar degeneration, was isolated from a human brain cDNA library . The interaction between PKN and PCD17 was also determined by the in vitro binding analysis . PCD17 was coimmunoprecipitated with PKN from the lysate of COS7 cells transfected with both expression constructs for PKN and the amino-terminal region of PCD17 . PCD17 was phosphorylated by PKN, and the extent of this phosphorylation was enhanced by addition of 40 microM arachidonic acid . The amino-terminal region of PCD17 could form a homodimer in vitro, and PCD17 fused to the Gal4 DNA binding domain showed the transcriptional transactivation of the chloramphenicol acetyltransferase reporter gene linked to 5 Gal4 binding sites and minimal promoter in rat C6 glioma cells . These results suggest the participation of PCD17 in gene expression and lead to a clue for elucidating the PKN signaling pathway from the cytosol to the nucleus .

Mol Cell Biol, 1998 Jul, 18(7), 4315 - 23
Interactions among Drosophila nuclear envelope proteins lamin, otefin, and YA; Goldberg M et al.; The nuclear envelope plays many roles, including organizing nuclear structure and regulating nuclear events . Molecular associations of nuclear envelope proteins may contribute to the implementation of these functions . Lamin, otefin, and YA are the three Drosophila nuclear envelope proteins known in early embryos . We used the yeast two-hybrid system to explore the interactions between pairs of these proteins . The ubiquitous major lamina protein, lamin Dm, interacts with both otefin, a peripheral protein of the inner nuclear membrane, and YA, an essential, developmentally regulated protein of the nuclear lamina . In agreement with this interaction, lamin and otefin can be coimmunoprecipitated from the vesicle fraction of Drosophila embryos and colocalize in nuclear envelopes of Drosophila larval salivary gland nuclei . The two-hybrid system was further used to map the domains of interaction among lamin, otefin, and YA . Lamin's rod domain interacts with the complete otefin protein, with otefin's hydrophilic NH2-terminal domain, and with two different fragments derived from this domain . Analogous probing of the interaction between lamin and YA showed that the lamin rod and tail plus part of its head domain are needed for interaction with full-length YA in the two-hybrid system . YA's COOH-terminal region is necessary and sufficient for interaction with lamin . Our results suggest that interactions with lamin might mediate or stabilize the localization of otefin and YA in the nuclear lamina . They also suggest that the need for both otefin and lamin in mediating association of vesicles with chromatin might reflect the function of a protein complex that includes these two proteins.

Mol Cell Biol, 1998 Jul, 18(7), 4188 - 96
Developmental specificity of the interaction between the locus control region and embryonic or fetal globin genes in transgenic mice with an HS3 core deletion; Navas PA et al.; The human beta-globin locus control region (LCR) consists of five erythroid-lineage-specific DNase I-hypersensitive sites (HSs) and is required for activation of the beta-globin locus chromatin domain and globin gene expression . Each DNase I-HS of the LCR consists of a highly conserved core element and flanking sequences . To analyze the functional role of the core elements of the HSs, we deleted a 234-bp fragment encompassing the core of HS3 (HS3c) from a beta-globin locus residing on a 248-kb beta-locus yeast artificial chromosome and analyzed its function in F2 progeny of transgenic mice . Human epsilon-globin gene expression was absent at day 10 and severely reduced in the day 12 embryonic erythropoiesis of mice lacking HS3c . In contrast, gamma-globin gene expression was normal in embryonic erythropoiesis but it was absent in definitive erythropoiesis in the fetal liver . These results indicate that the core element of HS3 is necessary for epsilon-globin gene transcription in embryonic cells and for gamma-globin gene transcription in definitive cells . Normal gamma-globin gene expression in embryonic cells and the absence of gamma-globin gene expression in definitive cells show that different HSs interact with gamma-globin gene promoters in these two stages of development . Such results provide direct evidence for developmental stage specificity of the interactions between the core elements of HSs and the promoters of the globin genes.

Mol Cell Biol, 1998 Jul, 18(7), 4131 - 40
Tyrosine 1101 of Tie2 is the major site of association of p85 and is required for activation of phosphatidylinositol 3-kinase and Akt; Kontos CD et al.; Tie2 is an endothelium-specific receptor tyrosine kinase that is required for both normal embryonic vascular development and tumor angiogenesis and is thought to play a role in vascular maintenance . However, the signaling pathways responsible for the function of Tie2 remain unknown . In this report, we demonstrate that the p85 subunit of phosphatidylinositol 3-kinase (PI3-kinase) associates with Tie2 and that this association confers functional lipid kinase activity . Mutation of tyrosine 1101 of Tie2 abrogated p85 association both in vitro and in vivo in yeast . Tie2 was found to activate PI3-kinase in vivo as demonstrated by direct measurement of increases in cellular phosphatidylinositol 3-phosphate and phosphatidylinositol 3, 4-bisphosphate, by plasma membrane translocation of a green fluorescent protein-Akt pleckstrin homology domain fusion protein, and by downstream activation of the Akt kinase . Activation of PI3-kinase was abrogated in these assays by mutation of Y1101 to phenylalanine, consistent with a requirement for this residue for p85 association with Tie2 . These results suggest that activation of PI3-kinase and Akt may in part account for Tie2's role in both embryonic vascular development and pathologic angiogenesis, and they are consistent with a role for Tie2 in endothelial cell survival.

Mol Cell Biol, 1998 Jul, 18(7), 3974 - 82
The Mdm2 oncoprotein interacts with the cell fate regulator Numb; Juven-Gershon T et al.; The Mdm2 oncoprotein is a well-known inhibitor of the p53 tumor suppressor, but it may also possess p53-independent activities . In search of such p53-independent activities, the yeast two-hybrid screen was employed to identify Mdm2-binding proteins . We report that in vitro and in transfected cells, Mdm2 can associate with Numb, a protein involved in the determination of cell fate . This association causes translocation of overexpressed Numb into the nucleus and leads to a reduction in overall cellular Numb levels . Through its interaction with Numb, Mdm2 may influence processes such as differentiation and survival . This could potentially contribute to the altered properties of tumor cells which overexpress Mdm2.

Genes Dev, 1998 Jun 15, 12(12), 1847 - 57
Miranda mediates asymmetric protein and RNA localization in the developing nervous system; Schuldt AJ et al.; Neuroblasts undergo asymmetric stem cell divisions to generate a series of ganglion mother cells (GMCs) . During these divisions, the cell fate determinant Prospero is asymmetrically partitioned to the GMC by Miranda protein, which tethers it to the basal cortex of the dividing neuroblast . Interestingly, prospero mRNA is similarly segregated by the dsRNA binding protein, Staufen . Here we show that Staufen interacts in vivo with a segment of the prospero 3' UTR . Staufen protein and prospero RNA colocalize to the apical side of the neuroblast at interphase, but move to the basal side during prophase . Both the apical and basal localization of Staufen are abolished by the removal of a conserved domain from the carboxyl terminus of the protein, which interacts in a yeast two-hybrid screen with Miranda protein . Furthermore, Miranda colocalizes with Staufen protein and prospero mRNA during neuroblast divisions, and neither Staufen nor prospero RNA are localized in miranda mutants . Thus Miranda, which localizes Prospero protein, also localizes prospero RNA through its interaction with Staufen protein.

FEBS Lett, 1998 May 15, 427(3), 330 - 6
Human RanBP3, a group of nuclear RanGTP binding proteins; Mueller L et al.; A group of novel human Ran-binding proteins, RanBP3, was identified using the yeast two-hybrid system via Ran-mediated interaction with the nucleotide exchange factor RCC1 . Several open reading frames, representing putative alternatively spliced products, were established by cDNA cloning . Two of them, RanBP3-a and RanBP3-b, encode nuclear hydrophilic proteins of 499 and 562 amino acid residues . The sequences contain FXFG motifs, characteristic of a subgroup of nucleoporins, and a C-terminal domain showing similarity to the Ran-binding protein RanBP1 . These proteins are localized in the nucleus, preferentially bind RanGTP and may be nuclear effectors of the Ran pathway.

Virology, 1998 Jun 5, 245(2), 313 - 22
Characterization of the interactions among vaccinia virus transcription factors G2R, A18R, and H5R; Black EP et al.; Prior genetic analysis suggests that there may exist an interaction between the products of the vaccinia virus genes A18R, a putative negative transcription elongation factor, and G2R, a putative positive transcription elongation factor . In addition, affinity purification of polyhistidine-tagged G2R protein overexpressed in vaccinia virus-infected cells, reported here, results in copurification of the vaccinia H5R protein, previously characterized as a late viral transcription factor . We have therefore used several methods to screen further for interactions among the G2R, A18R, and H5R proteins . Methods include copurification or co-immunoprecipitation of proteins overexpressed during vaccinia virus infection, activation of the gal 4 promoter by gal 4 fusions in the yeast two-hybrid system, and co-immunoprecipitation of proteins synthesized in vitro in a rabbit reticulocyte lysate . The results reveal interactions which include all possible pairwise combinations of the three proteins G2R, A18R, and H5R; however, not all possible permutations of the interactions are observed and the interactions are not observed in all environments tested . The results suggest that the vaccinia virus proteins G2R, A18R, and H5R interact as part of a higher order transcription complex.

Biotechnology (N Y), 1995 Nov, 13(11), 1185 - 90
Biotechnology of breadmaking: unraveling and manipulating the multi-protein gluten complex; Shwry PR et al.; Breadmaking is one of humankind's oldest technologies, being established some 4,000 years ago . The ability to make leavened bread depends largely on the visco-elastic properties conferred to wheat doughs by the gluten proteins . These allow the entrapment of carbon dioxide released by the yeast, giving rise to a light porous structure . One group of gluten proteins, the high molecular weight (HMW) subunits, are largely responsible for gluten elasticity, and variation in their amount and composition is associated with differences in elasticity (and hence quality) between various types of wheat . These proteins form elastomeric polymers stabilized by inter-chain disulphide bonds, and detailed studies of their structures have led to models for the mechanism of elasticity . This work has also provided a basis for direct improvement of wheat quality by transformation with additional HMW subunit genes.

Proc Natl Acad Sci U S A, 1998 Jun 23, 95(13), 7445 - 50
Protein kinase mutants of human ATR increase sensitivity to UV and ionizing radiation and abrogate cell cycle checkpoint control; Wright JA et al.; In fission yeast, the rad3 gene product plays a critical role in sensing DNA structure defects and activating damage response pathways . A structural homologue of rad3 in humans (ATR) has been identified based on sequence similarity in the protein kinase domain . General information regarding ATR expression, protein kinase activity, and cellular localization is known, but its function in human cells remains undetermined . In the current study, the ATR protein was examined by gel filtration of protein extracts and was found to exist predominantly as part of a large protein complex . A kinase-inactivated form of the ATR gene was prepared by site-directed mutagenesis and was used in transfection experiments to probe the function of this complex . Introduction of this kinase-dead ATR into a normal fibroblast cell line, an ATM-deficient fibroblast line derived from a patient with ataxia-telangiectasia, or a p53 mutant cell line all resulted in significant losses in cell viability . Clones expressing the kinase-dead ATR displayed increased sensitivity to x-rays and UV and a loss of checkpoint control . We conclude that ATR functions as a critical part of a protein complex that mediates responses to ionizing and UV radiation in human cells . These responses include effects on cell viability and cell cycle checkpoint control.

Proc Natl Acad Sci U S A, 1998 Jun 23, 95(13), 7322 - 6
Chromatin components as part of a putative transcriptional repressing complex; Lehming N et al.; The Drosophila HMG1-like protein DSP1 was identified by its ability to inhibit the transcriptional activating function of Dorsal in a promoter-specific fashion in yeast . We show here that DSP1 as well as its mammalian homolog hHMG2 bind to the mammalian protein SP100B and that SP100B in turn binds to human homologs of HP1 . The latter is a Drosophila protein that is involved in transcriptional silencing . Each of these proteins represses transcription when tethered to DNA in mammalian cells . These results suggest how heterochromatin proteins might be recruited to specific sites on DNA with resultant specific effects on gene expression.

Development, 1998 Jun, 125(11), 2159 - 69
The Dorsal-related immunity factor (Dif) can define the dorsal-ventral axis of polarity in the Drosophila embryo; Stein D et al.; In Drosophila embryos, dorsal-ventral polarity is defined by a signal transduction pathway that regulates nuclear import of the Dorsal protein . Dorsal protein's ability to act as a transcriptional activator of some zygotic genes and a repressor of others defines structure along the dorsal-ventral axis . Dorsal is a member of a group of proteins, the Rel-homologous proteins, whose activity is regulated at the level of nuclear localization . Dif, a more recently identified Drosophila Rel-homologue, has been proposed to act as a mediator of the immune response in Drosophila . In an effort to understand the function and regulation of Rel-homologous proteins in Drosophila, we have expressed Dif protein in Drosophila embryos derived from dorsal mutant mothers . We found that the Dif protein was capable of restoring embryonic dorsal-ventral pattern elements and was able to define polarity correctly with respect to the orientation of the egg shell . This, together with the observation that the ability of Dif to restore a dorsal-ventral axis depended on the signal transduction pathway that normally regulates Dorsal, suggests that Dif protein formed a nuclear concentration gradient similar to that seen for Dorsal . By studying the expression of Dorsal target genes we found that Dif could activate the zygotic genes that Dorsal activates and repress the genes repressed by Dorsal . Differences in the expression of these target genes, as well as the results from interaction studies carried out in yeast, suggest that Dif is not capable of synergizing with the basic helix-loop-helix transcription factors with which Dorsal normally interacts, and thereby lacks an important component of Dorsal-mediated pattern formation.

Am J Hum Genet, 1998 Jul, 63(1), 218 - 24
Direct evidence for suppression of recombination within two pericentric inversions in humans: a new sperm-FISH technique; Jaarola M et al.; Crossover within a pericentric inversion produces reciprocal recombinant chromosomes that are duplicated/deficient for all chromatin distal to the breakpoints . In view of this fact, a new technique is presented for estimating the frequency of recombination within pericentric inversions . YAC probes were selected from within the q- and p-arm flanking regions of two human inversions, and two-color FISH analysis was performed on sperm from heterozygous inversion carriers . A total of 6,006 sperm were analyzed for chromosome 1 inversion (p31q12), and 3,168 were analyzed for chromosome 8 inversion (p23q22) . Both inversions displayed suppression of crossing-over, although the amount of suppression differed between the two inversions . The recombination frequency of 13.1% recorded for chromosome 8 inversion was similar to the frequency of 11.4% previously estimated by the human/hamster-fusion method . For chromosome 1 inversion, the recombination frequency of 0 . 4% reported here was below the limits of detection of the fusion technique . The simplicity of the FISH technique and the ease of scoring facilitate analysis of a sample-population size much larger than previously had been possible.

Pediatr Dent, 1998 May-Jun, 20(3), 162 - 8
Detection of fungal organisms in saliva from HIV-infected children: a preliminary cytologic analysis; Hicks MJ et al.; PURPOSE: Fungal infections in HIV-infected individuals are associated with advancement of disease . In pediatric HIV infection, symptomatic children have a significantly higher incidence of clinical candidiasis and persistent drug-resistant candidiasis than do asymptomatic HIV-infected children . The purpose of this preliminary cytologic study was to determine the prevalence of fungal organisms in whole unstimulated saliva from children with vertically acquired HIV infection . METHODS: The subjects included 27 HIV-infected and 11 HIV-exposed, but uninfected, children . Whole unstimulated saliva was obtained for cytologic evaluation (hematoxylin and eosin, silver stains) with selected samples evaluated by electron microscopy . RESULTS: Yeast and hyphae were identified cytologically in 19% of HIV-infected (22% symptomatic HIV-infected, 11% asymptomatic HIV-infected) and 9% of HIV-exposed, but uninfected, children . Fungal organisms were found more frequently in HIV-infected with moderate (18%) and severe (27%) suppression . Fungi were more frequent with antiretroviral therapy (22%) vs no antiretroviral therapy (0%) and no antifungal therapy (20%) vs . antifungal therapy (7%) . Yeast and hyphal fungal forms are more prevalent in symptomatic HIV-infection with moderate and severe suppression, and those receiving antiretroviral agents, but no antifungal medications . CONCLUSION: Fungal organisms in the saliva may reflect oral carriage or mucosal colonization, which may influence the development of clinically significant candidiasis in these immunocompromised children.

Plant Cell, 1998 Jun, 10(6), 1055 - 68
Dissection of the fusarium I2 gene cluster in tomato reveals six homologs and one active gene copy; Simons G et al.; The I2 locus in tomato confers resistance to race 2 of the soil-borne fungus Fusarium oxysporum f sp lycopersici . The selective restriction fragment amplification (AFLP) positional cloning strategy was used to identify I2 in the tomato genome . A yeast artificial chromosome (YAC) clone covering approximately 750 kb encompassing the I2 locus was isolated, and the AFLP technique was used to derive tightly linked AFLP markers from this YAC clone . Genetic complementation analysis in transgenic R1 plants using a set of overlapping cosmids covering the I2 locus revealed three cosmids giving full resistance to F . o . lycopersici race 2 . These cosmids shared a 7-kb DNA fragment containing an open reading frame encoding a protein with similarity to the nucleotide binding site leucine-rich repeat family of resistance genes . At the I2 locus, we identified six additional homologs that included the recently identified I2C-1 and I2C-2 genes . However, cosmids containing the I2C-1 or I2C-2 gene could not confer resistance to plants, indicating that these members are not the functional resistance genes . Alignments between the various members of the I2 gene family revealed two significant variable regions within the leucine-rich repeat region . They consisted of deletions or duplications of one or more leucine-rich repeats . We propose that one or both of these leucine-rich repeats are involved in Fusarium wilt resistance with I2 specificity.

J Biol Chem, 1998 Jun 19, 273(25), 15528 - 32
The uncoupling protein UCP1 does not increase the proton conductance of the inner mitochondrial membrane by functioning as a fatty acid anion transporter; Gonzalez-Barroso MM et al.; The activity of the brown fat uncoupling protein (UCP1) is regulated by purine nucleotides and fatty acids . Although the inhibition by nucleotides is well established, the activation by fatty acids is still controversial . It has been reported that the ADP/ATP carrier, and possibly other members of the mitochondrial carrier family, mediate fatty acid uncoupling of mitochondria from a variety of sources by facilitating the transbilayer movement of the fatty acid anion . Brown fat mitochondria are known to be more sensitive to fatty acid uncoupling, a property that has been assigned to the presence of UCP1 . We have analyzed the transport properties of UCP1 and conclude that fatty acids are not essential for UCP1 function, although they increase its uncoupling activity . In order to establish the difference between the proposed carrier-mediated uncoupling and that exerted through UCP1, we have studied the facility with which fatty acids uncouple respiration in mitochondria from control yeast and strains expressing UCP1 or the mutant Cys-304 --> Gly . The concentration of free palmitate required for half-maximal activation of respiration in UCP1-expressing mitochondria is 80 or 40 nM for the mutant protein . These concentrations have virtually no effect on the respiration of mitochondria from control yeast and are nearly 3 orders of magnitude lower than those reported for carrier-mediated uncoupling . We propose that there exist two modes of fatty acid-mediated uncoupling; nanomolar concentrations activate proton transport through UCP1, but only if their concentrations rise to the micromolar range do they become substrates for nonspecific carrier-mediated uncoupling.

J Biol Chem, 1998 Jun 26, 273(26), 16434 - 41
Isolation and characterization of a novel coactivator protein, NCoA-62, involved in vitamin D-mediated transcription; Baudino TA et al.; The vitamin D receptor (VDR) forms a heterodimeric complex with retinoid X receptor (RXR) and binds to vitamin D-responsive promoter elements to regulate the transcription of specific genes or gene networks . The precise mechanism of transcriptional regulation by the VDR.RXR heterodimer is not well understood, but it may involve interactions of VDR.RXR with transcriptional coactivator or corepressor proteins . Here, a yeast two-hybrid strategy was used to isolate proteins that selectively interacted with VDR and other nuclear receptors . One cDNA clone designated NCoA-62, encoded a 62, 000-Da protein that is highly related to BX42, a Drosophila melanogaster nuclear protein involved in ecdysone-stimulated gene expression . Yeast two-hybrid studies and in vitro protein-protein interaction assays using glutathione S-transferase fusion proteins demonstrated that NCoA-62 formed a direct protein-protein contact with the ligand binding domain of VDR . Coexpression of NCoA-62 in a vitamin D-responsive transient gene expression system augmented 1, 25-dihydroxyvitamin D3-activated transcription, but it had little or no effect on basal transcription or gal4-VP16-activated transcription . NCoA-62 also interacted with retinoid receptors, and its expression enhanced retinoic acid-, estrogen-, and glucocorticoid-mediated gene expression . These data indicate that NCoA-62 may be classified into an emerging set of transcriptional coactivator proteins that function to facilitate vitamin D- and other nuclear receptor-mediated transcriptional pathways.

J Gen Virol, 1998 Jun, 79 ( Pt 6), 1399 - 403
Identification of regions of bovine respiratory syncytial virus N protein required for binding to P protein and self-assembly; Krishnamurthy S et al.; The interaction of bovine respiratory syncytial virus (BRSV) nucleocapsid protein (N) with itself and phosphoprotein (P) was investigated using the yeast two-hybrid system . N-P interaction was abolished by any of a series of internal deletions or deletions at the C terminus . In contrast, removal of up to 32 amino acids from the N terminus had little effect . Interestingly, while removal of the C-terminal 32 amino acids ablated interaction, it was largely restored by a second deletion removing up to 32 amino acids from the N terminus . Many of these interactions of the BRSV N protein demonstrated a pattern that was similar to those occurring in the N protein of related viruses . N-N interaction was abolished by any of the internal deletions; however, removal of up to 32 amino acids from the N terminus or C terminus was tolerated and increased the strength of the interaction between the two N proteins.

J Biol Chem, 1998 Jun 26, 273(26), 15933 - 9
The PR domain of the Rb-binding zinc finger protein RIZ1 is a protein binding interface and is related to the SET domain functioning in chromatin-mediated gene expression; Huang S et al.; The PR domain, first noted as the PRDI-BF1-RIZ1 homologous region, defines a sub-class of zinc finger genes that appear to function as negative regulators of tumorigenesis . This family includes the MDS1-EVI1 gene inactivated in myeloid leukemia, the PRDI-BF1/BLIMP1 transcription repressor of c-myc involved in driving B-cell differentiation, and the RIZ gene, which encodes proteins capable of binding to the retinoblastoma tumor suppressor protein (Rb) . The PR domain of MDS1-EVI1 is disrupted by translocations linked to myeloid leukemia, resulting in the activation of the PR-minus oncogenic product EVI1 . Remarkably similar to MDS1-EVI1, RIZ gene also normally produces two protein products of different length, and the smaller protein RIZ2 lacks the PR domain of RIZ1 but is otherwise identical to RIZ1 . These observations raise considerable interest to determine the function of PR . We show here that RIZ1 PR domain mediates protein-protein interaction . Recombinant fusion proteins of PR can bind to in vitro translated RIZ1 and RIZ2 proteins . The binding can be disrupted by amino acid substitutions at conserved residues of PR, suggesting that binding is specific . Of the three conserved exons of PR, the first two appear dispensable for binding, whereas the third exon is required . A region in the carboxyl terminus of RIZ proteins was mapped to be necessary and sufficient for PR binding . We also found that the PR domain shares significant sequence identity to the SET domain present in chromosomal proteins that function in modulating gene expression from yeast to mammals . Our data suggest that the PR domain is a derivative of SET domain and may function as protein binding interface in the regulation of chromatin-mediated gene expression.

Genomics, 1998 May 15, 50(1), 109 - 11
Physical mapping of the evolutionary boundary between human chromosomes 21 and 22 on mouse chromosome 10; Cole SE et al.; Adjacent regions of mouse Chromosome 10 (MMU10) show conserved synteny with human chromosome 22 (HSA22) and the telomeric region of HSA21 . Physical mapping on MMU10 using YAC fragmentation and PAC contig analyses demonstrates that Prmt2 has a position consistent with its human homolog, HRMT1L1, being telomeric to S100B on HSA21 . This result establishes Prmt2 as the new proximal boundary of the region of conserved synteny between MMU10 and HSA21 and predicts that it is the most telomeric gene known on HSA21 . Physical mapping refines the positions and order of HSA22 homologs Mmp11, Mif, and Ddt, demonstrates the orientation of S100b on the mouse chromosome, and localizes the junction of conserved synteny between HSA21 and HSA22 on MMU10 . Comparative mapping in this region is important for defining gene structure and dosage imbalance in Down syndrome (DS), for developing animal models of DS, and for understanding processes of chromosome evolution .

Genomics, 1998 May 15, 50(1), 44 - 52
Identification of sequence-tagged transcripts differentially expressed within the human hematopoietic hierarchy; Claudio JO et al.; Hematopoiesis is regulated by a complex gene expression program . To gain further insight into the molecular mechanisms underlying this process in humans, we sampled the transcriptional activity of the CD34+ hematopoietic progenitor line KG1a by single-pass sequencing the 5' ends of 1018 clones from a unidirectional cDNA library . Searches of public databases with the resulting expressed sequence tags (ESTs) identified 101 clones that showed no sequence similarity to any of the existing entries and that were therefore considered to derive from previously undescribed genes . Of the remaining 917 ESTs, 553 (a total of 485 distinct transcripts) corresponded to known genes . A further 279 KG1a ESTs matched or exhibited sequence similarity to ESTs or genomic sequences from humans and other species . Among the latter were putative human orthologs of developmental and cell cycle control genes from Caenorhabditis elegans, Drosophila, and yeast, as well as genes whose predicted amino acid sequences showed similarity to mammalian transcription factors . Hybridization of selected novel KG1a ESTs to globally amplified cDNAs prepared from single primary human hematopoietic precursors and homogeneous populations of terminally maturing hematopoietic cells revealed transcripts that are expressed preferentially at a specific stage or in a particular lineage within the hematopoietic hierarchy . Thus, included in the KG1a EST dataset are candidates for new human genes that may play roles in hematopoietic differentiative progression and lineage commitment .

Genomics, 1998 May 15, 50(1), 34 - 43
The human homologue of the Drosophila tailless gene (TLX): characterization and mapping to a region of common deletion in human lymphoid leukemia on chromosome 6q21; Jackson A et al.; Deletion of the long arm of chromosome 6 (6q) is one of the most common chromosomal abnormalities in human hematological malignancies . Two distinct regions of minimal deletion have been identified by loss of heterozygosity studies at 6q25 to 6q27 (RMD-1) and at 6q21 to 6q23 (RMD-2), suggesting the presence of one or more tumor suppressor genes . We have cloned sequences within RMD-2 and screened for novel genes using a combination of direct sequencing, cDNA library screening, and exon trapping . Sequences generated from a cosmid fragment, mapping within RMD-2, showed homology to the Drosophila tailless gene (tll) . The human homologue of the Drosophila tailless gene (human tlx; MGMW-approved symbol, TLX) was subsequently cloned from a fetal brain cDNA library . The gene is a member of the steroid nuclear receptor superfamily and is homologous to tll genes from other species that are involved in brain development . TLX is predominately expressed in the brain and maps to RMD-2 at 6q21 between DNA markers FYN and D6S447, in a YAC clone that also contains marker D6S246 . The contributions of this gene to human B-cell leukemia and to brain development are unknown at present .

Chem Res Toxicol, 1998 May, 11(5), 535 - 43
Mechanism for benomyl action as a mitochondrial aldehyde dehydrogenase inhibitor in mice; Staub RE et al.; Benomyl (a non-thio fungicide) inhibits hepatic mitochondrial low-Km aldehyde dehydrogenase (mALDH or ALDH2) in ip-treated mice by 50% (IC50) at 7.0 mg/kg, which is surprisingly the same potency range as that for several dithiocarbamate fungicides (and the related alcohol abuse drug disulfiram) and thiocarbamate herbicides previously known for their alcohol-sensitizing action . The mechanism by which benomyl inhibits mALDH was therefore examined, first by comparing the metabolism of benomyl with the aforementioned mono- and dithiocarbamates and second by evaluating the inhibitory potency of the benomyl metabolites . Benomyl in ip-treated mice is converted, via butyl isocyanate, S-(N-butylcarbamoyl)glutathione, and S-(N-butylcarbamoyl)cysteine, to S-methyl N-butylthiocarbamate (MBT), identified as a transient metabolite in liver . MBT is >10-fold more potent than benomyl or butyl isocyanate as an in vivo mALDH inhibitor and is also more potent than the intermediary S-(N-butylcarbamoyl) conjugates . Benomyl and MBT inhibit mouse hepatic mALDH in vitro with IC50s of 0.77 and 8.7 microM, respectively . The potency of MBT is greatly enhanced by fortification of the mitochondria with NADPH alone or plus microsomes giving IC50s of 0.50 and 0.23 microM, respectively . This activation of MBT is almost completely blocked by the cytochrome P450 inhibitor N-benzylimidazole but not by several other cytochrome P450 inactivators . MBT (probably following bioactivation) inhibits mALDH in vivo with an IC50 of 0.3 mg/kg . Two candidate activation products were synthesized for potency determinations . N-Hydroxy MBT (prepared via the trimethylsilyl derivative) was not detected as an MBT metabolite; its low potency also rules against N-hydroxylation as the activation process . MBT sulfoxide, from oxidation of MBT with magnesium monoperoxyphthalate in water, is one of the most potent inhibitors known for mALDH and yeast ALDH in vitro (IC50 0.08-0.09 microM) . These findings are consistent with a six-step bioactivation of benomyl, via the metabolites above and N-butylthiocarbamic acid, with MBT as the penultimate and MBT sulfoxide as the ultimate inhibitor of mALDH.

Mol Cell Biol, 1998 Jun, 18(6), 3572 - 9
Interaction of mouse polycomb-group (Pc-G) proteins Enx1 and Enx2 with Eed: indication for separate Pc-G complexes; van Lohuizen M et al.; The Polycomb group (Pc-G) constitutes an important, functionally conserved group of proteins, required to stably maintain inactive homeobox genes repressed during development . Drosophila extra sex combs (esc) and its mammalian homolog embryonic ectoderm development (eed) are special Pc-G members, in that they are required early during development when Pc-G repression is initiated, a process that is still poorly understood . To get insight in the molecular function of Eed, we searched for Eed-interacting proteins, using the yeast two-hybrid method . Here we describe the specific in vivo binding of Eed to Enx1 and Enx2, two mammalian homologs of the essential Drosophila Pc-G gene Enhancer-of-zeste {E(z)} . No direct biochemical interactions were found between Eed/Enx and a previously characterized mouse Pc-G protein complex, containing several mouse Pc-G proteins including mouse polyhomeotic (Mph1) . This suggests that different Pc-G complexes with distinct functions may exist . However, partial colocalization of Enx1 and Mph1 to subnuclear domains may point to more transient interactions between these complexes, in support of a bridging role for Enx1.

Mol Cell Biol, 1998 Jun, 18(6), 3266 - 77
Identification of primary initiation sites for DNA replication in the hamster dihydrofolate reductase gene initiation zone; Kobayashi T et al.; Mammalian replication origins appear paradoxical . While some studies conclude that initiation occurs bidirectionally from specific loci, others conclude that initiation occurs at many sites distributed throughout large DNA regions . To clarify this issue, the relative number of early replication bubbles was determined at 26 sites in a 110-kb locus containing the dihydrofolate reductase (DHFR)-encoding gene in CHO cells; 19 sites were located within an 11-kb sequence containing ori-beta . The ratio of approximately 0.8-kb nascent DNA strands to nonreplicated DNA at each site was quantified by competitive PCR . Nascent DNA was defined either as DNA that was labeled by incorporation of bromodeoxyuridine in vivo or as RNA-primed DNA that was resistant to lambda-exonuclease . Two primary initiation sites were identified within the 12-kb region, where two-dimensional gel electrophoresis previously detected a high frequency of replication bubbles . A sharp peak of nascent DNA occurred at the ori-beta origin of bidirectional replication where initiation events were 12 times more frequent than at distal sequences . A second peak occurred 5 kb downstream at a previously unrecognized origin (ori-beta') . Thus, the DHFR gene initiation zone contains at least three primary initiation sites (ori-beta, ori-beta', and ori-gamma), suggesting that initiation zones in mammals, like those in fission yeast, consist of multiple replication origins.

Mol Cell Biol, 1998 Jun, 18(6), 3245 - 56
RACK1, a receptor for activated C kinase and a homolog of the beta subunit of G proteins, inhibits activity of src tyrosine kinases and growth of NIH 3T3 cells; Chang BY et al.; To isolate and characterize proteins that interact with the unique domain and SH3 and SH2 domains of Src and potentially regulate Src activity, we used the yeast two-hybrid assay to screen a human lung fibroblast cDNA library . We identified RACK1, a receptor for activated C kinase and a homolog of the beta subunit of G proteins, as a Src-binding protein . Using GST-Src fusion proteins, we determined that RACK1 binds to the SH2 domain of Src . Coimmunoprecipitation of Src and RACK1 was demonstrated with NIH 3T3 cells . Purified GST-RACK1 inhibited the in vitro kinase activity of Src in a concentration-dependent manner . GST-RACK1 (2 microM) inhibited the activities of purified Src and Lck tyrosine kinases by 40 to 50% but did not inhibit the activities of three serine/threonine kinases that we tested . Tyrosine phosphorylation on many cellular proteins decreased in 293T cells that transiently overexpressed RACK1 . Src activity and cell growth rates decreased by 40 to 50% in NIH 3T3 cells that stably overexpressed RACK1 . Flow cytometric analyses revealed that RACK1-overexpressing cells do not show an increased rate of necrosis or apoptosis but do spend significantly more time in G0/G1 than do wild-type cells . Prolongation of G0/G1 could account for the increased doubling time of RACK1-overexpressing cells . We suggest that RACK1 exerts its effect on the NIH 3T3 cell cycle in part by inhibiting Src activity.

Mol Cell Biol, 1998 Jun, 18(6), 3130 - 9
Characterization of ABF-1, a novel basic helix-loop-helix transcription factor expressed in activated B lymphocytes; Massari ME et al.; Proteins of the basic helix-loop-helix (bHLH) family are required for a number of different developmental pathways, including neurogenesis, lymphopoiesis, myogenesis, and sex determination . Using a yeast two-hybrid screen, we have identified a new bHLH transcription factor, ABF-1, from a human B-cell cDNA library . Within the bHLH region, ABF-1 shows a remarkable conservation with other HLH proteins, including tal-1, NeuroD, and paraxis . Its expression pattern is restricted to a subset of lymphoid tissues, Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines, and activated human B cells . ABF-1 is capable of binding an E-box element either as a homodimer or as a heterodimer with E2A . Furthermore, a heterodimeric complex containing ABF-1 and E2A can be detected in EBV-immortalized lymphoblastoid cell lines . ABF-1 contains a transcriptional repression domain and is capable of inhibiting the transactivation capability of E47 in mammalian cells . ABF-1 represents the first example of a B-cell-restricted bHLH protein, and its expression pattern suggests that ABF-1 may play a role in regulating antigen-dependent B-cell differentiation.

J Biol Chem, 1998 May 15, 273(20), 12443 - 50
Identification of a novel AMP-activated protein kinase beta subunit isoform that is highly expressed in skeletal muscle; Thornton C et al.; The AMP-activated protein kinase (AMPK) is a member of a growing family of related kinases, including the SNF1 complex in yeast, which respond to nutritional stress . AMPK is a heterotrimeric complex of a catalytic subunit (alpha) and two regulatory subunits (beta and gamma), and proteins related to all three subunits have been identified in the SNF1 complex . We have used the two-hybrid system in order to identify proteins interacting with the catalytic subunit (alpha2) . Using this approach, we have isolated a novel AMPKbeta isoform, which we designate AMPKbeta2 . The N-terminal region of beta2 differs significantly from that of the previously characterized isoform (beta1), suggesting that this region could play a role in isoform-specific AMPK activity . Comparison of the C-terminal sequences of beta1 and beta2 with their related proteins in yeast identifies two highly conserved regions predicted to be involved in binding of the alpha and gamma subunits . The expression of beta1 and beta2 was examined in a number of tissues, revealing that the beta1 isoform is highly expressed in liver with low expression in skeletal muscle, whereas the opposite pattern is observed for the beta2 isoform . These results suggest that the beta isoforms have tissue-specific roles, which may involve altered responses to upstream signaling and/or downstream targeting of the AMPK complex.

Blood, 1998 May 15, 91(10), 3593 - 600
The human intrinsic factor-vitamin B12 receptor, cubilin: molecular characterization and chromosomal mapping of the gene to 10p within the autosomal recessive megaloblastic anemia (MGA1) region; Kozyraki R et al.; Uptake of vitamin B12 (cyanocobalamin) is facilitated by the cobalamin-binder gastric intrinsic factor (IF), which recognizes a 460-kD receptor, cubilin, present in the epithelium of intestine and kidney . Surface plasmon resonance analysis of ligand-affinity-purified human cubilin demonstrated a high-affinity calcium- and cobalamin-dependent binding of IF-cobalamin . Complete cDNA cloning of the human receptor showed a 3597 amino acid peripheral membrane protein with 69% identity to rat cubilin . Amino-terminal sequencing of the receptor indicates that the cDNA sequence encodes a precursor protein undergoing proteolytic processing due to cleavage at a recognition site (Arg7-Glu8-Lys9-Arg) for the trans-Golgi proteinase furin . Using fluorescence in situ hybridization, radiation hybrid mapping, and screening of YAC clones, the human cubilin gene was mapped between the markers D10S1661 and WI-5445 on the short arm of chromosome 10 . This is within the autosomal recessive megaloblastic anemia (MGA1) 6-cM region harboring the unknown recessive-gene locus of juvenile megaloblastic anemia caused by intestinal malabsorption of cobalamin (Imerslund-Grasbeck's disease) . In conclusion, the present molecular and genetic information on human cubilin now provides circumstantial evidence that an impaired synthesis, processing, or ligand binding of cubilin is the molecular background of this hereditary form of megaloblastic anemia.

Cutis, 1998 Apr, 61(4), 217 - 9
Routine histologic examination for the diagnosis of onychomycosis: an evaluation of sensitivity and specificity; Machler BC et al.; Clinical differentiation of dermatophyte infection from dystrophic changes due to psoriasis may be challenging . Typically, potassium hydroxide (KOH) preparations, fungal culture, and occasionally, nail unit biopsy specimens are utilized to help differentiate between the two . These tests are often time-consuming and may yield false-negative results . Increasing regulation of the office laboratory has caused some physicians to forgo this testing, which was previously routine . We investigated the utility of routine histologic examination of nail clippings in differentiating onychomycosis from psoriatic onychodystrophy . Twenty-three distal nail clipping specimens (twelve specimens from patients with onychodystrophy of unknown cause and eleven control specimens from nails with known cause) were evaluated by routine histology and periodic acid-Schiff (PAS) staining . Of the dystrophic cases, four were demonstrated to be onychomycosis by the presence of hyphae on histologic evaluation and by culture, whereas only three of these cases yielded positive results on KOH examination . Eight cases of onychodystrophy were due to psoriasis . Yeast forms were detected on one case of psoriatic onychodystrophy that demonstrated yeast growth on culture . In our study, routine histologic examination with PAS staining was equal to culture and superior to KOH preparation in leading to the correct diagnosis of dermatophyte infection . In addition, the diagnosis of psoriasis of the nail plate was detected accurately by routine histologic examination . Routine histologic examination with PAS staining is a rapid, simple, and reliable test in the evaluation of onychodystrophy.

Nat Biotechnol, 1996 Jul, 14(7), 845 - 51
High-avidity human IgG kappa monoclonal antibodies from a novel strain of minilocus transgenic mice; Fishwild DM et al.; Human immunoglobulin transgenic mice provide a method of obtaining human monoclonal antibodies (Mabs) using conventional hybridoma technology . We describe a novel strain of human immunoglobulin transgenic mice and the use of this strain to generate multiple high-avidity human sequence IgG kappa Mabs directed against a human antigen . The light chain transgene is derived in part from a yeast artificial chromosome clone that includes nearly half of the germline human V kappa region . In addition, the heavy-chain transgene encodes both human mu and human gamma 1 constant regions, the latter of which is expressed via intratransgene class switching . We have used these animals to isolate human IgG kappa Mabs that are specific for the human T-cell marker CD4, have high binding avidities, and are immunosuppressive in vitro . The human Mab-secreting hybridomas display properties similar to those of wild-type mice including stability, growth, and secretion levels . Mabs with four distinct specificities were derived from a single transgenic mouse, consistent with an extensive diversity in the primary repertoire encoded by the transgenes.

Gene, 1998 Jun 15, 213(1-2), 101 - 6
A Dictyostelium discoideum homologue to Tcp-1 is essential for growth and development; Iijima M et al.; Tcp-1 (t-complex polypeptide 1 gene) was first identified in the mouse as relevant for tail-less and embryonic lethal phenotypes . Since then, its homologous sequences have been isolated in several other species, and the yeast Tcp-1 has been shown to encode a molecular chaperon for actin and tubulin . In a random sample of genes expressed in the gamete of Dictyostelium discoideum (Dd), we encountered a sequence containg the TCP1 motifs . The complete ORF of the gene (DdTcp-1) showed more than 60% similarity to TCP-1 of several organisms, including human . DdTcp-1 was found to be expressed in both sexually mature and immature cells at the growth phase . Although the sexual process itself was not affected, antisense interference of this gene resulted in severe retardation of cell growth, leading to the complete cessation of division . In addition, the antisense transformants stopped asexual development at the finger stage . These results suggest an important function of DdTcp-1 in growth and development of this organism .

Am J Hum Genet, 1998 Jun, 62(6), 1312 - 9
Amerindian pyruvate carboxylase deficiency is associated with two distinct missense mutations; Carbone MA et al.; We characterized the pyruvate carboxylase (PC) gene by PCR amplification, subcloning, and sequencing . The coding region has 19 exons and 18 introns spanning approximately 16 kb of genomic DNA . Screening both the cDNA and the gene of individuals with the simple A form of PC deficiency revealed an 1828G-->A missense mutation in 11 Ojibwa and 2 Cree patients and a 2229G-->T transversion mutation in 2 brothers of Micmac origin . Carrier frequency may be as high as 1/10 in some groupings . The two point mutations are located in a region of homology conserved among yeast, rat, and human PC, in the vicinity of the carboxylation domain of the enzyme . These data provide the first characterization of the human PC gene structure, the identification of common pathogenic mutations, and the demonstration of a founder effect in the Ojibwa and Cree patients.

J Antimicrob Chemother, 1998 May, 41(5), 567 - 70
Susceptibility of Aspergillus strains from culture collections to amphotericin B and itraconazole; Rath PM; Susceptibility testing of 27 Aspergillus reference strains belonging to five species was performed using the microdilution broth method with yeast nitrogen broth and RPMI-1640 . Similar results were found using the two media . The strains of Aspergillus fumigatus (n = 8) and Aspergillus niger (n = 4) had MICs of amphotericin B in the range 0.125-0.5 mg/L . In contrast, nine out of 13 strains of Aspergillus flavus and Aspergillus nidulans had MICs in the range 2-16 mg/L . All strains had MICs of itraconazole in the range 0.03-0.5 mg/L . It is concluded that both media can be used for testing . Susceptibility testing to amphotericin B is recommended in clinical settings.

Eur J Clin Nutr, 1998 May, 52(5), 363 - 7
Plasma selenoprotein P levels of healthy males in different selenium status after oral supplementation with different forms of selenium; Persson-Moschos M et al.; OBJECTIVE: To assess changes in selenoprotein P levels in plasma from subjects who had received oral supplements of different selenium forms . DESIGN: The same study group participated in two similar selenium supplementation trials, Trial I in 1981 (Levander et al, 1983) and Trial II in 1987 (Alfthan et al, 1991) . During Trial II the mean baseline intake of selenium in Finland was higher compared to that during Trial I (100 and 40 microg/d, respectively), due to a nationwide supplementation of fertilisers which started in 1985 . SUBJECTS: Fifty healthy Finnish men, 36-60 y old . Intervention: The study group received daily placebo or oral supplements consisting of 200 microg selenium as selenium-enriched yeast, sodium selenate or selenium-enriched wheat (Trial I) or selenium-enriched yeast, sodium selenate or sodium selenite (Trial II) . The duration of supplementation periods was 11 (Trial I) and 16 (Trial II) weeks . RESULTS: In Trial I the mean plasma selenoprotein P values in all the supplemented groups increased significantly, approaching a plateau at 2 weeks and reaching maxima at 4 weeks (mean increase 34%, P < 0.05) . In Trial II the mean selenoprotein P levels of the supplemented groups were not significantly different from each other or from the placebo group at the start or at any time point of the supplementation period . CONCLUSIONS: At a low selenium status the selenoprotein P levels increased in a similar fashion after supplementation with different forms of selenium, but at a high selenium status no significant effects of supplementation with the same amount of selenium were observed . No differences in selenoprotein P levels were observed for inorganic and organic selenium supplements.

Cell, 1998 May 29, 93(5), 731 - 40
Sec6/8 complex is recruited to cell-cell contacts and specifies transport vesicle delivery to the basal-lateral membrane in epithelial cells; Grindstaff KK et al.; In budding yeast, the Sec6/8p complex is essential for generating cell polarity by specifying vesicle delivery to the bud tip . We show that Sec6/8 homologs are components of a cytosolic, approximately 17S complex in nonpolarized MDCK epithelial cells . Upon initiation of calcium-dependent cell-cell adhesion, approximately 70% of Sec6/8 is rapidly (t(1/2) approximately 3-6 hr) recruited to sites of cell-cell contact . In streptolysin-O-permeabilized MDCK cells, Sec8 antibodies inhibit delivery of LDL receptor to the basal-lateral membrane, but not p75NTR to the apical membrane . These results indicate that lateral membrane recruitment of the Sec6/8 complex is a consequence of cell-cell adhesion and is essential for the biogenesis of epithelial cell surface polarity.

Gene, 1998 May 12, 211(2), 251 - 7
Rainbow trout Sox24, a novel member of the Sox family, is a transcriptional regulator during oogenesis; Kanda H et al.; We isolated a cDNA clone encoding a novel SRY-type HMG box (Sox) protein, designated Sox24, from a rainbow trout ovary cDNA library . On the basis of the HMG box amino acid sequence, Sox24 can be categorized into the same subgroup of Sox proteins as SOX4, SOX11, and SOX22 . The proteins in this group also share a highly conserved sequence at the C-terminus . The Sox24 mRNA is expressed at high levels in the ovary, and in-situ hybridization localized its expression to oocytes . The recombinant protein containing the Sox24 HMG box region bound to an AACAAT sequence strongly in a gel retardation assay . Upon co-transfection into CHO cells, the full-length Sox24 transactivated transcription from a reporter plasmid through the AACAAT binding motif . We used GAL4/Sox24 chimeras with the DNA binding domain of yeast GAL4 at the N-terminus to map the transactivation function to the C-terminal region, which included the conserved sequence . These results suggest that Sox24 plays a role as a transcriptional regulator during oogenesis.

Int J Cancer, 1998 Jun 10, 76(6), 797 - 800
Absence of p53 gene mutations in a tumor panel representative of pilocytic astrocytoma diversity using a p53 functional assay; Ishii N et al.; Although p53-gene mutations occur with significant frequency in diffuse low-grade and high-grade astrocytomas, and are postulated to play an important role in tumorigenesis in these cases, the role of the p53 gene in pilocytic astrocytomas remains unclear . Published data using DNA-based assays for p53-gene analysis in these tumors have shown contradictory results in mutation frequency (0-14%) . It is not known whether these heterogeneous results stem from the biological diversity of this tumor group or from technical problems . To re-evaluate p53-gene status in pilocytic tumors, we analyzed 18 tumors chosen to represent the clinical and biological heterogeneity of this tumor type with respect to anatomical location, patient age, gender, ethnic origin (Caucasian or Japanese) and the concomitant occurrence of neurofibromatosis type 1 (NF1) . All primary tumors were histologically diagnosed as pilocytic astrocytoma (WHO grade I), except for one anaplastic pilocytic astrocytoma (WHO grade III) which developed in an NF1 patient and recurred as glioblastoma multiforme (WHO grade IV) . p53 mutations were detected using an assay in yeast which tests the transcriptional activity of p53 proteins synthesized from tumor mRNA-derived p53-cDNA templates . None of 18 tumors, including 3 NF1-related tumors, showed p53-gene mutations between and including exons 4 and 11 . We conclude that p53-gene mutations are extremely rare findings in pilocytic astrocytomas, and are absent even in those exceptional cases in which malignant progression of such tumors has occurred.

Nucleic Acids Res, 1998 Jul 1, 26(13), 3202 - 7
U2 and U6 snRNA genes in the microsporidian Nosema locustae: evidence for a functional spliceosome; Fast NM et al.; The removal of introns from pre-messenger RNA is mediated by the spliceosome, a large complex composed of many proteins and five small nuclear RNAs (snRNAs) . Of the snRNAs, the U6 and U2 snRNAs are the most conserved in sequence, as they interact extensively with each other and also with the intron, in several base pairings that are necessary for splicing . We have isolated and sequenced the genes encoding both U6 and U2 snRNAs from the intracellularly parasitic microsporidian Nosema locustae . Both genes are expressed . Both RNAs can be folded into secondary structures typical of other known U6 and U2 snRNAs . In addition, the N.locustae U6 and U2 snRNAs have the potential to base pair in the functional intermolecular interactions that have been characterized by extensive analyses in yeast and mammalian systems . These results indicate that the N.locustae U6 and U2 snRNAs may be functional components of an active spliceosome, even though introns have not yet been found in microsporidian genes.

Nucleic Acids Res, 1998 Jul 1, 26(13), 3119 - 26
A highly specific terminal uridylyl transferase modifies the 3'-end of U6 small nuclear RNA; Trippe R et al.; HeLa cell extracts contain significant amounts of terminal uridylyl transferase (TUTase) activity . In a template-independent reaction with labeled UTP, these enzymes are capable of modifying a broad spectrum of cellular RNA molecules in vitro . However, fractionation of cell extracts by gel filtration clearly separated two independent activities . In addition to a non-specific enzyme, an additional terminal uridylyl transferase has been identified that is highly specific for cellular and in vitro synthesized U6 small nuclear RNA (snRNA) molecules . This novel TUTase enzyme was also able to select as an efficient substrate U6 snRNA species from higher eucaryotes . In contrast, no labeling was detectable with purified fission yeast RNA . Using synthetic RNAs containing different amounts of transcribed 3'-end UMP residues, high resolution gel electrophoresis revealed that U6 snRNA species with three terminal U nucleotides served as the optimal substrate for the transferase reaction . The 3'-end modification of the optimal synthetic substrate was identical to that observed with endogenous U6 snRNA isolated from HeLa cells . Therefore, we conclude that the specific addition of UMP residues to 3'-recessed U6 snRNA molecules reflects a recycling process, ensuring the functional regeneration for pre-mRNA splicing of this snRNA.

EMBO J, 1998 Jun 15, 17(12), 3484 - 94
Binary specification of nonsense codons by splicing and cytoplasmic translation; Thermann R et al.; Premature translation termination codons resulting from nonsense or frameshift mutations are common causes of genetic disorders . Complications arising from the synthesis of C-terminally truncated polypeptides can be avoided by 'nonsense-mediated decay' of the mutant mRNAs . Premature termination codons in the beta-globin mRNA cause the common recessive form of beta-thalassemia when the affected mRNA is degraded, but the more severe dominant form when the mRNA escapes nonsense-mediated decay . We demonstrate that cells distinguish a premature termination codon within the beta-globin mRNA from the physiological translation termination codon by a two-step specification mechanism . According to the binary specification model proposed here, the positions of splice junctions are first tagged during splicing in the nucleus, defining a stop codon operationally as a premature termination codon by the presence of a 3' splicing tag . In the second step, cytoplasmic translation is required to validate the 3' splicing tag for decay of the mRNA . This model explains nonsense-mediated decay on the basis of conventional molecular mechanisms and allows us to propose a common principle for nonsense-mediated decay from yeast to man.

EMBO J, 1998 Jun 15, 17(12), 3258 - 68
Mapping the interaction between GRASP65 and GM130, components of a protein complex involved in the stacking of Golgi cisternae; Barr FA et al.; The nature of the complex containing GRASP65, a membrane protein involved in establishing the stacked structure of the Golgi apparatus, and GM130, a putative Golgi matrix protein and vesicle docking receptor, was investigated . Gel filtration revealed that GRASP65 and GM130 interact in detergent extracts of Golgi membranes under both interphase and mitotic conditions, and that this complex can bind to the vesicle docking protein p115 . Using in vitro translation and site-directed mutagenesis in conjunction with immunoprecipitation, the binding site for GRASP65 on GM130 was mapped to the sequence xxNDxxxIMVI-COOH at the C-terminus of GM130, a region known to be required for its localization to the Golgi apparatus . The same approach was used to show that the binding site for GM130 on GRASP65 maps to amino acids 189-201, a region conserved in the mammalian and yeast proteins and reminiscent of PDZ domains . Using green fluorescent protein (GFP)-tagged reporter constructs, it was shown that one essential function of the interaction between GRASP65 and GM130 is in the correct targeting of the two proteins to the Golgi apparatus.

EMBO J, 1998 Jun 15, 17(12), 3251 - 7
Role of the proteasome in membrane extraction of a short-lived ER-transmembrane protein; Mayer TU et al.; Selective degradation of proteins at the endoplasmic reticulum (ER-associated degradation) is thought to proceed largely via the cytosolic ubiquitin-proteasome pathway . Recent data have indicated that the dislocation of short-lived integral-membrane proteins to the cytosolic proteolytic system may require components of the Sec61 translocon . Here we show that the proteasome itself can participate in the extraction of an ER-membrane protein from the lipid bilayer . In yeast mutants expressing functionally attenuated proteasomes, degradation of a short-lived doubly membrane-spanning protein proceeds rapidly through the N-terminal cytosolic domain of the substrate, but slows down considerably when continued degradation of the molecule requires membrane extraction . Thus, proteasomes engaged in ER degradation can directly process transmembrane proteins through a mechanism in which the dislocation of the substrate and its proteolysis are coupled . We therefore propose that the retrograde transport of short-lived substrates may be driven through the activity of the proteasome.

Exp Eye Res, 1998 May, 66(5), 625 - 44
Primary sequence, secondary structure, gene structure, and assembly properties suggests that the lens-specific cytoskeletal protein filensin represents a novel class of intermediate filament protein; Hess JF et al.; The ocular lens fiber cell assembles a novel cytoskeletal element, the Beaded Filament, from CP49 and filensin, two proteins expressed only in the differentiated lens fiber cell . We report the primary sequence, secondary structural analysis, gene structure and Yeast Two Hybrid interaction data for human filensin, and develop a consensus model of filensin from the human and previously reported bovine and chicken filensin sequences . This consensus model, combined with gene structure and Yeast Two Hybrid studies establish that filensin is a member of the Intermediate Filament family of proteins . Specifically, filensin exhibits (1) divergence at amino acid sequence motifs otherwise highly conserved among intermediate filament proteins, (2) a loss of 29 amino acids from the central rod domain which is unique among cytoplasmic intermediate filament proteins, (3) an absence of sequence identity with any existing class of intermediate filament protein, (4) a gene structure unique among intermediate filament family, (5) an inability to dimerize with representatives of Type I, II, and III intermediate filament proteins . Thus, at each level of analysis, we find that filensin is similar to the consensus model of intermediate filament proteins, supporting our conclusion that filensin's relatedness to the IF family is not the consequence of convergent evolution . However, filensin also shows unique or extreme distinctions from the consensus intermediate filament protein at each level of analysis, indicating that filensin constitutes a novel class of IF protein . Some of filensin's unique features are incompatible with current models of IF assembly . Analysis of filensin gene structure suggests that the 29 amino acid reduction in the central rod domain was not the result of a single splice site mutation, the mechanism suggested for the transition between nuclear lamins and cytoplasmic intermediate filament proteins.

DNA Res, 1998 Feb 28, 5(1), 19 - 23
Contig map of the Parkinson's disease region on 4q21-q23; Lavedan C et al.; We have constructed a yeast artificial chromosome contig (YAC) map of human chromosome 4q21-q23 across the Parkinson's disease region by combining molecular and fluorescence in situ hybridization techniques . This map contains 55 YACs and 51 molecular markers, including 23 polymorphic markers . We have also isolated one P1 and 33 bacterial artificial chromosomes located within this contig . Plasmid libraries were generated from 11 of these BAC and P1 clones, and 614 random plasmid clones were sequenced for a total of about 200 kb . This contig allowed us to precisely determine the location of 18 transcripts within the D4S2460-D4S2986 interval, including the alpha-synuclein gene found to be mutated in some families with Parkinson's disease.

Eur J Cell Biol, 1998 Apr, 75(4), 309 - 20
Epithelial and fibroblastoid cells contain numerous cell-type specific putative microtubule-regulating proteins, among which are ezrin and fodrin; Shestakova E et al.; Upon cell junction formation, the microtubules of polarizing epithelial cells become reorganized by unknown signaling mechanisms and regulating proteins . Microtubule-associated (MAPs) and other types of proteins are likely to be involved in this process, but most of these are unknown . Such proteins are called here collectively microtubule-regulating proteins (MRPs) . As a first step towards their characterization, we used co-sedimentation of cytosolic proteins of MDCK cells and A72, a dog fibroblastoid line, with an excess of taxol-stabilized MTs, to obtain a cell fraction enriched in putative MRPs ("MRPs") . Additional tests have led to the inventory of around 40 "MRPs" among the 80 proteins present in the microtubule pellet . We also found that "MRPs" are recovered in higher amounts from MDCK cytosol, and that half of these are cell-type specific . These results corroborate data from yeast cells and insect eggs, and show that in mammalian somatic cells too, a large number of proteins seems to be involved in microtubule regulation, and that different cell types use a specific set of MRPs . "MRPs" found in both cell types are the intermediate chain of cytoplasmic dynein, Arp1, the major subunit of the dynactin complex, and CLIP-170 . Two MDCK-specific "MRPs" were identified as the actin-binding proteins ezrin and alpha-fodrin . These results are discussed with regard to a possible involvement of ezrin and fodrin in morphogenetic interactions of microtubules with the membrane cytoskeleton in polarizing epithelia upon junction formation.

Plant J, 1998 Apr, 14(2), 143 - 6
Sensing trehalose biosynthesis in plants; Goddijn O et al.; A most unexpected finding in research on plant carbohydrate metabolism is the recent discovery that angiosperms encode genes whose products are involved in trehalose metabolism . The presence and functionality of such genes has been elegantly shown by expressing Arabidopsis-derived trehalose phosphate synthase and trehalose phosphate phosphatase genes in yeast mutants lacking these enzymatic activities . Homologue sequences have now been cloned from a number of different plant species suggesting that the capacity to synthesise trehalose is ubiquitous in angiosperms . Except for Myrothamnus flabellifolius, trehalose biosynthesis has never been observed in tissues of higher plants, probably due to the presence of high levels of trehalase activity . The function of trehalose metabolism in plants is still a mystery . One of the postulated functions of trehalose metabolism in yeast is in the control of glucose repression and a similar function in sugar sensing can be proposed for plants as well.

J Eukaryot Microbiol, 1998 May-Jun, 45(3), 347 - 51
Mechanisms of microsporidial cell division: ultrastructural study on Encephalitozoon hellem; Bigliardi E et al.; The mitotic process in microsporidian Encephalitozoon hellem, a known human pathogen, has been studied with the aim of elucidating some ultrastructural aspects of its nuclear division . The presence of a nuclear spindle, of "electrondense spindle plaques" associated with the nuclear envelope and of cytoplasmic double walled vesicles are reported . We suggest that these "electrondense spindle plaques" serve as foci for intranuclear and cytoplasmic microtubule arrangements, similar to the microtubule organizing centers within the centrosomes of animal cells . The extent to which the microsporidial division process is comparable with that of more familiar eukaryotes such as yeast cells is discussed.

In Vivo, 1998 Mar-Apr, 12(2), 155 - 8
Proteasomal RNase activity in human epidermis; Horikoshi T et al.; The proteasome is a cytoplasmic high-molecular-weight structure composed of several smaller protein and RNA subunits . It has been associated with non-lysosomal pathways of intracellular degradation, expressing multicatalytic proteinase activities and specific RNase activity . By standard methods, we have isolated andpartially purified proteasomes from human epidermis . We obtained the expected multiple 24-32 kDa subunits by SDS-PAGE, and evidence of RNA . Proteasomes degraded casein, as well as chromogens for t-PA and trypsin but not for chymotrypsin, these proteolytic activities overlap, but do not coincide with those observed in other organs . We found that human epidermal 28 S and 18 S rRNAs were degraded, but yeast RNA was not . By means of zymography, we demonstrated, for the first time, that RNase activity persists after dissociation of the proteasome on the gel and that it co-localizes to the same range of molecular weight subunits as the proteinase activity.

Oncogene, 1998 May 14, 16(19), 2541 - 7
The Bmi-1 oncoprotein interacts with dinG and MPh2: the role of RING finger domains; Hemenway CS et al.; Experimentally-induced mutations in the C3HC4 RING finger domain of the Bmi-1 oncoprotein block its ability to induce lymphomas in mice . In this report, the role of the Bmi-1 RING finger in mediating protein-protein interactions is examined using the yeast two-hybrid system . Bmi-1 interacts directly with the RING finger protein dinG/RING1B . Heterodimerization of the two proteins requires the intact RING finger structures of both Bmi-1 and dinG . Although the RING finger domains are necessary for dimerization, they are not sufficient for this process as residues outside the C3HC4 motif are also required . Thus, binding specificity may be partly conferred by residues outside the RING motif . Both Bmi-1 and dinG interact with the Polyhomeotic protein MPh2 through binding domains apart from the RING finger . The data suggest a model whereby Bmi-1, dinG, and MPh2 form a stable heterotrimeric complex in which each protein contributes to the binding of the others.

Ecotoxicol Environ Saf, 1998 May-Jun, 40(1-2), 160 - 5
Cytoskeleton alterations by tributyltin (TBT) in tunicate phagocytes; Cima F et al.; The effects of tributyltin chloride (TBT) on cytoskeletal components, as possible cell targets of toxicity, were examined on cultured hemocytes of the colonial ascidian Botryllus schlosseri by means of indirect immunofluorescence . The immunotoxic effect of 10 microM TBT (sublethal concentration) consists of (1) inhibition of yeast phagocytosis, Ca2+ ATPase activity, and respiratory burst; (2) increase in intracellular Ca2+ concentration; and (3) alterations in cell morphology . After 60 min, TBT-exposed amebocytes become spherical, withdrawing their long pseudopodia, and lose motility . Their microfilaments assemble in clusters around the peripheric cytoplasm, indicating massive disassembly, with the exception of unaltered adhesion plaques . Analogously, their microtubules reveal extensive disaggregation, being scattered in the cytoplasm and not recognizable as single filaments, whereas the microtubule organizing center (MTOC) is still visible . Treatment together with 20 micrograms/ml calmodulin (CaM) can partially restore the cytoskeleton architecture . These results suggest a relationship between TBT and Ca2+ homeostasis in ascidian hemocytes . By interfering with Ca2+ ATPase activity through CaM inhibition, either directly or indirectly, TBT induces an excess of intracellular Ca2+ accumulation, which first causes internal disorganization of cytoskeletal proteins and consequently inhibition of phagocytosis, beginning from chemotaxis and particle adhesion.

Cell Immunol, 1998 Feb 25, 184(1), 45 - 50
Modulation by interferon-gamma of the production and gene expression of IL-1 receptor antagonist in human neutrophils; McDonald PP et al.; In this report, we show that interferon-gamma (IFN-gamma) modulates the production of IL-1ra in activated human neutrophils . In lipopolysaccharide-stimulated cells, IFN-gamma increased the release of IL-1ra without modulating IL-1ra mRNA accumulation; under these conditions, IFN-gamma only marginally enhanced IL-1ra de novo synthesis, while IL-1ra was more efficiently secreted . In response to the formylated peptide, fMLP, neutrophils released small but significant amounts of IL-1ra, yet without an increase in IL-1ra mRNA over constitutive levels . Following IFN-gamma treatment, however, the fMLP-elicited IL-1ra production was greatly potentiated, and this was accompanied by a transient increased accumulation of IL-1ra mRNA . Finally, opsonized yeast particles were found to induce IL-1ra formation at late incubation times, and prior treatment of neutrophils with IFN-gamma moderately enhanced this response . Collectively, our results demonstrate that in neutrophils activated by different classes of agonists, IFN-gamma modulates the release of IL-1ra by acting through distinct mechanisms.

J Virol, 1998 Jul, 72(7), 5502 - 9
Derivation and functional characterization of a consensus DNA binding sequence for the tas transcriptional activator of simian foamy virus type 1; Kang Y et al.; Although DNA binding sites specific for the Bel-1 and Tas transcriptional activators, encoded, respectively, by the human and simian foamy viruses, have been mutationally defined, they show little evident sequence identity . As a result, the sequence determinants for DNA binding by both Bel-1 and Tas have remained unclear . Here, we report the use of a novel in vivo randomization and selection strategy to identify a Tas DNA binding site consensus . This approach takes advantage of the fact that Tas can effectively activate gene expression in yeast cells via a Tas DNA binding site derived from the simian foamy virus type 1 (SFV-1) internal promoter . The defined Tas DNA binding site consensus extends over approximately 25 bp and contains a critical core sequence of approximately 5 bp . Positions adjacent to this core sequence, while clearly also subject to selection, show a significantly higher level of sequence variation . Surprisingly, the wild-type SFV-1 internal promoter Tas DNA binding site fails to conform to the consensus at several positions . Further analysis demonstrated that the consensus sequence bound Tas more effectively than did the wild-type sequence in vitro and could mediate an enhanced Tas response in vivo when substituted into the SFV-1 internal promoter context . These findings explain the limited sequence identity observed for mutationally defined Tas or Bel-1 response elements and should facilitate the identification of Tas DNA target sites located elsewhere in the SFV-1 genome.

Proc Natl Acad Sci U S A, 1998 Jun 9, 95(12), 7220 - 4
Identification of a family of zinc transporter genes from Arabidopsis that respond to zinc deficiency; Grotz N et al.; Millions of people worldwide suffer from nutritional imbalances of essential metals like zinc . These same metals, along with pollutants like cadmium and lead, contaminate soils at many sites around the world . In addition to posing a threat to human health, these metals can poison plants, livestock, and wildlife . Deciphering how metals are absorbed, transported, and incorporated as protein cofactors may help solve both of these problems . For example, edible plants could be engineered to serve as better dietary sources of metal nutrients, and other plant species could be tailored to remove metal ions from contaminated soils . We report here the cloning of the first zinc transporter genes from plants, the ZIP1, ZIP2, and ZIP3 genes of Arabidopsis thaliana . Expression in yeast of these closely related genes confers zinc uptake activities . In the plant, ZIP1 and ZIP3 are expressed in roots in response to zinc deficiency, suggesting that they transport zinc from the soil into the plant . Although expression of ZIP2 has not been detected, a fourth related Arabidopsis gene identified by genome sequencing, ZIP4, is induced in both shoots and roots of zinc-limited plants . Thus, ZIP4 may transport zinc intracellularly or between plant tissues . These ZIP proteins define a family of metal ion transporters that are found in plants, protozoa, fungi, invertebrates, and vertebrates, making it now possible to address questions of metal ion accumulation and homeostasis in diverse organisms.

Blood, 1998 Jun 15, 91(12), 4457 - 63
Frequent dysregulation of the c-maf proto-oncogene at 16q23 by translocation to an Ig locus in multiple myeloma; Chesi M et al.; Dysregulation of oncogenes by translocation to an IgH (14q32) or IgL (kappa, 2p11 or lambda, 22q11) locus is a frequent event in the pathogenesis of B-cell tumors . Translocations involving an IgH locus and a diverse but nonrandom array of chromosomal loci occur in most multiple myeloma (MM) tumors even though the translocations often are not detected by conventional cytogenetic analysis . In a continuing analysis of translocations in 21 MM lines, we show that the novel, karyotypically silent t(14;16)(q32.3;q23) translocation is present in 5 MM lines, with cloned breakpoints from 4 lines dispersed over an approximately 500-kb region centromeric to the c-maf proto-oncogene at 16q23 . Another line has a t(16;22)(q23;q11), with the breakpoint telomeric to c-maf, so that the translocation breakpoints in these 6 lines bracket c-maf . Only these 6 lines overexpress c-maf mRNA . As predicted for dysregulation of c-maf by translocation, there is selective expression of one c-maf allele in 2 informative lines with translocations . This is the first human tumor in which the basic zipper c-maf transcription factor is shown to function as an oncogene.

Blood, 1998 Jun 15, 91(12), 4419 - 26
Fusion of Huntingtin interacting protein 1 to platelet-derived growth factor beta receptor (PDGFbetaR) in chronic myelomonocytic leukemia with t(5;7)(q33;q11.2); Ross TS et al.; We report the fusion of the Huntingtin interactin protein 1 (HIP1) gene to the platelet-derived growth factor betareceptor (PDGFbetaR) gene in a patient with chronic myelomonocytic leukemia (CMML) with a t(5;7)(q33;q11.2) translocation . Southern blot analysis of patient bone marrow cells with a PDGFbetaR gene probe demonstrated rearrangement of the PDGFbetaR gene . Anchored polymerase chain reaction using PDGFbetaR primers identified a chimeric transcript containing the HIP1 gene located at 7q11.2 fused to the PDGFbetaR gene on 5q33 . HIP1 is a 116-kD protein recently cloned by yeast two-hybrid screening for proteins that interact with Huntingtin, the mutated protein in Huntington's disease . The consequence of t(5;7)(q33;q11.2) is an HIP1/PDGFbetaR fusion gene that encodes amino acids 1 to 950 of HIP1 joined in-frame to the transmembrane and tyrosine kinase domains of the PDGFbetaR . The reciprocal PDGFbetaR/HIP1 transcript is not expressed . HIP1/PDGFbetaR is a 180-kD protein when expressed in the murine hematopoietic cell line, Ba/F3, and is constitutively tyrosine phosphorylated . Furthermore, HIP1/PDGFbetaR transforms the Ba/F3 cells to interleukin-3-independent growth . These data are consistent with an alternative mechanism for activation of PDGFbetaR tyrosine kinase activity by fusion with HIP1, leading to transformation of hematopoietic cells, and may implicate Huntingtin or HIP1 in the pathogenesis of hematopoietic malignancies.

J Biol Chem, 1998 Jun 12, 273(24), 15061 - 8
The human proteinase-activated receptor-3 (PAR-3) gene . Identification within a Par gene cluster and characterization in vascular endothelial cells and platelets; Schmidt VA et al.; Proteolytically activated receptors (PARs) represent an emerging subset of seven transmembrane G protein-coupled receptors that mediate cell activation events by receptor cleavage at distinct scissile bonds located within receptor amino termini . Differential genomic blotting using a yeast artificial chromosome known to contain the PAR-1 and PAR-2 genes identified the PAR-3 gene within a PAR gene cluster spanning approximately 100 kilobases at 5q13 . The PAR-3 gene is relatively small (approximately 12 kilobases); and, like the PAR-1 and PAR-2 genes, it displays a two-exon structure, with the majority of the coding sequence and the proteolytic cleavage site contained within the larger second exon . Sequence analysis of the 5'-flanking region demonstrates that the promoter is TATA-less, similar to that seen with PAR-1, with the identification of nucleic acid motifs potentially involved in transcriptional gene regulation, including AP-1, GATA, and octameric sequences . PAR-3 transcripts were apparent in human vascular endothelial cells, although at considerably lower levels than those of PAR-1 and not significantly modulated by the endothelial cell stimulus tumor necrosis factor-alpha . Likewise, although PAR-3 mRNA was evident in human platelets, receptor cell surface expression was modest (approximately 10%) compared with that of PAR-1 . Thus, although PAR-3 is postulated to represent a second thrombin receptor, its modest endothelial cell and platelet expression suggest that PAR-3 activation by alpha-thrombin is less relevant for physiological responses in these mature cells . Rather, given its disparately greater expression in megakaryocytes (and megakaryocyte-like human erythroleukemia cells), a regulatory role in cellular development (by protease activation) could be postulated.

J Biol Chem, 1998 Jun 12, 273(24), 14796 - 804
Identification of transcriptional activation and repression domains in human CCAAT/enhancer-binding protein epsilon; Williamson EA et al.; Human CCAAT/enhancer-binding protein epsilon (C/EBPepsilon), a new member of the C/EBP family, significantly up-regulates both the mim-1 and human myeloperoxidase promoters, suggesting an important role for C/EBPepsilon in the transcriptional regulation of a subset of myeloid-specific genes . To elucidate the structure and function of C/EBPepsilon in transcriptional activation, amino acid residues 1-115, 147-249, or 1-249 of C/EBPepsilon were fused to the yeast GAL4 DNA binding domain . These expression vectors were cotransfected with a chloramphenicol acetyltransferase reporter gene and, in all cell lines tested, only the GAL-C/EBPepsilon-(1-115) fusion protein significantly activated expression from the chloramphenicol acetyltransferase reporter gene . Sixteen deletion mutants of C/EBPepsilon mapped the transactivation domain to amino acids 1-18 at the N terminus and revealed the presence of a transcription repression element between amino acid residues 116 and 162 . Expression vectors containing the repression domain of C/EBPepsilon strongly inhibited gene transcription from TK, SV40, and adenoviral major late promoters bearing GAL4 binding sites . Fusion of this repression domain to the VP16 activation domain inhibited the transactivation function of VP16 . Deletion of this repression domain increased gene transcription from a neutrophil elastase promoter-luciferase reporter . Taken together, these data suggest that C/EBPepsilon regulates transcription by utilizing both activation and repression functions.

J Biol Chem, 1998 Jun 12, 273(24), 14767 - 71
AMP-activated protein kinase inhibits the glucose-activated expression of fatty acid synthase gene in rat hepatocytes; Foretz M et al.; Although it is now clearly established that a number of genes involved in glucose and lipid metabolism are up-regulated by high glucose concentrations in both liver and adipose tissue, the signaling pathway arising from glucose to the transcriptional machinery is still poorly understood . We have analyzed the regulation of fatty acid synthase gene expression by glucose in cultured rat hepatocytes . Glucose (25 mM) induces an activation of the transcription of the fatty acid synthase gene, and this effect is markedly reduced by incubation of the cells with okadaic acid, an inhibitor of protein phosphatases 1 and 2A . A similar reduction in glucose-activated fatty acid synthase gene expression is obtained by incubation with 5-amino-imidazolecarboxamide riboside, a cell-permeable activator of the AMP-activated protein kinase . Taken together, these results indicate that the glucose-induced expression of the fatty acid synthase gene involves a phosphorylation/dephosphorylation mechanism and suggest that the AMP-activated protein kinase plays an important role in this process . This is the first evidence that implicates the AMP-activated protein kinase in the regulation of gene expression . AMP-activated protein kinase is the mammalian analog of SNF1, a kinase involved in yeast in the transcriptional regulation of genes by glucose.

Genes Chromosomes Cancer, 1998 Jul, 22(3), 186 - 99
Chromosome region 8p11-p21: refined mapping and molecular alterations in breast cancer; Adelaide J et al.; Several genes, most of them unknown, of the short arm of chromosome 8 are involved in malignant diseases . Numerous studies have implicated a portion of the 8p11-p21 region as the location of one or more tumor suppressor genes involved in a variety of human cancers, including breast cancer . We and others have reported linkage analyses suggesting the presence of a putative breast cancer susceptibility gene . Furthermore, several oncogenes of the 8p11-p12 region are involved in reciprocal translocations in myeloproliferative and myelodysplastic disorders and in amplification in breast cancer . To facilitate the analysis of the 8p11-p21 region and the cloning of candidate oncogenes and tumor suppressor genes, a high-resolution physical and transcriptional map was established with 39 yeast artificial chromosomes and 94 markers, including so-called sequence-tagged sites and expressed sequence-tagged sites derived from either known genes or expressed sequence tags corresponding to unidentified transcripts . In addition, four novel transcripts were identified and localized precisely within the map . This transcription map provides a detailed description of gene order for the 8p11-p21 region and will be helpful in the identification of candidate genes for diseases . From this basis, we refined the mapping of two types of molecular alterations that occur at 8p11-p21 in sporadic breast cancers, i.e., amplification and deletion.

Am J Hum Genet, 1998 Apr, 62(4), 785 - 91
Mutations in the liver glycogen phosphorylase gene (PYGL) underlying glycogenosis type VI; Burwinkel B et al.; Deficiency of glycogen phosphorylase in the liver gives rise to glycogen-storage disease type VI (Hers disease; MIM 232700) . We report the identification of the first mutations in PYGL, the gene encoding the liver isoform of glycogen phosphorylase, in three patients with Hers disease . These are two splice-site mutations and two missense mutations . A mutation of the 5' splice-site consensus of intron 14 causes the retention of intron 14 and the utilization of two illegitimate 5' splice sites, whereas a mutation of the 3' splice-site consensus of intron 4 causes the skipping of exon 5 . Two missense mutations, N338S and N376K, both cause nonconservative replacements of amino acids that are absolutely conserved even in yeast and bacterial phosphorylases . We also report corrections of the PYGL coding sequence, sequence polymorphisms, and a partial PYGL gene structure with introns in the same positions as in PYGM, the gene of the muscle isoform of phosphorylase . Our findings demonstrate that PYGL mutations cause Hers disease, and they may improve laboratory diagnosis of deficiencies of the liver phosphorylase system.

Am J Hum Genet, 1998 Apr, 62(4), 925 - 36
Molecular cytogenetic evidence for a common breakpoint in the largest inverted duplications of chromosome 15; Wandstrat AE et al.; Chromosomes from 20 patients were used to delineate the breakpoints of inverted duplications of chromosome 15 (inv dup{15}) that include the Prader-Willi syndrome/Angelman syndrome (PWS/AS) chromosomal region (15q11-q13) . YAC and cosmid clones from 15q11-q14 were used for FISH analysis, to detect the presence or absence of material on each inv dup(15) . We describe two types of inv dup(15): those that break between D15S12 and D15S24, near the distal boundary of the PWS/AS chromosomal region, and those that share a breakpoint immediately proximal to D15S1010 . Among the latter group, no breakpoint heterogeneity could be detected with the available probes, and one YAC (810f11) showed a reduced signal on each inv dup(15), compared with that on normal chromosomes 15 . The lack of breakpoint heterogeneity may be the result of a U-type exchange involving particular sequences on either homologous chromosomes or sister chromatids . Parent-of-origin studies revealed that, in all the cases analyzed, the inv dup(15) was maternal in origin.

Hum Mol Genet, 1998 Jun, 7(6), 959 - 67
Androgen receptor YAC transgenic mice carrying CAG 45 alleles show trinucleotide repeat instability; La Spada AR et al.; X-linked spinal and bulbar muscular atrophy (SBMA) is caused by a CAG repeat expansion in the first exon of the androgen receptor (AR) gene . Disease-associated alleles (37-66 CAGs) change in length when transmitted from parents to offspring, with a significantly greater tendency to shift size when inherited paternally . As transgenic mice carrying human AR cDNAs with 45 and 66 CAG repeats do not display repeat instability, we attempted to model trinucleotide repeat instability by generating transgenic mice with yeast artificial chromosomes (YACs) carrying AR CAG repeat expansions in their genomic context . Studies of independent lines of AR YAC transgenic mice with CAG 45 alleles reveal intergenerational instability at an overall rate of approximately 10% . We also find that the 45 CAG repeat tracts are significantly more unstable with maternal transmission and as the transmitting mother ages . Of all the CAG/CTG repeat transgenic mice produced to date the AR YAC CAG 45 mice are unstable with the smallest trinucleotide repeat mutations, suggesting that the length threshold for repeat instability in the mouse may be lowered by including the appropriate flanking human DNA sequences . By sequence-tagged site content analysis and long range mapping we determined that one unstable transgenic line has integrated an approximately 70 kb segment of the AR locus due to fragmentation of the AR YAC . Identification of the cis -acting elements that permit CAG tract instability and the trans -acting factors that modulate repeat instability in the AR YAC CAG 45 mice may provide insights into the molecular basis of trinucleotide repeat instability in humans.

J Cell Biol, 1998 May 4, 141(3), 675 - 87
Xgrip109: a gamma tubulin-associated protein with an essential role in gamma tubulin ring complex (gammaTuRC) assembly and centrosome function; Martin OC et al.; Previous studies indicate that gamma tubulin ring complex (gammaTuRC) can nucleate microtubule assembly and may be important in centrosome formation . gammaTuRC contains approximately eight subunits, which we refer to as Xenopus gamma ring proteins (Xgrips), in addition to gamma tubulin . We found that one gammaTuRC subunit, Xgrip109, is a highly conserved protein, with homologues present in yeast, rice, flies, zebrafish, mice, and humans . The yeast Xgrip109 homologue, Spc98, is a spindle-pole body component that interacts with gamma tubulin . In vertebrates, Xgrip109 identifies two families of related proteins . Xgrip109 and Spc98 have more homology to one family than the other . We show that Xgrip109 is a centrosomal protein that directly interacts with gamma tubulin . We have developed a complementation assay for centrosome formation using demembranated Xenopus sperm and Xenopus egg extract . Using this assay, we show that Xgrip109 is necessary for the reassembly of salt-disrupted gammaTuRC and for the recruitment of gamma tubulin to the centrosome . Xgrip109, therefore, is essential for the formation of a functional centrosome.

J Biol Chem, 1998 May 8, 273(19), 11745 - 51
Cloning and expression of a novel K+ channel regulatory protein, KChAP; Wible BA et al.; Voltage-gated K+ (Kv) channels are important in the physiology of both excitable and nonexcitable cells . The diversity in Kv currents is reflected in multiple Kv channel genes whose products may assemble as multisubunit heteromeric complexes . Given the fundamental importance and diversity of Kv channels, surprisingly little is known regarding the cellular mechanisms regulating their synthesis, assembly, and metabolism . To begin to dissect these processes, we have used the yeast two-hybrid system to identify cytoplasmic regulatory molecules that interact with Kv channel proteins . Here we report the cloning of a novel gene encoding a Kv channel binding protein (KChAP, for K+ channel-associated protein), which modulates the expression of Kv2 channels in heterologous expression system assays . KChAP interacts with the N termini of Kvalpha2 subunits, as well as the N termini of Kvalpha1 and the C termini of Kvbeta subunits . Kv2.1 and KChAP were coimmunoprecipitated from in vitro translation reactions supporting a direct interaction between the two proteins . The amplitudes of Kv2 . 1 and Kv2.2 currents are enhanced dramatically in Xenopus oocytes coexpressing KChAP, but channel kinetics and gating are unaffected . Although KChAP binds to Kv1.5, it has no effect on Kv1.5 currents . We suggest that KChAP may act as a novel type of chaperone protein to facilitate the cell surface expression of Kv2 channels.

Mech Dev, 1998 May, 73(2), 169 - 82
Gene regulatory functions of Drosophila fish-hook, a high mobility group domain Sox protein; Ma Y et al.; In this study we investigate the gene regulatory functions of Drosophila Fish-hook (Fish), a high mobility group (HMG) Sox protein that is essential for embryonic segmentation . We show that the Fish HMG domain binds to the vertebrate Sox protein consensus DNA binding sites, AACAAT and AACAAAG, and that this binding induces an 85 degrees DNA bend . In addition, we use a heterologous yeast system to show that the NH2-terminal portion of Fish protein can function as a transcriptional activator . Fish directly regulates the expression of the pair rule gene, even-skipped (eve), by binding to multiple sites located in downstream regulatory regions that direct formation of eve stripes 1, 4, 5, and 6 . Fish may function along with the Drosophila POU domain proteins Pdm-1 and Pdm-2 to regulate eve transcription, as genetic interactions were detected between fish and pdm mutants . Finally, we determined that Fish protein is expressed in a dynamic pattern throughout embryogenesis, and is present in nuclear and cytoplasmic compartments .

J Cell Biol, 1998 Jun 1, 141(5), 1193 - 205
Microinjection of antibody to Mad2 protein into mammalian cells in mitosis induces premature anaphase; Gorbsky GJ et al.; In yeast, the Mad2 protein is required for the M phase arrest induced by microtubule inhibitors, but the protein is not essential under normal culture conditions . We tested whether the Mad2 protein participates in regulating the timing of anaphase onset in mammalian cells in the absence of microtubule drugs . When microinjected into living prophase or prometaphase PtK1 cells, anti-Mad2 antibody induced the onset of anaphase prematurely during prometaphase, before the chromosomes had assembled at the metaphase plate . Anti-Mad2 antibody-injected cells completed all aspects of anaphase including chromatid movement to the spindle poles and pole-pole separation . Identical results were obtained when primary human keratinocytes were injected with anti-Mad2 antibody . These studies suggest that Mad2 protein function is essential for the timing of anaphase onset in somatic cells at each mitosis . Thus, in mammalian somatic cells, the spindle checkpoint appears to be a component of the timing mechanism for normal mitosis, blocking anaphase onset until all chromosomes are aligned at the metaphase plate.

Development, 1998 May, 125(9), 1647 - 57
The CArG boxes in the promoter of the Arabidopsis floral organ identity gene APETALA3 mediate diverse regulatory effects; Tilly JJ et al.; APETALA3 is a MADS box gene required for normal development of the petals and stamens in the Arabidopsis flower . Studies in yeast, mammals and plants demonstrate that MADS domain transcription factors bind with high affinity to a consensus sequence called the CArG box . The APETALA3 promoter contains three close matches to the consensus CArG box sequence . To gain insights into the APETALA3 regulatory circuitry, we have analyzed the APETALA3 promoter using AP3::uidA(GUS) fusions . 496 base pairs of APETALA3 promoter sequence 5' to the transcriptional start directs GUS activity in the same temporal and spatial expression pattern as the APETALA3 RNA and protein in wild-type flowers . A synthetic promoter consisting of three tandem repeats of a 143 base pair sequence directs reporter gene activity exclusively to petals and stamens in the flower . We have analyzed the role of the CArG boxes by site-specific mutagenesis and find that the three CArG boxes mediate discrete regulatory effects . Mutations in CArG1 result in a decrease in reporter expression suggesting that CArG1 is the binding site for a positively acting factor or factors . Mutations in CArG2 result in a decrease in reporter expression in petals, but the expression pattern in stamens is unchanged . By contrast, mutations in CArG3 result in an increase in the level of reporter gene activity during early floral stages suggesting that CArG3 is the binding site for a negatively acting factor.

Cancer Res, 1998 Jun 1, 58(11), 2456 - 60
Identification of two common regions of allelic loss in chromosome arm 12q in human pancreatic cancer; Kimura M et al.; Using the method of microsatellite analysis, we studied 40 tissues with pancreatic ductal adenocarcinoma and identified two commonly deleted regions on the long arm of chromosome 12 . One (region A) was found between D12S81 and D12S1719 at 12q21 at a frequency of 67.5%, and the other (region B) was located between D12S360 and D12S78 at 12q22-q23.1 at a frequency of 60%; the latter was reported previously (M . Kimura, et al . Genes Chromosomes Cancer, 17: 88-93, 1996) . The results of microsatellite analyses were verified by fluorescence in situ hybridization . We further analyzed 19 pancreatic cancer cell lines by fluorescence in situ hybridization and found that 10 of them showed allelic loss at D12S81 and 6 showed allelic loss at D12S360 . Yeast artificial chromosome contigs were constructed to cover the deleted regions . Region B was completely covered by a 650-kb yeast artificial chromosome clone . The frequently deleted regions in chromosome 12q in pancreatic cancer that were identified here may provide new avenues for isolating novel tumor suppressor genes.

Genetics, 1998 Jun, 149(2), 663 - 75
Members of the Arabidopsis actin gene family are widely dispersed in the genome; McKinney EC et al.; Plant genomes are subjected to a variety of DNA turnover mechanisms that are thought to result in rapid expansion and presumable contraction of gene copy number . The evolutionary history of the 10 actin genes in Arabidopsis thaliana is well characterized and can be traced to the origin of vascular plant genomes . Knowledge about the genomic position of each actin gene may be the key to tracing landmark genomic duplication events that define plant families or genera and facilitate further mutant isolation . All 10 actin genes were mapped by following the segregation of cleaved amplified polymorphisms between two ecotypes and identifying actin gene locations among yeast artificial chromosomes . The Arabidopsis actin genes are widely dispersed on four different chromosomes (1, 2, 3, and 5) . Even the members of three closely related and recently duplicated pairs of actin genes are unlinked . Several other cytoskeletal genes (profilins, tubulins) that might have evolved in concert with actins were also mapped, but showed few patterns consistent with that evolutionary history . Thus, the events that gave rise to the actin gene family have been obscured either by the duplication of very small genic fragments or by extensive rearrangement of the genome.

Biochem J, 1998 Apr 15, 331 ( Pt 2), 607 - 13
Structure of the human hexokinase type I gene and nucleotide sequence of the 5' flanking region; Ruzzo A et al.; This study reports the precise intron/exon boundaries and intron/exon composition of the human hexokinase type I gene . A yeast artificial chromosome containing the hexokinase type I gene was isolated from the yeast artificial chromosome library of the Centre d'Etude du Polymorphisme Humaine . A cosmid sublibrary was created and direct sequencing of the individual cosmids was used to provide the exon/intron organization . The human hexokinase type I gene was found to be composed of 18 exons ranging in size from 63 to 305 bp . Intron 1 is at least 15 kb in length, whereas intron 2 spans at least 10 kb . Overall, the length of the 17 introns ranges from 104 to greater than 15 kb . The entire coding region is contained in at least 75 kb of the gene . The structure of the gene reveals a remarkable conservation of the size of the exons compared with glucokinase and hexokinase type II . Isolation of the 5' flanking region of the gene revealed a 75-90% identity with the rat sequence . Direct evidence of an alternative red-blood-cell-specific exon 1 located upstream of the 5' flanking region of the gene is also provided.

Biochem J, 1998 Apr 15, 331 ( Pt 2), 387 - 93
Nucleolar protein p120 contains an arginine-rich domain that binds to ribosomal RNA; Gustafson WC et al.; Human proliferation-associated protein p120 has previously been shown to localize to the nucleolus, and several functional domains of p120 have been elucidated . By using a nitrocellulose filter binding assay and a Northwestern blotting procedure this study shows that recombinant p120 binds to an rRNA fragment in vitro with a dissociation constant of 4 nM . The specific RNA-binding region of p120 (residues 1-57) was identified with glutathione S-transferase-fused p120 deletion constructs and Northwestern blotting procedures . This RNA-binding region of p120, which includes the nucleolar localization signal of p120, is similar to the arginine-rich RNA-binding regions found in other RNA-binding proteins such as HIV Rev and Tat . Experiments in vivo with HeLa cell nucleolar extracts showed that p120 was associated with the 60-80S pre-ribosomal particles . This association is disrupted by treatment with either RNase A or buffer of high ionic strength . These results suggest that p120 might be involved in rRNA/ribosome maturation, consistent with the role of the yeast homologue Nop2p in rRNA biogenesis.

Bioessays, 1998 Apr, 20(4), 317 - 27
In vivo gap repair in Drosophila: a one-way street with many destinations; Lankenau DH et al.; While it has long been possible to study the process of recombination in yeast and other single-celled organisms, it has been difficult to distinguish between pathways of meiotic and mitotic recombination in multicellular eukaryotes . The experimental system described here bridges the historically separated fields of Genetic Recombination and DNA Repair in Drosophila . It is now feasible to study the repair of unique double-strand breaks induced in the Drosophila genome by the excision of a P-transposable element or by cleavage at an introduced endonuclease recognition sequence . This repair can be studied in both somatic cells and mitotically dividing germ cells . The repair of these breaks occurs mainly by copying sequence from a template located anywhere in the karyoplasm, and occurs in both male and female flies . This system, which was the first of its kind in metazoan organisms, is now being used for gene targeting in Drosophila . This review summarizes results that provide new insights into the process of gap repair in Drosophila and outline some recent experiments that demonstrate the power of the gene targeting technique.

Mol Hum Reprod, 1998 Apr, 4(4), 325 - 31
Interstitial and terminal deletion of chromosome Y in a male individual with cryptozoospermia; Duell T et al.; A constitutional de-novo deletion of the long arm of the Y chromosome was detected by standard cytogenetic analysis in a 38-year old male who, except for small testes and cryptozoospermia, was phenotypically normal . The deletion was further characterized by fluorescent in-situ hybridization (FISH) and digital image analysis using contigs of overlapping yeast artificial chromosome (YAC) clones, spanning almost the entire Y chromosome . These results showed that the deletion involved a large interstitial segment on the proximal long arm of the Y chromosome (Yq11.1-->Yq11.22) as well as a more distal portion of the Y chromosome, including the entire heterochromatic region (Yq11.23-->qter) . The breakpoints as determined by the YAC probes were defined within the published Vergnaud intervals so that region 6B and 6C was mostly retained . However, the AZFc region harbouring the DAZ locus on distal subinterval 6F was lost in the deletion, making the absence of this region the most probable location for the patient's infertility . The data underline the usefulness of FISH as an alternative technique to conventional banding for the refined detection of chromosome Y deletions/rearrangements.

Nat Genet, 1998 Jun, 19(2), 199 - 202
Telomere elongation by hnRNP A1 and a derivative that interacts with telomeric repeats and telomerase; LaBranche H et al.; Telomeric DNA of mammalian chromosomes consists of several kilobase-pairs of tandemly repeated sequences with a terminal 3' overhang in single-stranded form . Maintaining the integrity of these repeats is essential for cell survival; telomere attrition is associated with chromosome instability and cell senescence, whereas stabilization of telomere length correlates with the immortalization of somatic cells . Telomere elongation is carried out by telomerase, an RNA-dependent DNA polymerase which adds single-stranded TAGGGT repeats to the 3' ends of chromosomes . While proteins that associate with single-stranded telomeric repeats can influence tract lengths in yeast, equivalent factors have not yet been identified in vertebrates . Here, it is shown that the heterogeneous nuclear ribonucleoprotein A1 participates in telomere biogenesis . A mouse cell line deficient in A1 expression harbours telomeres that are shorter than those of a related cell line expressing normal levels of A1 . Restoring A1 expression in A1-deficient cells increases telomere length . Telomere elongation is also observed upon introduction of exogenous UP1, the amino-terminal fragment of A1 . While both A1 and UP1 bind to vertebrate single-stranded telomeric repeats directly and with specificity in vitro, only UP1 can recover telomerase activity from a cell lysate . These findings establish A1/UP1 as the first single-stranded DNA binding protein involved in mammalian telomere biogenesis and suggest possible mechanisms by which UP1 may modulate telomere length.

Nat Genet, 1998 Jun, 19(2), 179 - 81
Positional cloning of the gene for Nijmegen breakage syndrome; Matsuura S et al.; Nijmegen breakage syndrome (NBS), also known as ataxia-telangiectasia (AT) variant, is an autosomal recessive disorder characterized by microcephaly, growth retardation, severe combined immunodeficiency and a high incidence of lymphoid cancers . Cells from NBS patients display chromosome instability, hypersensitivity to ionizing radiation and abnormal cell-cycle regulation after irradiation, all of which are characteristics shared with AT . Recently, the NBS locus was mapped at 8q21 by two independent approaches, complementation studies and linkage analysis . Here, we report the positional cloning of the NBS gene, NBS1, from an 800-kb candidate region . The gene comprises 50 kb and encodes a protein of 754 amino acids . The amino-terminal region of the protein shows weak homology to the yeast XRS2, MEK1, CDS1 and SPK1 proteins . The gene is expressed at high levels in the testes, suggesting that it might be involved in meiotic recombination . We detected the same 5-bp deletion in 13 individuals, and conclude that it is likely to be a founder mutation.

Immunity, 1998 May, 8(5), 647 - 56
Interactions between HIV1 Nef and vacuolar ATPase facilitate the internalization of CD4; Lu X et al.; CD4 is the primary receptor for the human immunodeficiency virus (HIV) . Nef is an accessory protein of HIV that decreases the expression of CD4 on the surface of infected cells . In this study, we identified the Nef binding protein 1 (NBP1), which interacts specifically with Nef in vitro and in vivo . Since it shares sequence similarity with the catalytic subunit of the vacuolar ATPase (V-ATPase) and complements the loss of this VMA13 gene in yeast, NBP1 is the human homolog of Vma13p . Direct interactions between Nef and NBP1 were correlated with the ability of Nef to internalize CD4 . The expression of the antisense NBP1 abrogated these effects . We conclude that NBP1 helps to connect Nef with the endocytic pathway.

Oncogene, 1998 May 7, 16(18), 2311 - 9
FRA7G extends over a broad region: coincidence of human endogenous retroviral sequences (HERV-H) and small polydispersed circular DNAs (spcDNA) and fragile sites; Huang H et al.; FRA7G is an aphidicolin-inducible common fragile site at human chromosomal band 7q31.2 . This region is frequently altered in a number of different tumor types including prostate, breast, and ovarian cancer . It has also been hypothesized that this region contains an important tumor suppressor gene which is mutated during the development of these cancers or an oncogene which is amplified . We previously used a FISH-based approach to isolate YAC clones which spanned FRA7G . In this report, we describe the isolation and restriction endonuclease mapping of three overlapping P1 clones which cover FRA7G and the region frequently altered in the different cancers . FISH-based analysis of these clones reveals that aphidicolin-induced breakage in the FRA7G region occurs over a region of at least 300 Kb in length . We have also localized a previously sequenced BAC clone to this region . The sequence obtained from this clone reveals the presence of an endogenous retroviral sequence (HERV-H) in the midst of the FRA7G region as well as sequences with homology to small polydispersed circular DNAs (spcDNAs) . Thus for the first two cloned common fragile sites, FRA7G and FRA3B, there is an association with both spcDNAs and hot-spots for viral integration.

Plant Mol Biol, 1998 May, 37(1), 53 - 66
Inositide signalling in Chlamydomonas: characterization of a phosphatidylinositol 3-kinase gene; Molendijk AJ et al.; Phosphoinositide (PI) 3-kinases, which phosphorylate the D-3 position of the inositol ring, function in several different signalling pathways . The phosphatidylinositol (PtdIns)-specific PI 3-kinase of yeast (Vps34p) is part of a receptor signalling protein complex associated with the trans-Golgi membranes, whereas PI 3-kinases that phosphorylate polyphosphoinositides in animal cells form a major receptor-controlled signalling pathway in the plasma membrane . Recent studies have indicated the presence of active PLC, PLD, and PI 3-kinase-dependent signalling systems in the unicellular green alga Chlamydomonas, and PtdIns-3P in Chlamydomonas shows a particularly high rate of turnover . Here we report the cloning of the Chlamydomonas Vps34p, and some characterisation of its properties, regulation and localisation . A single-copy 12 kb gene was present . The corresponding protein of 122 kDa had full-length homology with Vps34ps from other species, but it contained a novel spacer-like insert region of 148 amino acid residues between homology region 2 (HR2) and the C-terminal catalytic core domain, and three other shorter putative inserts . Available cDNAs were used to assemble a pBluescript clone expressing a recombinant protein which had PtdIns-specific 3-kinase activity . However, an unexpected observation was that recombinant proteins containing the complete catalytic core, but lacking HR2, had no lipid kinase activity, pointing to a previously unsuspected role for this domain, possibly in substrate binding . VPS34 mRNA and protein levels, as determined by RNAse protection assays and by immunological methods respectively, were low in all cell stages that were examined . Western blotting of subcellular fractions revealed that most of Vps34p in cell lysates of cw-15 (a cell wall-deficient mutant) could be recovered in a NP-40-resistant 100000 x g pellet, suggesting that the enzyme may have a location different from that found in higher plants.

Genes Cells, 1998 Mar, 3(3), 189 - 202
Characterization of the C . elegans gap-2 gene encoding a novel Ras-GTPase activating protein and its possible role in larval development; Hayashizaki S et al.; BACKGROUND: The Ras signalling pathway plays several important roles in the development of the nematode Caenorhabditis elegans . So far, two types of Ras-GTPase activating proteins (Ras-GAPs) have been identified in this organism . To aid the study of the regulation and function of the Ras pathway, we set out to isolate a new GAP gene from C . elegans by transcomplementation of the fission yeast gap1 mutant . RESULTS: We isolated a C . elegans cDNA that encoded a protein which was similar to, but not exactly homologous with mammalian p120 Ras-GAP . This gene, named gap-2, generated at least nine distinct mRNA species through transcription from different promoters and subsequent alternative splicing involving 25 exons . These isoforms were differentially expressed among tissues . A deletion of gap-2 caused no obvious phenotype by itself, but a loss of gap-2 function could suppress larval lethality in both let-23 and let-60 reduction-of-function mutants, in which the Ras activity was lowered . CONCLUSIONS: C . elegans gap-2 encodes a novel Ras-GAP, which is similar to vertebrate p120 but which may constitute a new GAP subfamily . gap-2 mRNA isoforms arise by an unusually extensive variation in initiation sites and associated alternative splicing, and each isoform may play a distinct role in specific tissues . GAP-2 appears to function as a negative regulator of LET-60 Ras during larval development.

Genes Cells, 1998 Mar, 3(3), 135 - 44
Double-strand break repair mediated by DNA end-joining; Tsukamoto Y et al.; DNA double-strand breaks formed by ionizing irradiation or other stresses are repaired by homologous recombination or DNA end-joining . This review focuses on the mechanism of double-strand break repair mediated by DNA end-joining, in which many factors have recently been identified . After DNA double-strand breakage, DNA end-joining takes place between the DNA ends that have nonhomologous sequences or very short regions ofhomology . The broken DNA is repaired if the DNA end-joining occurs in the same molecule, while it causes chromosome aberrations such as deletions, insertions, translocations and inversions if it occurs between different molecules . Rad50 and its relatives, Ku-proteins, DNA ligase VI and silencing factors, are involved in DNA end-joining in yeast and mammalian cells . These findings led us to propose a model in which the formation of a heterochromatin-like complex at broken ends is an important element in DNA end-joining.

Semin Immunol, 1998 Apr, 10(2), 127 - 32
HMG box containing transcription factors in lymphocyte differentiation; Schilham MW et al.; The identification of the mammalian sex-determining gene Sry has led to the discovery of a large family of related ('HMG box') transcription factors that control developmental events in yeast, C . elegans, Drosophila and vertebrates . In lymphocyte differentiation, several HMG box proteins play a decisive role . Sox-4 is important for very early B-cell differentiation, while TCF-1/LEF-1 play a crucial role in early thymocyte development . TCF/LEF proteins have recently been found to constitute a downstream component of the Wingless/Wnt signal transduction pathway . In flies, this pathway controls segment polarity; in Xenopus it controls the definition of the body axis . Deregulation of the pathway occurs in several human tumors . These insights in the molecular events that are involved in TCF/LEF function in these organisms may eventually lead to the understanding of the function of these HMG box proteins in lymphoid development.

Microbiol Mol Biol Rev, 1998 Jun, 62(2), 465 - 503
Molecular genetics of the RNA polymerase II general transcriptional machinery; Hampsey M; Transcription initiation by RNA polymerase II (RNA pol II) requires interaction between cis-acting promoter elements and trans-acting factors . The eukaryotic promoter consists of core elements, which include the TATA box and other DNA sequences that define transcription start sites, and regulatory elements, which either enhance or repress transcription in a gene-specific manner . The core promoter is the site for assembly of the transcription preinitiation complex, which includes RNA pol II and the general transcription fctors TBP, TFIIB, TFIIE, TFIIF, and TFIIH . Regulatory elements bind gene-specific factors, which affect the rate of transcription by interacting, either directly or indirectly, with components of the general transcriptional machinery . A third class of transcription factors, termed coactivators, is not required for basal transcription in vitro but often mediates activation by a broad spectrum of activators . Accordingly, coactivators are neither gene-specific nor general transcription factors, although gene-specific coactivators have been described in metazoan systems . Transcriptional repressors include both gene-specific and general factors . Similar to coactivators, general transcriptional repressors affect the expression of a broad spectrum of genes yet do not repress all genes . General repressors either act through the core transcriptional machinery or are histone related and presumably affect chromatin function . This review focuses on the global effectors of RNA polymerase II transcription in yeast, including the general transcription factors, the coactivators, and the general repressors . Emphasis is placed on the role that yeast genetics has played in identifying these factors and their associated functions.

EMBO J, 1998 May 15, 17(10), 2961 - 9
Cell cycle modulation of protein-DNA interactions at a human replication origin; Abdurashidova G et al.; We followed the variations of protein-DNA interactions occurring in vivo over the early firing replication origin located near the human lamin B2 gene, in IMR-90 cells synchronized in different moments of the cell cycle . In G0 phase cells no protection is present; as the cells progress in G1 phase an extended footprint covering over 100 bp appears, particularly marked at the G1/S border . As the cells enter S phase the protection shrinks to 70 bp and remains unchanged throughout this phase . In mitosis the protection totally disappears, only to reappear in its extended form as the cells move into the next G1 . These variations are reminiscent of those corresponding to the formation of the pre- and post-replicative complexes described in yeast and Xenopus cells.

EMBO J, 1998 May 15, 17(10), 2736 - 47
p53-inducible human homologue of Drosophila seven in absentia (Siah) inhibits cell growth: suppression by BAG-1; Matsuzawa S et al.; The Drosophila seven in absentia (sina) gene is required for R7 photoreceptor cell formation during Drosophila eye development, where it functions within the Ras/Raf pathway and targets other proteins for degradation via associations with a ubiquitin-conjugating enzyme . Recently, a mammalian sina homologue was reported to be a p53-inducible gene in a myeloid leukemia cell line . To explore the function of human SINA-homologous (Siah) proteins, expression plasmids encoding Siah-1A were transiently transfected into 293 epithelial cells and GM701 fibroblast cells, resulting in growth arrest without induction of apoptosis . We discovered that BAG-1, a ubiquitin-like Hsp70/Hsc70-regulating protein, is a negative regulator of Siah-1A . Siah-1A was identified as a BAG-1-binding protein via yeast two-hybrid methods . Specific interaction of BAG-1 with Siah-1A was also demonstrated by in vitro binding experiments using glutathione S-transferase fusion proteins and co-immunoprecipitation studies . Siah-1A-induced growth arrest in 293 and GM701 cells was abolished by co-transfection of wild-type BAG-1 with Siah-1A but not by a C-terminal deletion mutant of BAG-1 that fails to bind Siah-1A . Over-expression of BAG-1 significantly inhibited p53-induced growth arrest in 293 cells without preventing p53 transactivation of reporter gene plasmids . BAG-1 also prevented growth arrest following UV-irradiation-induced genotoxic injury without interfering with accumulation of p53 protein or p21(waf-1) expression . BAG-1 functions downstream of p53-induced gene expression to inhibit p53-mediated suppression of cell growth, presumably by suppressing the actions of Siah-1A . We suggest that Siah-1A may be an important mediator of p53-dependent cell-cycle arrest and demonstrate that Siah-1A is directly inhibited by BAG-1.

J Steroid Biochem Mol Biol, 1998 Mar, 64(5-6), 239 - 44
Selenium regulates gene expression for estrogen sulfotransferase and alpha 2U-globulin in rat liver; Yang Q et al.; Dietary intake of the essential trace element selenium (Se) regulates expression of genes for selenoproteins and certain non-Se-containing proteins . However, these proteins do not account for all of Se's biological effects . The objective of this work was to identify additional genes whose expression is regulated by Se . Identification of these genes may reveal new functions for Se or define mechanisms for its biological effects . Weanling male Sprague-Dawley rats were fed a Torula yeast-based Se-deficient basal diet or the same diet supplemented with 0.5 mg Se/kg diet as sodium selenite for 13 weeks . Total RNA was used as template for RNA fingerprinting . Two differentially expressed cDNA fragments were identified and cloned . The first had 99% nucleotide identity with rat liver estrogen sulfotransferase (EST) isoform-6 . The second had 99% nucleotide sequence identity with rat liver alpha 2u-globulin . The mRNA levels for both were markedly reduced in Se deficiency . Laser densitometry showed that EST mRNA in Se deficiency was 7.3% of that in Se-adequate rat liver . The level of alpha 2u-globulin mRNA in Se-deficient rat liver was only 12.6% of that in Se-adequate rat liver . These results indicate that dietary Se may play a role in steroid hormone metabolism in rat liver.

Biochimie, 1998 Mar, 80(3), 235 - 46
Bis(2,6-dioxopiperazines), catalytic inhibitors of DNA topoisomerase II, as molecular probes, cardioprotectors and antitumor drugs; Andoh T; Bis(2,6-dioxopiperazines) and other catalytic inhibitors of mammalian DNA topoisomerase II have recently been found in natural and synthetic compounds . These compounds target the enzyme within the cell and inhibit various genetic processes involving the enzyme such as DNA replication and chromosome dynamics and thus proved to be good probes for the functional analyses of the enzyme in a variety of eucaryotes from yeast to mammals . Catalytic inhibitors were shown to be antagonists against topoisomerase II poisons under some conditions, but to be synergistic under others . Bis(2,6-dioxopiperazines) have a potential to overcome cardiac toxicity caused by potent antitumor anthracycline antibiotics such as doxorubicin and daunorubicin . ICRF-187, +enantiomer of racemic ICRF-159, has been used in EU countries as cardioprotector in cancer clinics . Furthermore, bis(2,6-dioxopiperazines) enhance the efficacy of antitumor topoisomerase II poisons, e.g . anthracycline antibiotics such as daunorubicin and doxorubicin, by reducing their side effects and by allowing dose escalation of the antitumor drugs in preclinical and clinical settings . Besides bis(2,6-dioxopiperazines) per se having antitumor activity, and one of their derivatives, MST-16 or sobuzoxane, bis(N1-isobutyloxycarbonyloxymethyl-2,6-dioxopiperazine), has been developed in Japan and used in clinics as anticancer drug for malignant lymphomas and adult T-cell leukemia (ATL) . Further developments of bis(2,6-dioxopiperazines) as antimetastatic agents are expected.

Genomics, 1998 May 1, 49(3), 371 - 7
Physical and genetic maps of the deafwaddler region on distal mouse Chr 6; McKee-Johnson JW et al.; The deafwaddler (dfw) mutation, displaying motor ataxia and profound deafness, arose spontaneously in a C3H/HeJ colony and was mapped previously to distal mouse Chr 6 . In this study, a high-resolution genetic map was generated by positioning 10 microsatellite markers and 5 known genes on a 968-meioses intersubspecific backcross segregating for dfw {(CAST/Ei(-)+/+ x C3HeB/ FeJ-dfw/dfw) x C3HeB/FeJ-dfw/dfw}, giving the following marker order and sex-averaged distances: D6Mit64-(0.10 + 0.10 cM)-Pang-(1.24 + 0.36 cM)-Itpr1-(0.62 + 0.25 cM)-D6Mit108-(0.52 + 0.23 cM)-D6Mit54-(0.21 + 0.15 cM)-D6Mit23, D6Mit107, D6Mit328-(0.72 + 0.27 cM)-D6Mit11-(0.21 + 0.15 cM)-dfw-(0.93 + 0.31 cM)-Gat4, D6Mit55-(0.10 + 0.10 cM)-D6Mit63-(0.31 + 0.18 cM)-Syn2-(0.62 + 0.25 cM)-D6Mit44 (Rho) . Female and male genetic maps are similar immediately surrounding the dfw locus, but show marked differences in other areas . A yeast artificial chromosome-based physical map suggests that the closest markers flanking the dfw locus, D6Mit11 (proximal) and Gat4, D6Mit55 (distal), are contained within 650-950 kb . The human homologues of the flanking loci Itpr1 (proximal) and Syn2 (distal) map to chromosome 3p25-p26, suggesting that the human homologue of the dfw gene is located within this same region.

Genomics, 1998 May 1, 49(3), 351 - 62
A detailed physical and transcriptional map of the region of chromosome 20 that is deleted in myeloproliferative disorders and refinement of the common deleted region; Bench AJ et al.; Acquired deletions of the long arm of chromosome 20 are the most common chromosomal abnormality seen in polycythemia vera and are also associated with other myeloid malignancies . Such deletions are believed to mark the site of one or more tumor suppressor genes, loss of which perturbs normal hematopoiesis . A common deleted region (CDR) has previously been identified on 20q . We have now constructed the most detailed physical map of this region to date--a YAC contig that encompasses the entire CDR and spans 23 cM (11 Mb) . This contig contains 140 DNA markers and 65 unique expressed sequences . Our data represent a first step toward a complete transcriptional map of the CDR . The high marker density within the physical map permitted two complementary approaches to reducing the size of the CDR . Microsatellite PCR refined the centromeric boundary of the CDR to D20S465 and was used to search for homozygous deletions in 28 patients using 32 markers . No such deletions were detected . Genetic changes on the remaining chromosome 20 may therefore be too small to be detected or may occur in a subpopulation of cells.

Virology, 1998 May 25, 245(1), 120 - 7
The adeno-associated virus Rep78 major regulatory protein binds the cellular TATA-binding protein in vitro and in vivo; Hermonat PL et al.; Rep78 is the major regulatory protein of adenoassociated virus (AAV) . Rep78 is able to transcriptionally regulate all three of AAV's promoters, as well as a variety of heterologous promoters . In an attempt to understand the mechanism of action by which Rep78 is able to regulate gene expression, we are investigating Rep78's possible protein-protein interaction with basal transcription factors . One such critical basal transcription factor is the human TATA binding protein, TBP . TBP is a core factor required for the assemblage of the transcription initiation complex, TFIID . In this report an in vitro interaction between Rep78 and TBP was demonstrated in three different assay systems, including West(far)-Western analysis, electrophoretic mobility shift assay-supershift, and coimmunoprecipitation . Furthermore, using the yeast GAL4 two-hybrid system, an in vivo interaction between Rep78 and TBP was also demonstrated . Further still, the amino half of Rep78 is shown to be needed for Rep78-TBP interaction . Mutations within this region of Rep78 are known to be defective for transcriptional regulatory ability, suggesting a biological role for this interaction . Thus, Rep78 may regulate transcription through binding and regulating TBP's numerous interactions . Furthermore, as Rep78 is known to bind at least one other transcription factor (Sp 1) and likely others, Rep78 may function as a TBP-associated factor in an altered TFIID-like complex.

Science, 1998 May 15, 280(5366), 1091 - 4
COI1: an Arabidopsis gene required for jasmonate-regulated defense and fertility; Xie DX et al.; The coi1 mutation defines an Arabidopsis gene required for response to jasmonates, which regulate defense against insects and pathogens, wound healing, and pollen fertility . The wild-type allele, COI1, was mapped to a 90-kilobase genomic fragment and located by complementation of coi1-1 mutants . The predicted amino acid sequence of the COI1 protein contains 16 leucine-rich repeats and an F-box motif . It has similarity to the F-box proteins Arabidopsis TIR1, human Skp2, and yeast Grr1, which appear to function by targeting repressor proteins for removal by ubiquitination.

Proc Natl Acad Sci U S A, 1998 Apr 28, 95(9), 5027 - 32
A cytoplasmic protein, bystin, interacts with trophinin, tastin, and cytokeratin and may be involved in trophinin-mediated cell adhesion between trophoblast and endometrial epithelial cells; Suzuki N et al.; Trophinin and tastin form a cell adhesion molecule complex that potentially mediates an initial attachment of the blastocyst to uterine epithelial cells at the time of implantation . Trophinin and tastin, however, do not directly bind to each other, suggesting the presence of an intermediary protein . The present study identifies a cytoplasmic protein, named bystin, that directly binds trophinin and tastin . Bystin consists of 306 amino acid residues and is predicted to contain tyrosine, serine, and threonine residues in contexts conforming to motifs for phosphorylation by protein kinases . Database searches revealed a 53% identity of the predicted peptide sequence with the Drosophila bys (mrr) gene . Direct protein-protein interactions of trophinin, tastin, and bystin analyzed by yeast two-hybrid assays and by in vitro protein binding assays indicated that binding between bystin and trophinin and between bystin and tastin is enhanced when cytokeratin 8 and 18 are present as the third molecule . Immunocytochemistry of bystin showed that bystin colocalizes with trophinin, tastin, and cytokeratins in a human trophoblastic teratocarcinoma cell, HT-H . It is therefore possible that these molecules form a complex and thus are involved in the process of embryo implantation.

Proc Natl Acad Sci U S A, 1998 Apr 28, 95(9), 5021 - 6
A distinct cyclin-dependent kinase-activating kinase of Arabidopsis thaliana; Umeda M et al.; The activation of cyclin-dependent kinases (CDKs) requires phosphorylation of a threonine residue within the T-loop catalyzed by CDK-activating kinases (CAKs) . Thus far no functional CAK homologue has been reported in plants . We screened an Arabidopsis cDNA expression library for complementation of a budding yeast CAK mutant . A cDNA, cak1At, was isolated that suppressed the CAK mutation in budding yeast, and it also complemented a fission yeast CAK mutant . cak1At encodes a protein related to animal CAKs . The CAK similarity was restricted to the conserved kinase domains, leading to classification of Cak1At as a distinct CDK in the phylogenetic tree . Immunoprecipitates with the anti-Cak1At antibody phosphorylated human CDK2 at the threonine residue (T160) within the T-loop and activated its activity to phosphorylate histone H1 . Whereas CAKs in animals and fission yeast are involved in regulation of the cell cycle and basal transcription by phosphorylating the carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II, Cak1At did not phosphorylate the CTD . An Arabidopsis CTD-kinase isolated separately from Cak1At was shown to interact with the yeast protein p13(suc1), but it had no CDK2-kinase activity . Therefore, the CTD of RNA polymerase II is probably phosphorylated by a Cdc2-related kinase distinct from Cak1At . cak1At is a single-copy gene in Arabidopsis and is highly expressed in proliferating cells of suspension cultures.

J Biol Chem, 1998 May 1, 273(18), 11392 - 9
Golgi localization and functional expression of human uridine diphosphatase; Wang TF et al.; A full-length E(ecto)-ATPase (Plesner, L . (1995) Int . Rev . Cytol . 158, 141-214) cDNA was cloned from a human brain cDNA library; it encodes a 610-amino acid protein that contains two putative transmembrane domains . Heterologous expression of this protein in COS-7 cells caused a significant increase in intracellular membrane-bound nucleoside phosphatase activity . The activity was highest with UDP as substrate and was stimulated by divalent cations in the following order: Ca2+ >> Mg2+ > Mn2+ . The results of immunofluorescence staining indicate that this protein is located in the Golgi apparatus . UDP hydrolysis was increased in the presence of Triton X-100 or alamethicin, an ionophore that facilitates movement of UDP across the membrane, suggesting that the active site of this UDPase is on the luminal side of the Golgi apparatus . This is the first identification of a mammalian Golgi luminal UDPase gene . Computer-aided sequence analysis of the EATPase superfamily indicates that the human UDPase is highly similar to two hypothetical proteins of the nematode Caenorhabditis elegans and to an unidentified 71.9-kDa yeast protein and is less related to the previously identified yeast Golgi GDPase.

J Biol Chem, 1998 May 1, 273(18), 11274 - 80
Mutations in the double-stranded RNA-activated protein kinase insert region that uncouple catalysis from eIF2alpha binding; Cai R et al.; The interferon-induced, double-stranded RNA (dsRNA)-activated protein kinase, PKR, inhibits protein synthesis via phosphorylation of the alpha subunit of the translation initiation factor eIF2 . A kinase insert region N-terminal of PKR kinase subdomain V, which is conserved among eIF2alpha kinases, has been proposed to determine substrate specificity of these kinases . To investigate the function of this kinase insert region, selective PKR mutants were generated, and kinase activities and eIF2alpha affinities were analyzed in vitro . The in vivo function was investigated by growth inhibitory assays in yeast and translational assays in COS cells . Among the 13 mutations, 5 lost kinase activity and 3 exhibited less than 30% of wild-type eIF2alpha binding activity . The deletion of the conserved sequence (amino acids 362-370) resulted in a protein that had no kinase activity and only about 25% of wild-type eIF2alpha binding, suggesting that this sequence is not only required for PKR kinase activity but also is important for substrate interaction . It was determined that the hydrophobicity of the conserved sequence of PKR is required for kinase activity but is not crucial for eIF2alpha binding . The amino acid residue Glu-367 in the conserved motif was shown to be directly involved in substrate binding but was not important for kinase activity . These results suggest that the activation of PKR is not a prerequisite for its binding to the substrate and that the conserved motif in subdomain V contributes to the interaction of PKR and eIF2alpha.

J Biol Chem, 1998 May 1, 273(18), 11075 - 81
Regions of association between the alpha and the beta subunit of the gastric H,K-ATPase; Melle-Milovanovic D et al.; A binding and a yeast two-hybrid analysis were carried out on the gastric H,K-ATPase to determine interactive regions of the extracytoplasmic domains of the alpha and beta subunits of this P type ATPase . Wheat germ agglutinin fractionation of fluorescein 5-maleimide-labeled tryptic fragments of detergent-solubilized H, K-ATPase showed that a fragment Leu855 to Arg922 of the alpha subunit was bound to the beta subunit . The yeast two-hybrid system showed that the region containing only a part of the seventh transmembrane segment, the loop, and part of the eighth transmembrane segment was capable of giving positive interaction signals with the ectodomain of the beta subunit . The sequence in the extracytoplasmic loop close to the eighth transmembrane segment, namely Arg898 to Thr928, was identified as being the site of interaction using this method . We deduced that the sequence Arg898 to Arg922 in the alpha subunit has strong interaction with the extracytoplasmic domain of the beta subunit . Again, using yeast two-hybrid analysis, two different sequences in the beta subunit Gln64 to Asn130 and Ala156 to Arg188 were identified as association domains in the extracytoplasmic sequence of the beta subunit . These data enable identification of major associative regions of the alpha-beta subunits of the H,K-ATPase.

J Biol Chem, 1998 May 1, 273(18), 10939 - 47
Identification and characterization of a novel 9.2-kDa membrane sector-associated protein of vacuolar proton-ATPase from chromaffin granules; Ludwig J et al.; Vacuolar proton-translocating ATPase (holoATPase and free membrane sector) was isolated from bovine chromaffin granules by blue native polyacrylamide gel electrophoresis . A 5-fold excess of membrane sector over holoenzyme was determined in isolated chromaffin granule membranes . M9.2, a novel extremely hydrophobic 9.2-kDa protein comprising 80 amino acids, was detected in the membrane sector . It shows sequence and structural similarity to Vma21p, a yeast protein required for assembly of vacuolar ATPase . A second membrane sector-associated protein (M8-9) was identified and characterized by amino-terminal protein sequencing.

J Biol Chem, 1998 May 1, 273(18), 10831 - 4
Steroid receptor coactivator-1 interacts with the p50 subunit and coactivates nuclear factor kappaB-mediated transactivations; Na SY et al.; Steroid receptor coactivator-1 (SRC-1) specifically bound to the transcription factor NFkappaB subunit p50 but not to p65 as demonstrated by the yeast two hybrid tests and glutathione S-transferase pull down assays . The p50-binding site was localized to a subregion of SRC-1 (amino acids 759-1141) that encompasses the previously described CBP-p300-binding domain . In mammalian cells, SRC-1 potentiated the NFkappaB-mediated transactivations in a dose-dependent manner . Coexpression of p300 further enhanced this SRC-1-potentiated level of transactivations, consistent with the recent findings in which CBP and p300 were shown to be transcription coactivators of the p65 subunit (Perkins, N . D., Felzien, L . K., Betts, J . C., Leung, K., Beach, D . H., and Nabel, G . J . (1997) Science 275, 523-527; Gerritsen, M . E., Williams, A . J., Neish, A . S . , Moore, S., Shi, Y., and Collins, T . (1997) Proc . Acad . Natl . Sci . U . S . A . 94, 2927-2932) . These results suggest that at least two distinct coactivator molecules may cooperate to regulate the NFkappaB-dependent transactivations in vivo and SRC-1, originally identified as a coactivator for the nuclear receptors, may constitute a more widely used coactivation complex.

J Mol Evol, 1998 May, 46(5), 571 - 88
Phylogenetic and functional classification of mitogen- and stress-activated protein kinases; Kultz D; All currently sequenced stress-activated protein kinases (SAPKs), extracellular signal-regulated kinases (ERKs), and other mitogen-activated protein kinases (MAPKs) were analyzed by sequence alignment, phylogenetic tree construction, and three-dimensional structure modeling in order to classify members of the MAPK family . Based on this analysis the MAPK family was divided into three subgroups (SAPKs, ERKs, and MAPK3) that consist of at least nine subfamilies . Members of a given subfamily were exclusively from animals, plants, or yeast/fungi . A single signature sequence, {LIVM}{TS}XX{LIVM}XT{RK}{WY}YRXPX{LIVM} {LIVM}, was identified that is characteristic for all MAPKs and sufficient to distinguish MAPKs from other members of the protein kinase superfamily . This signature sequence contains the phosphorylation site and is located on loop 12 of the three-dimensional structure of MAPKs . I also identified signature sequences that are characteristic for each of the nine subfamilies of MAPKs . By modeling the three-dimensional structure of three proteins for each MAPK subfamily based on the resolved atomic structures of rat ERK2 and murine p38, it is demonstrated that amino acids conserved in all MAPKs are located primarily in the center of the protein around the catalytic cleft . I conclude that these residues are important for maintaining proper folding into the gross structure common to all MAPKs . On the other hand, amino acids conserved in a given subfamily are located mainly in the periphery of MAPKs, indicating their possible importance for defining interactions with substrates, activators, and inhibitors . Within these subfamily-specific regions, amino acids were identified that represent unique residues occurring in only a single subfamily and their location was mapped in three-dimensional structure models . These unique residues are likely to be crucial for subfamily-specific interactions of MAPKs with substrates, inhibitors, or activators and, therefore, represent excellent targets for site-directed mutagenesis experiments.

EMBO J, 1998 Apr 1, 17(7), 2067 - 78
Drosophila CtBP: a Hairy-interacting protein required for embryonic segmentation and hairy-mediated transcriptional repression; Poortinga G et al.; hairy is a Drosophila pair-rule segmentation gene that functions genetically as a repressor . To isolate protein components of Hairy-mediated repression, we used a yeast interaction screen and identified a Hairy-interacting protein, the Drosophila homolog of the human C-terminal-binding protein (CtBP) . Human CtBP is a cellular phosphoprotein that interacts with the C-terminus of the adenovirus E1a oncoprotein and functions as a tumor suppressor . dCtBP also interacts with E1a in a directed yeast two-hybrid assay . We show that dCtBP interacts specifically and directly with a small, previously uncharacterized C-terminal region of Hairy . dCtBP activity appears to be specific to Hairy of the Hairy/Enhancer of split {E(spl)}/Dpn basic helix-loop-helix protein class . We identified a P-element insertion within the dCtBP transcription unit that fails to complement alleles of a known locus, l(3)87De . We demonstrate that dCtBP is essential for proper embryonic segmentation by analyzing embryos lacking maternal dCtBP activity . While Hairy is probably not the only segmentation gene interacting with dCtBP, we show dose-sensitive genetic interactions between dCtBP and hairy mutations.

EMBO J, 1998 Apr 1, 17(7), 2042 - 54
FacB, the Aspergillus nidulans activator of acetate utilization genes, binds dissimilar DNA sequences; Todd RB et al.; The facB gene is required for acetate induction of acetamidase (amdS) and the acetate utilization enzymes acetyl-CoA synthase (facA), isocitrate lyase (acuD) and malate synthase (acuE) in Aspergillus nidulans . The facB gene encodes a transcriptional activator with a GAL4-type Zn(II)2Cys6 zinc binuclear cluster DNA-binding domain which is shown to be required for DNA binding . In vitro DNA-binding sites for FacB in the 5' regions of the amdS, facA, acuD and acuE genes have been identified . Mutations in amdS FacB DNA-binding sites affected expression of an amdS-lacZ reporter in vivo and altered the affinity of in vitro DNA binding . This study shows that the FacB Zn(II)2Cys6 cluster binds to dissimilar sites which show similarity in form but not sequence with DNA-binding sites of other Zn(II)2Cys6 proteins . Sequences with homology to FacB sites are found in the 5' regions of genes regulated by the closely related yeast Zn(II)2Cys6 protein CAT8.

EMBO J, 1998 Apr 1, 17(7), 2033 - 41
Efficient synthesis, termination and release of RNA polymerase III transcripts in Xenopus extracts depleted of La protein; Lin-Marq N et al.; La proteins are conserved, abundant and predominantly nuclear phosphoproteins which bind to the 3'-U termini of newly synthesized RNA polymerase III transcripts . The human La protein has been implicated in the synthesis, termination and release of such transcripts . Here we examine the potential transcriptional properties of La in Xenopus laevis, using a homologous tRNA gene as template . Immunodepletion of La from cell-free extracts leads to the formation of tRNA precursors lacking 3'-U residues . This shortening can be uncoupled from RNA polymerase III transcription, indicating that it results from nuclease degradation rather than incomplete synthesis . Extracts containing <1% of the normal La protein content synthesize tRNA precursors just as well as complete extracts, with no change in termination efficiency, and the vast majority of these full-length transcripts are not associated with the template or with residual La protein . Hence, Xenopus La seems not to function as an initiation, termination or release factor for RNA polymerase III . Consistent with the recently discovered role of La in yeast tRNA maturation in vivo, recombinant Xenopus La prevents 3'-exonucleolytic degradation of tRNA precursors in vitro . A conserved RNA chaperone function may best explain the abundance of La in eukaryotic nuclei.

EMBO J, 1998 Apr 1, 17(7), 1930 - 40
Two distinct effectors of the small GTPase Rab5 cooperate in endocytic membrane fusion; Gournier H et al.; Using the yeast two-hybrid system, we have identified a novel 62 kDa coiled-coil protein that specifically interacts with the GTP-bound form of Rab5, a small GTPase that regulates membrane traffic in the early endocytic pathway . This protein shares 42% sequence identity with Rabaptin-5, a previously identified effector of Rab5, and we therefore named it Rabaptin-5beta . Like Rabaptin-5, Rabaptin-5beta displays heptad repeats characteristic of coiled-coil proteins and is recruited on the endosomal membrane by Rab5 in a GTP-dependent manner . However, Rabaptin-5beta has features that distinguish it from Rabaptin-5 . The relative expression levels of the two proteins varies in different cell types . Rabaptin-5beta does not heterodimerize with Rabaptin-5, and forms a distinct complex with Rabex-5, the GDP/GTP exchange factor for Rab5 . Immunodepletion of the Rabaptin-5beta complex from cytosol only partially inhibits early endosome fusion in vitro, whereas the additional depletion of the Rabaptin-5 complex has a stronger inhibitory effect . Fusion activity can mostly be recovered by addition of the Rabaptin-5 complex alone, but maximal fusion efficiency requires the presence of both Rabaptin-5 and Rabaptin-5beta complexes . Our results suggest that Rab5 binds to at least two distinct effectors which cooperate for optimal endocytic membrane docking and fusion.

Nucleic Acids Res, 1998 Apr 15, 26(8), 1965 - 73
Molecular and biochemical characterisation of DNA-dependent protein kinase-defective rodent mutant irs-20; Priestley A et al.; The catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) is a member of a sub-family of phosphatidylinositol (PI) 3-kinases termed PIK-related kinases . A distinguishing feature of this sub-family is the presence of a conserved C-terminal region downstream of a PI 3-kinase domain . Mutants defective in DNA-PKcs are sensitive to ionising radiation and are unable to carry out V(D)J recombination . Irs-20 is a DNA-PKcs-defective cell line with milder gamma-ray sensitivity than two previously characterised mutants, V-3 and mouse scid cells . Here we show that the DNA-PKcs protein from irs-20 cells can bind to DNA but is unable to function as a protein kinase . To verify the defect in irs-20 cells and provide insight into the function and expression of DNA-PKcs in double-strand break repair and V(D)J recombination we introduced YACs encoding human and mouse DNA-PKcs into defective mutants and achieved complementation of the defective phenotypes . Furthermore, in irs-20 we identified a mutation in DNA-PKcs that causes substitution of a lysine for a glutamic acid in the fourth residue from the C-terminus . This represents a strong candidate for the inactivating mutation and provides supportive evidence that the extreme C-terminal motif is important for protein kinase activity.

Nippon Rinsho, 1998 May, 56(5), 1102 - 7
{Structure and regulation mechanisms of telomerase}; Nakayama J et al.; Telomerase is a specialized type of reverse transcriptase which catalyzes the synthesis of telomeric DNA using intrinsic RNA as a template . The enzyme was originally found in a ciliate Tetrahymena, and has been extensively investigated using ciliates or budding yeast . In mammals, the enzyme is highly active in most cancer cells and germ cells, but is inactive in most somatic cells, suggesting the activation of telomerase may be important for the continued cell growth or progression of cancer cells . Recently, two protein components of the mammalian telomerase have been identified using homology to the sequencing data from unicellular eukaryotes . Interestingly, telomerase activity was induced by the expression of catalytic subunit, hTERT, in telomerase-negative normal fibroblast cells, indicating that it plays a key role in the activation of telomerase in cancer cells.

Trends Biochem Sci, 1998 May, 23(5), 174 - 8
Role of the CCAAT-binding protein CBF/NF-Y in transcription; Maity SN et al.; The CCAAT motif is one of the common promoter elements present in the proximal promoter of numerous mammalian genes transcribed by RNA polymerase II . CBF (also called NF-Y and CP1) consists of three different subunits and interacts specifically with the CCAAT motif . In each CBF subunit, the segment needed for formation of the CBF-DNA complex is conserved from yeast to human and, interestingly, the conserved segment of two CBF subunits, CBF-A and CBF-C, are homologous to the histone-fold motif of eukaryotic histones and archaebacterial histone-like protein HMf-2 . The histone fold motifs of CBF-A and CBF-C interact with each other to form a heterodimer that associates with CBF-B to form a heterotrimeric CBF molecule, which then binds to DNA.

Phytochemistry, 1998 Apr, 47(7), 1401 - 7
A new class of wine pigments generated by reaction between pyruvic acid and grape anthocyanins; Fulcrand H et al.; A new class of stable red pigments detected in grape pomace was analysed by electrospray ionisation mass spectrometry . They were shown to be pyruvic acid derivatives of genuine grape anthocyanins by synthesis experiments . The major product was identified by NMR (1H, NOE, HSQC, HMBC) experiments as the malvidin-3-monoglucoside pyruvic acid adduct . Its formation results from cyclisation between C-4 and the hydroxyl group at C-5 of the original flavylium moiety with the double bond of the enolic form of pyruvic acid, followed by dehydration and rearomatisation steps . This type of reaction leads to increased colour stability . Various yeast metabolites other than pyruvic acid were shown to react with grape anthocyanins following this mechanism, suggesting that it may be an important route of conversion into stable pigments during the maturation and ageing of wine.

Gene, 1998 Jun 8, 212(2), 315 - 22
Recombination trapping: an in-vivo approach to recover cDNAs encoded in YACs; Mazzarella R et al.; We have developed an approach to identify and localize cDNAs encoded by YACs . In this scheme, a YAC truncation vector containing a cDNA library is used to interrupt the YAC by homologous recombination in yeast . This approach generates YACs truncated at the site of recombination between the cDNA and the cognate YAC sequence and thus localizes the gene in the YAC . This method results in the production of a large percentage of true recombinants identifying gene encoding regions of the genome . This approach is shown to identify an unique EST sequence from a YAC in Xp22, the recently described transketolase-like gene in a YAC from Xq28 and a putative kinesin-like gene in Xq13 . This system should also be useful in the mapping of YACs by targeted integration . We have constructed a new telomere truncation vector, pGR8, which incorporates two selectable markers, HIS5 and LYS2 . This vector overcomes problems of previous vectors including: incompatibility with most YAC libraries, vector homology with the YAC arms and high backgrounds resulting from the use of a single selectible marker . A third counterselection with 5-fluoroorotic acid (5FOA) against yeast clones retaining the URA3 gene was also employed to reduce background further . Therefore, this vector and approach should be useful to the transcriptional analysis of YAC maps of any genome.

J Biol Chem, 1998 May 29, 273(22), 13892 - 7
Physical interaction of ApoE with amyloid precursor protein independent of the amyloid Abeta region in vitro; Hass S et al.; Variation at the APOE gene locus has been shown to affect the risk for Alzheimer's disease . To gain deeper insight into the postulated apoE-mediated amyloid formation, we have characterized the three common apoE isoforms (apoE2, apoE3, and apoE4) regarding their binding to amyloid precursor protein (APP) . We employed the yeast two-hybrid system and co-immunoprecipitation experiments in cell culture supernatants of COS-1 cells, ectopically expressing apoE isoforms and APP751 holoprotein or a COOH-terminal Abeta deletion mutant protein, designated APPtrunc . We found that all three apoE isoforms were able to bind APP751 holoprotein in an Abeta-independent fashion . The interacting domains could be mapped to the NH2 termini of APP (amino acids 1-207) and apoE (amino acids 1-191) . As a functional consequence of this novel APP751 ectodomain-mediated apoE binding, the secretion of soluble APP751 is differentially affected by distinct apoE isoforms in vitro, suggesting a new "chaperon-like" mechanism by which apoE isoforms may modulate APP metabolism and consequently the risk for Alzheimer's disease.

J Bone Miner Res, 1998 May, 13(5), 803 - 12
Inorganic polyphosphate in human osteoblast-like cells; Leyhausen G et al.; Significant amounts of inorganic polyphosphates and of polyphosphate-degrading exopolyphosphatase activity were detected in human mandibular-derived osteoblast-like cells . The amount of both soluble and insoluble long-chain polyphosphate in unstimulated osteoblast-like cells was higher than in human gingival cells, erythrocytes, peripheral blood mononuclear cells, and human blood plasma . The cellular content of polyphosphate in osteoblast-like cells strongly decreased after a combined treatment of the cells with the stimulators of osteoblast proliferation and differentiation, dexamethasone, beta-glycerophosphate, epidermal growth factor, and ascorbic acid . The amount of soluble long-chain polyphosphate, but not the amount of insoluble long-chain polyphosphate, further decreased after an additional treatment with 1alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3) . The decrease in polyphosphate content during treatment with dexamethasone, beta-glycerophosphate, epidermal growth factor, and ascorbic acid was accompanied by a decrease in exopolyphosphatase, pyrophosphatase, and alkaline phosphatase activity . However, additional treatment with 1,25(OH)2D3 resulted in an increase in these enzyme activities . Osteoblast-like cell exopolyphosphatase activity and exopolyphosphatase activity in yeast, rat tissues, and human leukemia cell line HL60 were inhibited by the bisphosphonates etidronate and, to a lesser extent, clodronate and pamidronate . From our results, we assume that inorganic polyphosphate may be involved in modulation of the mineralization process in bone tissue.

Curr Opin Genet Dev, 1998 Apr, 8(2), 226 - 32
Telomerase and chromosome end maintenance; Lingner J et al.; The past year has seen significant advances in our understanding of telomerase and other factors involved in chromosome end maintenance . The protein subunit of telomerase that provides the active site for telomeric DNA synthesis was identified in ciliates, yeast and mammals . It is structurally related to reverse transcriptase and thus represents the first member of this protein family with an essential cellular function . Telomere DNA-binding proteins that may mediate the interaction of telomerase with telomeres have been identified and further characterized in diverse eukaryotes . A further elucidation of telomeric DNA structure has influenced our view of how telomeres replicate.

Curr Opin Genet Dev, 1998 Apr, 8(2), 185 - 93
DNA damage checkpoints update: getting molecular; Weinert T; Eukaryotic checkpoint controls impose delays in the cell cycle in response to DNA damage or defects in DNA replication . Genetic and physiological studies in budding yeast have identified key genes and defined genetic pathways involved in checkpoint-mediated responses . Recent studies now lead to biochemical models that explain at least in part the arrest in G1 and delays during DNA replication after damage . Though progress in checkpoint controls has indeed been rapid, several observations identify puzzling aspects of checkpoint controls with few plausible explanations.

Br J Haematol, 1998 May, 101(2), 311 - 7
Functional analysis of the p53 protein in AIDS-related non-Hodgkin's lymphomas and polymorphic lymphoproliferations; Martin A et al.; Alteration of the tumour suppressor gene p53 is frequent in AIDS-related non-Hodgkin's lymphomas (AIDS-NHL), particularly in Burkitt's or Burkitt's-like lymphomas (BL/BLL) . Since mechanisms of inactivation other than mutations have been advanced, the transcriptional activity of the p53 protein was studied in a functional assay in yeast in a series of AIDS-NHL lesions and compared with their morphology, immunohistochemistry (IHC) and single-strand conformation polymorphism (SSCP) analysis detection of other p53 abnormalities, Epstein-Barr virus (EBV) status, MDM-2 oncoprotein expression and c-MYC rearrangement . Polymorphic lymphoproliferations (PL), identified as precursors of NHL in HIV-patients, were also analysed in attempt to detect p53 modifications related to clonal progression . The functional assay detected p53 mutants in 40% (12/ 30) of the tumours: 50% (6/12) of BL/BLL, 40% (4/10) of diffuse large cell lymphomas (DLCL) and 25% (2/8) of PL . An oligoclonal or monoclonal population was identified in the two PL cases with mutant p53 . An accumulation of the p53 protein was detected by IHC in 26% (8/30) of the tumours (five BL/BLL and three DLCL) and was associated with positive functional assay . In the 20 lesions tested by both of the screening methods for mutations, a p53 mutant pattern was detected in 55% of cases (11/20) and in 25% of cases (5/ 20) respectively with the functional assay and SSCP analysis of exons 5-8 . There was no inverse correlation between the detection of EBV genome and the presence of p53 mutations and no overexpression of MDM-2 protein for the whole series . In conclusion, the functional assay was more sensitive than IHC and SSCP for the detection of p53 mutations in tumour samples . The mutations identified in AIDS-NHL lesions inactivate the p53 protein and in PL they could represent a selection of an aggressive clone.

Mol Cell Biochem, 1998 May, 182(1-2), 31 - 48
Insulin signal transduction through protein kinase cascades; Avruch J; This review summarizes the evolution of ideas concerning insulin signal transduction, the current information on protein ser/thr kinase cascades as signalling intermediates, and their status as participants in insulin regulation of energy metabolism . Best characterized is the Ras-MAPK pathway, whose input is crucial to cell fate decisions, but relatively dispensable in metabolic regulation . By contrast the effectors downstream of PI-3 kinase, although less well elucidated, include elements indispensable for the insulin regulation of glucose transport, glycogen and cAMP metabolism . Considerable information has accrued on PKB/cAkt, a protein kinase that interacts directly with Ptd Ins 3'OH phosphorylated lipids, as well as some of the elements further downstream, such as glycogen synthase kinase-3 and the p70 S6 kinase . Finally, some information implicates other erk pathways (e.g . such as the SAPK/JNK pathway) and Nck/cdc42-regulated PAKs (homologs of the yeast Ste 20) as participants in the cellular response to insulin . Thus insulin recruits a broad array of protein (ser/thr) kinases in its target cells to effectuate its characteristic anabolic and anticatabolic programs.

FEBS Lett, 1998 May 8, 427(2), 241 - 6
Levels of expression of hRPB11, a core subassembly subunit of human RNA polymerase II, affect doxorubicin sensitivity and cellular differentiation; Bruno T et al.; We have previously shown that the human RNA polymerase II subunit 11 (hRPB11) is among the proteins specifically downregulated upon Doxorubicin (Dox) treatment of human cancer cell lines, and that Dox resistant clones derived upon drug selection express about 20% of the protein present in the original parental cell line . Given the prominent role that this subunit appears to have in eukaryotic cells, and the fact that its deletion causes lethality in yeast, we wanted to test the effect of the reintroduction of parental cell line levels of this subunit in Dox resistant colon cancer cells (LoVoDX) . Stable transfectants of LoVoDX expressing parental (LoVoH) levels of hRPB11 showed a reduced sensitivity to the drug without changing the response of these cells to other chemotherapeutic agents, confirming a specific inverse correlation between cellular Dox sensitivity anti-hRPB11 levels of expression . In addition we show here that the levels of expression of this same RNA polymerase II subunit directly affect cellular differentiation, reducing the rate of cell proliferation, clonogenicity and increasing the expression of E-cadherin, a marker of epithelial cell differentiation . As expected from cells with these characteristics, upon in vivo administration of these clones in nude mice, we detected a significant reduction in the size and time of appearance of the primary tumors and overall metastatic capability . Finally, the role played by hRPB11 in regulating the transcription of specific genes is underlined by transient transfection experiments that show transactivation of the E-cadherin promoter by this protein.

Nucleic Acids Res, 1998 Apr 1, 26(7), 1841 - 7
Interaction between the N-terminus of human topoisomerase I and SV40 large T antigen; Haluska P Jr et al.; We have attempted to identify human topoisomerase I-binding proteins in order to gain information regarding the cellular roles of this protein and the cytotoxic mechanisms of the anticancer drug camptothecin, which specifically targets topoisomerase I . In the course of this work we identified an interaction between the N-terminus of human topoisomerase I and the SV40 T antigen that is detectable in vitro using both affinity chromatography and co-immunoprecipitation . Additional results indicate that this interaction does not require intermediary DNA or stoichiometric quantities of other proteins . Furthermore, the interaction is detectable in vivo using a yeast two-hybrid assay . Two binding sites for T antigen are apparent on the topoisomerase I protein: one consisting of amino acids 1-139, the other present in the 383-765 region of the protein . Interestingly, nucleolin, which binds the 166-210 region of topoisomerase I, is able to bind an N-terminal fragment of topoisomerase I concurrently with T antigen . Taken together with our prior identification of nucleolin as a topoisomerase I-binding protein, the current results suggest that helicase-binding is a major role of the N-terminus of human topoisomerase I and that the resultant helicase-topoisomerase complex may function as a eukaryotic gyrase.

Cytogenet Cell Genet, 1997, 79(3-4), 293 - 7
Identification of positional candidates for neurological disorders on chromsome 13q14-->q22; Nothwang HG et al.; In the course of a research project aimed at the molecular characterization of balanced chromosome rearrangements associated with mental retardation (MR), several YACs spanning MR-associated chromosomal rearrangements in the 13q14-->q22 region were identified . To facilitate the search for relevant candidate genes, we have analyzed a total of 102 EST clones from this region . Sequence comparisons revealed that these 102 clones represent up to 72 distinct transcripts . When no physical mapping data were available, a minimal YAC contig was screened for each unique transcript by the polymerase chain reaction (PCR) or hybridization . Fifty-eight independent ESTs could be localized to YAC clones between the markers D13S1248 and D13S1201 . Several ESTs are located on YAC clones detecting chromosomal rearrangements in MR patients . One EST was mapped within the critical region for Rieger syndrome type 2, and three transcripts were identified in the region for the nocturnal enuresis type 1 . Some ESTs showed homologies to known genes, including the cadherin-related tumor suppressor gene from Drosophila, the yeast mitotic control protein DIS3, and the human alpha-2-macroglobulin receptor associated protein.

Protein Sci, 1998 May, 7(5), 1071 - 82
Refolding rate of stability-enhanced cytochrome c is independent of thermodynamic driving force; McGee WA et al.; N52I iso-2 cytochrome c is a variant of yeast iso-2 cytochrome c in which asparagine substitutes for isoleucine 52 in an alpha helical segment composed of residues 49-56 . The N52I substitution results in a significant increase in both stability and cooperativity of equilibrium unfolding, and acts as a "global suppressor" of destabilizing mutations . The equilibrium m-value for denaturant-induced unfolding of N52I iso-2 increases by 30%, a surprisingly large amount for a single residue substitution . The folding/unfolding kinetics for N52I iso-2 have been measured by stopped-flow mixing and by manual mixing, and are compared to the kinetics of folding/unfolding of wild-type protein, iso-2 cytochrome c . The results show that the observable folding rate and the guanidine hydrochloride dependence of the folding rate are the same for iso-2 and N52I iso-2, despite the greater thermodynamic stability of N52I iso-2 . Thus, there is no linear free-energy relationship between mutation-induced changes in stability and observable refolding rates . However, for N52I iso-2 the unfolding rate is slower and the guanidine hydrochloride dependence of the unfolding rate is smaller than for iso-2 . The differences in the denaturant dependence of the unfolding rates suggest that the N52I substitution decreases the change in the solvent accessible hydrophobic surface between the native state and the transition state . Two aspects of the results are inconsistent with a two-state folding/unfolding mechanism and imply the presence of folding intermediates: (1) observable refolding rate constants calculated from the two-state mechanism by combining equilibrium data and unfolding rate measurements deviate from the observed refolding rate constants; (2) kinetically unresolved signal changes ("burst phase") are observed for both N52I iso-2 and iso-2 refolding . The "burst phase" amplitude is larger for N52I iso-2 than for iso-2, suggesting that the intermediates formed during the "burst phase" are stabilized by the N52I substitution.

J Immunol, 1998 Jun 1, 160(11), 5596 - 604
Interaction of vaccinia virus complement control protein with human complement proteins: factor I-mediated degradation of C3b to iC3b1 inactivates the alternative complement pathway; Sahu A et al.; Vaccinia virus complement control protein (VCP) is a virulence determinant of vaccinia virus that helps protect the virus from the complement attack of the host . To characterize the interaction of VCP with C3 and C4 and understand the mechanism by which VCP inactivates complement, we have expressed VCP in a yeast expression system and compared the biologic activity of the purified protein to that of human factor H and complement receptor 1 (CR1) . Recombinant VCP bound to C3 and the proteolytically cleaved form of C3 (C3b), but not to the 135,300-m.w . fragment of C3 generated using elastase (C3c) and the 35,000-m.w . fragment of C3 generated using elastase (C3d) and inhibited both the classical and alternative pathways of complement activation . Although rVCP was less effective at inhibiting the alternative pathway than factor H or CR1, it was more effective than factor H at inhibiting the classical pathway . Unlike factor H, rVCP was unable discriminate between alternative pathway-mediated lysis of rabbit and sheep E . A comparison of the cofactor activity in factor I-mediated cleavage of C3b suggested that in contrast to factor H and CR1, which displayed cofactor activity for the three sites, rVCP displayed cofactor activity primarily for the first site, leading to generation of C3b cleaved by factor I between Arg1281-Ser1282 (iC3b1) . Its cofactor activity for C4b cleavages was similar to that of soluble complement receptor type 1 . Purification and functional analysis of iC3b1 showed that it was unable to interact with factor B to form the alternative pathway C3 convertase, C3b,Bb . These results suggest that the interaction of VCP with C3 is different from that of factor H and CR1 and that VCP-supported first cleavage of C3b by factor I is sufficient to render C3b nonfunctional.

Mol Gen Genet, 1998 Apr, 257(6), 672 - 80
Characterization of a Drosophila homologue of the 160-kDa subunit of the cleavage and polyadenylation specificity factor CPSF; Salinas CA et al.; Processing of the 3' end of mRNA precursors depends on several proteins . The multisubunit cleavage and polyadenylation specificity factor (CPSF) is required for cleavage of the mRNA precursor as well as polyadenylation . CPSF interacts with the cleavage stimulatory factor complex (CstF), and this interaction increases the specificity of binding . Following cleavage downstream of the AAUAAA site, CPSF and poly(A) polymerase (PAP) are required for efficient polyadenylation . Recently, it has been shown that 160-kDa subunit of CPSF interacts directly with the 77-kDa subunit of CstF, which is homologous to the product encoded by the Drosophila gene su(f), and with PAP . Here we report the cloning and characterization of a Drosophila homologue of CPSF-160 . The 1329-amino acid dCPSF protein exhibits about 45% and 20% sequence identity, respectively, to its mammalian and yeast counterparts over its entire length . We show that the CPSF homologue is expressed throughout development and that CPSF is essential for viability . Mutations in the cpsf gene did not alter the phenotype of homozygous su(f) mutations, suggesting that, for most genes, processing of 3' termini is not sensitive to small changes in cpsf and su(f) dosage.

Toxicon, 1998 Jan, 36(1), 207 - 15
Viper venom-induced inflammation and inhibition of free radical formation by pure compound (2-hydroxy-4-methoxy benzoic acid) isolated and purified from anantamul (Hemidesmus indicus R . BR) root extract; Alam MI et al.; The present investigation explored the possible venom neutralizing effect of a pure compound (2-hydroxy-4-methoxy benzoic acid) isolated and purified from the methanolic root extract of Hemidesmus indicus R.Rr . 2-OH-4-MeO benzoic acid possessed potent anti-inflammatory, antipyretic and antioxidant properties . The compound effectively neutralized inflammation induced by Vipera russelli venom in male albino mice and reduced cotton pellet-induced granuloma in rats . The compound produced a significant fall in body temperature in yeast-induced pyrexia in rats but did not change the normothermic body temperature . The compound effectively neutralized viper venom-induced changes in serum phosphatase and transaminase activity in male albino rats . It also neutralized free radical formation as estimated by TBAPS and superoxide dismutase activities . The antisnake venom activity of the pure compound is partly mediated through the above physiological process.

Proc Natl Acad Sci U S A, 1998 May 26, 95(11), 6425 - 9
A double-stranded RNA element from a hypovirulent strain of Rhizoctonia solani occurs in DNA form and is genetically related to the pentafunctional AROM protein of the shikimate pathway; Lakshman DK et al.; M2 is a double-stranded RNA (dsRNA) element occurring in the hypovirulent isolate Rhs 1A1 of the plant pathogenic basidiomycete Rhizoctonia solani . Rhs 1A1 originated as a sector of the virulent field isolate Rhs 1AP, which contains no detectable amount of the M2 dsRNA . The complete sequence (3,570 bp) of the M2 dsRNA has been determined . A 6.9-kbp segment of total DNA from either Rhs 1A1 or Rhs 1AP hybridizes with an M2-specific cDNA probe . The sequences of M2 dsRNA and of PCR products generated from Rhs 1A1 total DNA were found to be identical . Thus this report describes a fungal host containing full-length DNA copies of a dsRNA element . A major portion of the M2 dsRNA is located in the cytoplasm, whereas a smaller amount is found in mitochondria . Based on either the universal or the mitochondrial genetic code of filamentous fungi, one strand of M2 encodes a putative protein of 754 amino acids . The resulting polypeptide has all four motifs of a dsRNA viral RNA-dependent RNA polymerase (RDRP) and is phylogenetically related to the RDRP of a mitochondrial dsRNA associated with hypovirulence in strain NB631 of Cryphonectria parasitica, incitant of chestnut blight . This polypeptide also has significant sequence similarity with two domains of a pentafunctional polypeptide, which catalyzes the five central steps of the shikimate pathway in yeast and filamentous fungi.

Blood, 1998 Jun 1, 91(11), 4379 - 86
Molecular chaperone GRP94 binds to the Fanconi anemia group C protein and regulates its intracellular expression; Hoshino T et al.; The FAC protein encoded by the gene defective in Fanconi anemia (FA) complementation group C binds to at least three ubiquitous cytoplasmic proteins in vitro . We used here the complete coding sequence of FAC in a yeast two-hybrid screen to identify interacting proteins . The molecular chaperone GRP94 was isolated twice from a B-lymphocyte cDNA library . Binding was confirmed by coimmunoprecipitation of FAC and GRP94 from cytosolic, but not nuclear, lysates of transfected COS-1 cells, as well as from mouse liver cytoplasmic extracts . Deletion mutants of FAC showed that residues 103-308 were required for interaction with GRP94, and a natural splicing mutation within the IVS-4 of FAC that removes residues 111-148 failed to bind GRP94 . Ribozyme-mediated inactivation of GRP94 in the rat NRK cell line led to significantly reduced levels of immunoreactive FAC and concomitant hypersensitivity to mitomycin C, similar to the cellular phenotype of FA . Our results demonstrate that GRP94 interacts with FAC both in vitro and in vivo and regulates its intracellular level in a cell culture model . In addition, the pathogenicity of the IVS-4 splicing mutation in the FAC gene may be mediated in part by its inability to bind to GRP94.

Blood, 1998 Jun 1, 91(11), 4321 - 30
Characteristic pattern of chromosomal gains and losses in primary large B-cell lymphomas of the gastrointestinal tract; Barth TF et al.; In contrast to low-grade B-cell lymphomas originating in the gastrointestinal (GI) tract, only few cytogenetic data are available for the large cell, highly malignant variants . We studied 31 large B-cell lymphomas of the GI tract by comparative genomic hybridization (CGH) and fluorescence in situ hybridization using specific DNA probes (FISH) . The most frequent aberrations were gains of all or of parts of chromosomes 11 (11 cases), 12 (9 cases), 1q (4 cases), and 3q (4 cases) . Losses of parts of chromosome 6q and of parts of the short arm of chromosome 17 (6 cases each) were found most frequently . In four cases a total of seven high-level DNA amplifications was detected . In two of these cases, involvement of specific protooncogenes (REL and MYC) was shown . Some genetic aberrations seemed to be associated with an inferior clinical course: patients with >/=2 aberrations had a significantly shorter median survival . Furthermore, all patients with gains of all or parts of chromosome arm 1q and with high-level DNA amplifications as well as seven of nine patients with gains of all or parts of chromosome 12 died of lymphoma . In conclusion, the pattern of chromosomal gains and losses in large B-cell lymphomas was different from data reported for low-grade (MALT) lymphomas of the stomach and bowel, especially with respect to the high incidence of partial gains of chromosome arm 11q and of all or parts of chromosome 12 and the low frequency of polysomy 3 . In addition, our data suggest that chromosomal gains and losses detected by CGH and FISH may predict for the outcome of patients with this tumor entity.

J Virol, 1998 Jun, 72(6), 5189 - 97
Functional interaction of human immunodeficiency virus type 1 Vpu and Gag with a novel member of the tetratricopeptide repeat protein family; Callahan MA et al.; Viral protein U (Vpu) is a protein encoded by human immunodeficiency virus type 1 (HIV-1) that promotes the degradation of the virus receptor, CD4, and enhances the release of virus particles from cells . We isolated a cDNA that encodes a novel cellular protein that interacts with Vpu in vitro, in vivo, and in yeast cells . This Vpu-binding protein (UBP) has a molecular mass of 41 kDa and is expressed ubiquitously in human tissues at the RNA level . UBP is a novel member of the tetratricopeptide repeat (TPR) protein family containing four copies of the 34-amino-acid TPR motif . Other proteins that contain TPR motifs include members of the immunophilin superfamily, organelle-targeting proteins, and a protein phosphatase . UBP also interacts directly with HIV-1 Gag protein, the principal structural component of the viral capsid . However, when Vpu and Gag are coexpressed, stable interaction between UBP and Gag is diminished . Furthermore, overexpression of UBP in virus-producing cells resulted in a significant reduction in HIV-1 virion release . Taken together, these data indicate that UBP plays a role in Vpu-mediated enhancement of particle release.

J Biol Chem, 1998 Apr 24, 273(17), 10153 - 9
The crk3 gene of Leishmania mexicana encodes a stage-regulated cdc2-related histone H1 kinase that associates with p12; Grant KM et al.; A cdc2-related protein kinase gene, crk3, has been isolated from the parasitic protozoan Leishmania mexicana . Data presented here suggests that crk3 is a good candidate to be the leishmanial cdc2 homologue but that the parasite protein has some characteristics which distinguish it from mammalian cdc2 . crk3 is predicted to encode a 35.6-kDa protein with 54% sequence identity with the human cyclin-dependent kinase cdc2 and 78% identity with the Trypanosoma brucei CRK3 . The trypanosomatid CRK3 proteins have an unusual, poorly conserved 19-amino acid N-terminal extension not present in human cdc2 . crk3 is single copy, and there is 5-fold higher mRNA in the replicative promastigote life-cycle stage than in the non-dividing metacyclic form or mammalian amastigote form . A leishmanial suc-binding cdc2-related kinase (SBCRK) histone H1 kinase, has previously been described which binds the yeast protein, p13(suc1), and that has stage-regulated activity (Mottram J . C., Kinnaird, J., Shiels, B . R., Tait, A., and Barry, J . D . (1993) J . Biol . Chem . 268, 21044-21051) . CRK3 from cell extracts of the three life-cycle stages was found to bind p13(suc1) and the leishmanial homologue p12(cks1) . CRK3 fused with six histidines at the C terminus was expressed in L . mexicana and shown to have SBCRK histone H1 kinase activity . Depletion of histidine-tagged CRK3 from L . mexicana cell extracts, by Ni-nitrilotriacetic acid agarose selection, reduced histone H1 kinase activity binding to p13(suc1) . These data imply that crk3 encodes the kinase subunit of SBCRK . SBCRK and histidine-tagged CRK3 activities were inhibited by the purine analogue olomoucine with an IC50 of 28 and 42 microM, respectively, 5-6-fold higher than human p34(cdc2)/cyclinB.

J Mol Biol, 1998 Apr 10, 277(4), 839 - 57
TAPASIN, DAXX, RGL2, HKE2 and four new genes (BING 1, 3 to 5) form a dense cluster at the centromeric end of the MHC; Herberg JA et al.; TAPASIN, a gene recently shown to be required for antigen presentation through MHC class I molecules, is located 180 kbp centromeric of HLA-DP in a region linked to several diseases, and associated with altered developmental phenotypes in the mouse . We present the genomic analysis of a 70 kbp gene-dense segment flanking the TAPASIN locus, including sequence, structure and preliminary characterisation of seven additional genes . BING1 is a Zn finger gene containing a POZ motif . BING3 is similar to myosin regulatory light chain . BING4 shows homologies only to hypothetical yeast and Caenorhabditis elegans proteins . BING5 is found within an intron of BING4 on the complementary strand, and encodes a molecule with no homologies to database proteins . Another three genes were identified whose full sequence was not previously known; namely, RGL2, DAXX (BING2) and HKE2 . RGL2 encodes an effector of Ras, homologous to the mouse RalGDS protein, Rlf . DAXX encodes an effector of Fas that stimulates apoptosis through the Jun kinase (JNK) pathway . The location of DAXX is of interest given the linkage of autoimmune disease to the MHC and to apoptosis .

J Biochem (Tokyo), 1998 Mar, 123(3), 516 - 20
Oxidative refolding of bovine pancreatic RNases A and B promoted by Asn-glycans; Nishimura I et al.; It was previously revealed {Yamaguchi, H . and Uchida, M . (1996) J . Biochem . 120, 474-477} that both intra- and extramolecular high-mannose type Asn-glycans promote the renaturation of reductively denatured bovine pancreatic RNases A and B under oxidation conditions . To characterize the conformational changes of the polypeptides during the renaturation promoted by the intramolecular Asn-glycans, RNase B was compared with its nonglycosylated form, RNase A, as to the features of the regeneration from their reductively denatured species under Cu2+-catalyzed oxidation conditions . The refolding intermediates of RNase B, as compared with those of RNase A, seemed to contain much less impaired disulfide linkages . In agreement with this finding, the proper refolding of RNase B was much faster than that of RNase A, as revealed by the intrinsic fluorescence and 1-anilino-8-naphthalenesulfonate binding of the refolding intermediates . Such a promoting effect was also observed for extramolecular Asn-glycans of the complex as well as of the high-mannose type . In contrast, common mono-, oligo-, and polysaccharides, but not yeast mannan, exhibited much lower stimulatory effects on the oxidative refolding of RNase A.

Nucleic Acids Res, 1998 Apr 1, 26(7), 1731 - 40
Sequence-specific DNA recognition by the myb-like domain of the human telomere binding protein TRF1: a model for the protein-DNA complex; Konig P et al.; Telomeres consist of tandem arrays of short G-rich sequence motifs packaged by specific DNA binding proteins . In humans the double-stranded telomeric TTAGGG repeats are specifically bound by TRF1 and TRF2 . Although telomere binding proteins from evolutionarily distant species are not sequence homologues, they share a Myb-like DNA binding motif . Here we have used gel retardation, primer extension and DNase I footprinting analyses to define the binding site of the isolated Myb-like domain of TRF1 and present a three-dimensional model for its interaction with human telomeric DNA . Our results suggest that the Myb-like domain of TRF1 recognizes a binding site centred on the sequence GGGTTA and that its DNA binding mode is similar to that of the homeodomain-like motifs of the yeast telomere binding protein RAP1 . The implications of these findings for recognition of telomeric DNA in general are discussed.

J Cell Sci, 1998 Mar, 111 ( Pt 5), 557 - 72
The Xenopus protein kinase pEg2 associates with the centrosome in a cell cycle-dependent manner, binds to the spindle microtubules and is involved in bipolar mitotic spindle assembly; Roghi C et al.; By differential screening of a Xenopus laevis egg cDNA library, we have isolated a 2,111 bp cDNA which corresponds to a maternal mRNA specifically deadenylated after fertilisation . This cDNA, called Eg2, encodes a 407 amino acid protein kinase . The pEg2 sequence shows significant identity with members of a new protein kinase sub-family which includes Aurora from Drosophila and Ipl1 (increase in ploidy-1) from budding yeast, enzymes involved in centrosome migration and chromosome segregation, respectively . A single 46 kDa polypeptide, which corresponds to the deduced molecular mass of pEg2, is immunodetected in Xenopus oocyte and egg extracts, as well as in lysates of Xenopus XL2 cultured cells . In XL2 cells, pEg2 is immunodetected only in S, G2 and M phases of the cell cycle, where it always localises to the centrosomal region of the cell . In addition, pEg2 'invades' the microtubules at the poles of the mitotic spindle in metaphase and anaphase . Immunoelectron microscopy experiments show that pEg2 is located precisely around the pericentriolar material in prophase and on the spindle microtubules in anaphase . We also demonstrate that pEg2 binds directly to taxol stabilised microtubules in vitro . In addition, we show that the presence of microtubules during mitosis is not necessary for an association between pEg2 and the centrosome . Finally we show that a catalytically inactive pEg2 kinase stops the assembly of bipolar mitotic spindles in Xenopus egg extracts.

Eur J Immunol, 1998 May, 28(5), 1458 - 66
The 3' part of the immunoglobulin kappa locus of the mouse; Kirschbaum T et al.; A detailed restriction map of a 430-kb contig comprising the single Ckappa, the 5 Jkappa and the adjoining 22 Vkappa gene segments is presented . The first 12 Vkappa genes following the JkappaCkappa region belong to the Vkappa21 family, the subsequent ones to the closely related families Vkappa8 and Vkappal 9/ 28 . Previous difficulties in cloning all Vkappa21 genes can now be explained by the presence of a duplicated region in this part of the locus . The structure was established by analysis of yeast artificial chromosome, bacterial artificial chromosome and cosmid clones and by the so-called long template PCR technique . The distance between Ckappa and the proximal Vkappa21 gene is 22 kb and the average distances between the Vkappa genes are about 20 kb . Of the 12 Vkappa21 genes 5 were sequenced for the first time and 8 of the 12 genes were found to be expressed . Of the 10 Vkappa8 and Vkappa19/28 germline genes 9 are new; expression products of 8 of the 10 genes were known . The known 5', 3' polarities allow to specify for the 22 Vkappa genes whether they are rearranged to the JkappaCkappa element by a deletion or an inversion mechanism . Also the formation of interesting rearrangement products in classical cell lines as MPC11, MOPC41 and PC 7043 can be explained now . The non-Vkappa sequence L10 whose rearrangement by inversion has been described earlier (Hoechtl and Zachau, Nature 1983 . 302: 260-263) was now localized downstream of JkappaCkappa.

Prenat Diagn, 1998 Apr, 18(4), 399 - 403
High resolution chromosome analysis and in situ hybridization on amniotic fluid for diagnosis of a cryptic translocation; Guichet A et al.; We report a cryptic translocation ascertained in a family after the birth of a mentally retarded proband . High resolution chromosome examination revealed that the father had a subtle translocation between chromosome 5 and chromosome 13, 46, XY, t(5;13) (q35.2;q34) . Two specific, non-routine techniques were associated for prenatal diagnosis: high resolution cytogenetic studies on the amniotic fluid and fluorescent in situ hybridization with YACs as specific telomeric probes . The fetus had the same cryptic translocation as his father.

J Hosp Infect, 1998 Apr, 38(4), 283 - 95
The action of three antiseptics/disinfectants against enveloped and non-enveloped viruses; Wood A et al.; The antiviral action of chloroxylenol, benzalkonium chloride and cetrimide/chlorhexidine was assessed against a range of enveloped and non-enveloped human viruses using a suspension test method . Viral suspensions of 10(6)-10(7) pfu/TCID50 or sfu were prepared in each of the antiseptic/disinfectant solutions in the presence of a bovine serum/yeast extract mixture to simulate 'dirty conditions' . During incubation, aliquots were removed at predetermined timepoints up to 10 min to assess the kinetics of inactivation . Results indicate that all products were effective in inactivating the enveloped viruses herpes simplex virus type 1 and human immunodeficiency virus type 1, whilst being ineffective in inactivating human coronavirus, also enveloped, and the non-enveloped viruses . The exception to this was the benzalkonium chloride-based product (Dettol Hospital Concentrate) which was active against the non-enveloped human coxsackie virus . Four antiseptic/disinfectant solutions with chloroxylenol, benzalkonium chloride, cetrimide/chlorhexidine and povidone-iodine were also assessed for antiviral effect against human immunodeficiency virus in the presence of whole human blood . All four solutions proved to be effective within 1 min despite the cytotoxic nature of the compounds to the detection system.

Brain Res Mol Brain Res, 1998 May, 56(1-2), 192 - 9
The cell cycle gene SKP1 is regulated by light in postnatal rat brain; Uro-Coste E et al.; During the early postnatal phase of high neuronal plasticity, an altered visual input leads to great modifications of visual cortex organization {Y . Fregnac, M . Imbert, Development of neuronal selectivity in primary visual cortex of cat, Physiol . Rev., 64 (1984) 375-434; D.H . Hubel, T.N . Wiesel, S . LeVay, Plasticity of ocular dominance columns in monkey striate cortex, Philos . Trans . R . Soc . London, Ser . B, 278 (1977) 377-409.} . We used refined differential screening of an organized cDNA library to identify the genes that may participate in this plasticity . We isolated a candidate plasticity gene encoding for a 163 aa protein that is closely related to the human and yeast Skp1p, a key factor in cell cycle progression {C . Bai, K . Hofman, L . Ma, M . Goebl, J.W . Harper, S.J . Elledge, SKP1 connects cell cycle regulators to the ubiquitin proteolysis machinery through a novel motif, the F-box, Cell, 86 (1996) 263-274; C . Connelly, P . Hieter, Budding yeast SKP1 encodes an evolutionary conserved kinetochore protein required for cell cycle progression, Cell, 86 (1996) 275-285; H . Zhang, R . Kobayashi, K . Galaktionov, D . Beach, p19Skp1 and p45Skp2 are essential elements of the cyclin A-CDK2 S phase kinase, Cell, 82 (1995) 915-925.} . Northern blot analysis showed that the expression of SKP1 (Skp1p gene) dramatically decreased after 2 h of light stimulation in the visual cortex of young dark-reared rats . This down regulation lasted at least 72 h . It was specific for the critical period as we did not observe any significant regulation of SKP1 mRNA by light in adult dark-reared rat brain . The down regulation was observed in the superior colliculus but also in the frontal cortex and in the hippocampus . The fact that this down regulation was not restricted to the visual system, suggested that it could be produced by dark rearing-induced hormonal changes . The significance of SKP1 expression in the brain and its regulation are discussed .

Biochim Biophys Acta, 1998 Apr 23, 1384(1), 55 - 65
Ribonucleases from rat and bovine liver: purification, specificity and structural characterization; Zhao W et al.; The presence of four members of the pyrimidine-specific ribonuclease superfamily was demonstrated in rat liver . Three of them (RL1, RL2 and RL3) were purified and showed ribonuclease activity at pH 7.5 with yeast RNA as substrate . RL1 is identical to rat pancreatic ribonuclease (ribonuclease 1) . N-terminal sequence analysis showed the presence of the native protein and several N-terminally degraded components . RL2 and RL3 were N-terminally blocked proteins . After acidic cleavage or CNBr digestion, several parts of their sequences were determined . RL2 has high sequence similarity with neurotoxin-type ribonucleases (ribonucleases 2, 3 and 6) . The amino acid sequence of rat liver-type ribonuclease (ribonuclease 4) was determined from a liver cDNA library . It differs at about 20% of the amino acid positions from other mammalian liver-type ribonucleases . The sequence of a peptide of RL3 was identical to that derived from the cDNA sequence of the liver-type ribonuclease . A contaminant of the RL3 fraction had a high sequence similarity with mouse and other mammalian angiogenins . Bovine, porcine and rat liver-type ribonucleases showed a strong preference for poly(U) over poly(C) . This preference is a unique property of the liver-type enzymes of the ribonuclease superfamily.

Curr Biol, 1998 May 7, 8(10), 603 - 6
The lissencephaly gene product Lis1, a protein involved in neuronal migration, interacts with a nuclear movement protein, NudC; Morris SM et al.; Important clues to how the mammalian cerebral cortex develops are provided by the analysis of genetic diseases that cause cortical malformations {1-5} . People with Miller-Dieker syndrome (MDS) or isolated lissencephaly sequence (ILS) have a hemizygous deletion or mutation in the LIS1 gene {3,6}; both conditions are characterized by a smooth cerebral surface, a thickened cortex with four abnormal layers, and misplaced neurons {7,8} . LIS1 is highly expressed in the ventricular zone and the cortical plate {9,10}, and its product, Lis1, has seven WD repeats {3}; several proteins with such repeats have been shown to interact with other polypeptides, giving rise to multiprotein complexes {11} . Lis1 copurifies with platelet-activating factor acetylhydrolase subunits alpha 1 and alpha 2 {12}, and with tubulin; it also reduces microtubule catastrophe events in vitro {13} . We used a yeast two-hybrid screen to isolate new Lis1-interacting proteins and found a mammalian ortholog of NudC, a protein required for nuclear movement in Aspergillus nidulans {14} . The specificity of the mammalian NudC-Lis1 interaction was demonstrated by protein-protein interaction assays in vitro and by co-immunoprecipitation from mouse brain extracts . In addition, the murine mNudC and mLis1 genes are coexpressed in the ventricular zone of the forebrain and in the cortical plate . The interaction of Lis1 with NudC, in conjunction with the MDS and ILS phenotypes, raises the possibility that nuclear movement in the ventricular zone is tied to the specification of neuronal fates and thus to cortical architecture.

Curr Biol, 1998 May 7, 8(10), 583 - 6
Suppressor of fused links fused and Cubitus interruptus on the hedgehog signalling pathway; Monnier V et al.; The Hedgehog (Hh) family of signalling proteins {1} mediate inductive interactions either directly or by controlling the transcription of other secreted proteins through the action of Gli transcription factors, such as Cubitus interruptus (Ci) {2} . In Drosophila, the transcription of Hh targets requires the activation of the protein kinase Fused (Fu) and the inactivation of both Suppressor of fused (Su(fu)) and Costal-2 (Cos-2) {3} . Fu is required for Hh signalling in the embryo and in the wing imaginal disc and acts also as an antitumorigen in ovaries {4} . All fu- phenotypes are suppressed by the loss of function of Su(fu) {5} . Fu, Cos-2 and Ci are co-associated in vivo in large complexes that are bound to microtubules in a Hh-dependent manner {6,7} . Here we investigate the role of Su(fu) in the intracellular part of the Hh signalling pathway . Using the yeast two-hybrid method and an in vitro binding assay, we show that Su(fu), Ci and Fu can interact directly to form a trimolecular complex, with Su(fu) binding to both its partners simultaneously . Su(fu) and Ci also co-immunoprecipitate from embryo extracts . We propose that, in the absence of Hh signalling, Su(fu) inhibits Ci by binding to it and that, upon reception of the Hh signal, Fu is activated and counteracts Su(fu), leading to the activation of Ci.

Cell Calcium, 1998 Feb-Mar, 23(2-3), 115 - 21
New nuclear functions for calmodulin; Agell N et al.; The data reported here summarize a series of results which reveal new functions for nuclear calmodulin (CaM) . The addition of CaM inhibitors to cultures of proliferating NRK cells blocked the activity of the cyclin-dependent protein kinases 4 (cdk4) and 2 (cdk2), which are enzymes implicated in the progression of G1 and in the onset of DNA replication, respectively . CaM modulates the activity of cdk4 by regulating the nuclear location of both cdk4 and cyclin D, its associated regulatory subunit . By using CaM-affinity chromatography, we have recently identified two new nuclear CaM-binding proteins: (i) the protein La/SSB, which is an autoantigen implicated in several autoimmune diseases such as lupus erythematosus and Sjogren's syndrome (since La/SSB participates in the process of transcription mediated by RNA polymerase III, CaM could be involved in the regulation of this process); and (ii) the protein SAP145, a member of the spliceosome-associated proteins (SAPs) which is a subunit of the splicing factor SF3(b) . This finding suggests the involvement of CaM in pre-mRNA splicing . Finally, a screening for new CaM-binding proteins in the fission yeast performed by using the phage display analysis, revealed that several nucleolar-ribosomal proteins associate to CaM, suggesting that CaM modulates ribosomal assembly and/or function.

J Biol Chem, 1998 May 22, 273(21), 13297 - 306
The HOXC11 homeodomain protein interacts with the lactase-phlorizin hydrolase promoter and stimulates HNF1alpha-dependent transcription; Mitchelmore C et al.; The lactase-phlorizin hydrolase (LPH) gene is expressed specifically in the enterocytes of the small intestine . LPH levels are high in newborn mammals, but decrease after weaning . We have previously suggested that the promoter element CE-LPH1, located at -40 to -54, plays an important role in this down-regulation, because the DNA binding activity of a nuclear factor that binds to this site is present specifically in small intestinal extracts and is down-regulated after weaning . In an effort to clone CE-LPH1-binding factors, a yeast one-hybrid genetic selection was used, resulting in the isolation of a partial cDNA encoding the human homeodomain protein HOXC11 . The full-length HOXC11 sequence was obtained by rapid amplification of cDNA ends . It was shown in a yeast assay and by electrophoretic mobility shift assay that HOXC11 binds to the CE-LPH1 element with similar specificity to the endogenous intestinal factor . Two HOXC11 transcript sizes were identified by Northern blot analysis . The larger transcript (2.1 kilobase pairs) is likely to contain a translational start site in good context and is present in HeLa cells . The shorter 1.7-kilobase pair transcript, present in HeLa and Caco-2 cells, probably encodes a protein lacking 114 amino acids at the N-terminal end . Both forms of HOXC11 potentiate transcriptional activation of the LPH promoter by HNF1alpha . The expression of HOXC11 mRNA in human fetal intestine suggests a role in early intestinal development.

J Biol Chem, 1998 May 22, 273(21), 13143 - 9
New insights into the co-evolution of cytochrome c reductase and the mitochondrial processing peptidase; Brumme S et al.; The mitochondrial processing peptidase (MPP) is a heterodimeric enzyme that forms part of the cytochrome c reductase complex from higher plants . Mitochondria from mammals and yeast contain two homologous enzymes: (i) an active MPP within the mitochondrial matrix and (ii) an inactive MPP within the cytochrome c reductase complex . To elucidate the evolution of MPP, the cytochrome c reductase complexes from lower plants were isolated and tested for processing activity . Mitochondria were prepared from the staghorn fern Platycerium bifurcatum, from the horsetail Equisetum arvense, and from the colorless algae Polytomella, and cytochrome c reductase complexes were purified by a micro-isolation procedure based on Blue-native polyacrylamide gel electrophoresis and electroelution . This is the first report on the subunit composition of a respiratory enzyme complex from a fern or a horsetail . The cytochrome c reductase complexes from P . bifurcatum and E . arvense are shown to efficiently process mitochondrial precursor proteins, whereas the enzyme complex from Polytomella lacks proteolytic activity . An evolutionary model is suggested that assumes a correlation between the presence of an active MPP within the cytochrome c reductase complex and the occurrence of chloroplasts.

J Biol Chem, 1998 May 22, 273(21), 12841 - 5
Regulation of guanine nucleotide exchange through phosphorylation of eukaryotic initiation factor eIF2alpha . Role of the alpha- and delta-subunits of eiF2b; Kimball SR et al.; The guanine nucleotide exchange activity of eIF2B plays a key regulatory role in the translation initiation phase of protein synthesis . The activity is markedly inhibited when the substrate, i . e . eIF2, is phosphorylated on Ser51 of its alpha-subunit . Genetic studies in yeast implicate the alpha-, beta-, and delta-subunits of eIF2B in mediating the inhibition by substrate phosphorylation . However, the mechanism involved in the inhibition has not been defined biochemically . In the present study, we have coexpressed the five subunits of rat eIF2B in Sf9 cells using the baculovirus system and have purified the recombinant holoprotein to >90% homogeneity . We have also expressed and purified a four-subunit eIF2B complex lacking the alpha-subunit . Both the five- and four-subunit forms of eIF2B exhibit similar rates of guanine nucleotide exchange activity using unphosphorylated eIF2 as substrate . The five-subunit form is inhibited by preincubation with phosphorylated eIF2 (eIF2(alphaP)) and exhibits little exchange activity when eIF2(alphaP) is used as substrate . In contrast, eIF2B lacking the alpha-subunit is insensitive to inhibition by eIF2(alphaP) and is able to exchange guanine nucleotide using eIF2(alphaP) as substrate at a faster rate compared with five-subunit eIF2B . Finally, a double point mutation in the delta-subunit of eIF2B has been identified that results in insensitivity to inhibition by eIF2(alphaP) and exhibits little exchange activity when eIF2(alphaP) is used as substrate . The results provide the first direct biochemical evidence that the alpha- and delta-subunits of eIF2B are involved in mediating the effect of substrate phosphorylation.

J Biol Chem, 1998 May 22, 273(21), 12794 - 7
A point mutation in Galphao and Galphai1 blocks interaction with regulator of G protein signaling proteins; Lan KL et al.; Regulator of G protein-signaling (RGS) proteins accelerate GTP hydrolysis by Galpha subunits and are thought to be responsible for rapid deactivation of enzymes and ion channels controlled by G proteins . We wanted to identify and characterize Gi-family alpha subunits that were insensitive to RGS action . Based on a glycine to serine mutation in the yeast Galpha subunit Gpa1(sst) that prevents deactivation by Sst2 (DiBello, P . R., Garrison, T . R., Apanovitch, D . M., Hoffman, G., Shuey, D . J., Mason, K., Cockett, M . I., and Dohlman, H . G . (1998) J . Biol . Chem . 273, 5780-5784), site-directed mutagenesis of alphao and alphai1 was done . G184S alphao and G183S alphai1 show kinetics of GDP release and GTP hydrolysis similar to wild type . In contrast, GTP hydrolysis by the G --> S mutant proteins is not stimulated by RGS4 or by a truncated RGS7 . Quantitative flow cytometry binding studies show IC50 values of 30 and 96 nM, respectively, for aluminum fluoride-activated wild type alphao and alphai1 to compete with fluorescein isothiocyanate-alphao binding to glutathione S-transferase-RGS4 . The G --> S mutant proteins showed a greater than 30-100-fold lower affinity for RGS4 . Thus, we have defined the mechanism of a point mutation in alphao and alphai1 that prevents RGS binding and GTPase activating activity . These mutant subunits should be useful in biochemical or expression studies to evaluate the role of endogenous RGS proteins in Gi function.

Novartis Found Symp, 1998, 214, 233 - 44; discussion 244-50
Imprinting and gene silencing in mice and Drosophila; Brenton JD et al.; H19 and Igf2 are located within a large imprinting domain that confers monoallelic silencing of parental alleles . The silent paternal allele of H19 is hypermethylated and relatively resistant to nucleases . Using a 130 kb yeast artificial chromosome clone, appropriate imprinting of both H19 and Igf2 was observed at single insert loci in transgenic mice . Imprinting was also observed for H19-lacZ transgenes containing 4 kb of upstream sequence, but only at multicopy loci . The H19 RNA is therefore not essential for imprinting . When the H19-lacZ transgene was introduced into Drosophila, a 1.2 kb region was identified within the 4 kb upstream flank that functioned as a bi-directional silencer . This cis element is located within a region that is apparently necessary for imprinting in mice . These studies suggest an evolutionarily conserved mechanism for gene silencing in Drosophila and imprinting in mice . We propose a new model for imprinting of H19 and Igf2 in mice in which silencing of H19 is the default state, and activation of the maternal allele requires a specific activator element.

Novartis Found Symp, 1998, 214, 200 - 9; discussion 209-13, 228-32
Mammalian X chromosome inactivation; Jaenisch R et al.; X chromosome inactivation in mammals requires expression of the gene Xist, which maps to the X chromosome inactivation centre (Xic) and encodes an untranslated RNA . Truncation of Xist RNA by gene targeting is lethal for female embryos and prevents the inactivation of the X chromosome carrying the deletion . This indicates that Xist RNA is necessary for initiation and propagation of the inactivation process . Xist is transcribed from the inactive X and its expression is silenced by DNA methylation, suggesting that methylation is crucial for shielding the active X chromosome against the inactivation process . Gene transfer experiments using transgenes the size of yeast artificial chromosomes have determined that a 450 kb fragment of DNA carrying Xist acts as an inactivation centre and is sufficient for initiation, propagation and maintenance of the inactive state . The elements for counting and choosing X chromosomes are part of the transgene . We have shown that X inactivation is mediated by a post-translational mechanism, i.e . the stabilization of Xist RNA, rather than by the regulation of the Xist promoter.

Biochem Biophys Res Commun, 1998 May 8, 246(1), 222 - 7
Identification of mouse ULK1, a novel protein kinase structurally related to C . elegans UNC-51; Yan J et al.; A novel protein kinase related to the C . elegans serine/threonine kinase UNC-51 was cloned from mouse . The UNC-51-Like Kinase (ULK)1 is encoded by a cDNA of 1051 amino acids with calculated MW of 113 kDa . Comparison of the ULK1 and UNC-51 shows the highest conservation in the amino-terminal kinase domain, which is followed by a proline/serine-rich (PS) domain and a conserved carboxyl-terminal (C) domain . ULK1 mRNA is expressed in various tissues, and is mapped to mouse chromosome 5F and rat chromosome 12q16.3, by fluorescent in situ hybridization . HA-tagged ULK1 is expressed as a protein of approximately 150 kDa in COS7 cells and is auto-phosphorylated in vitro in its PS domain . We propose that ULK1, UNC-51 and a yeast protein kinase Apg1p comprise a novel subfamily of protein kinase, which is structurally conserved among eukaryotes.

Ann N Y Acad Sci, 1998 Apr 15, 842, 212 - 6
Confocal imaging of gene expression during hamster submandibular gland biogenesis; Fernandes R et al.; The data presented here provide evidence that the abundance of the ALG7 protein product, GPT, correlates with high proliferative activity during the postnatal development of the hamster SMG development, and that it becomes downregulated with differentiation . Based on our previous studies with yeast, changes in the level of ALG7 expression may be necessary for the events directing salivary cell polarization, migration, differentiation, and apoptosis at distinct developmental stages.

Ann N Y Acad Sci, 1998 Apr 15, 842, 91 - 9
Nucleotide sugars, nucleotide sulfate, and ATP transporters of the endoplasmic reticulum and Golgi apparatus; Berninsone P et al.; The lumina of the endoplasmic reticulum and Golgi apparatus are the subcellular sites where glycosylation, sulfation, and phosphorylation of secretory and membrane-bound proteins, proteoglycans, and lipids occur . Nucleotide sugars, nucleotide sulfate, and ATP are substrates in the above reactions and must first be translocated from the cytosol into the lumen of these organelles . Translocation of these nucleotide derivatives is mediated by highly specific transporters, which are antiporters with the corresponding nucleoside monophosphate, as shown by genetic and biochemical approaches in mammals and yeast . Studies with mammalian, yeast, and protozoa mutants have shown that a defect in a specific translocator results in selective impairments of glycosylation of proteins, lipids and proteoglycans in vivo . Several of these transporters have been purified, cloned, and found to encode very hydrophobic proteins with multitransmembrane domains . Experiments with yeast and mammalian cells demonstrate that these transporters play a regulatory role in posttranslational modifications.

Genes Chromosomes Cancer, 1998 Jun, 22(2), 105 - 13
Molecular cytogenetic analysis of consistent abnormalities at 8q12-q22 in breast cancer; Fejzo MS et al.; Studies using comparative genomic hybridization (CGH) indicate that portions of chromosome arm 8q from 8q12 to 8qter are present at an increased relative copy number in a broad range of solid tumors . In this study we define an approximately 1 Mb wide region that appears to be frequently abnormal in copy number or structure in breast cancer cell lines and primary tumors . This was accomplished by fluorescence in situ hybridization (FISH) with yeast artificial chromosomes (YACs) mapped to 8q2-q22 . Eleven breast cancer cell lines and ten primary tumors were analyzed . A minimal region of rearrangement was localized to the CEPH-YAC 928F9 in three breast cancer cell lines with unbalanced translocation breakpoints mapping in this region . Unbalanced translocations also were detected in two primary tumors mapping between CEPH-YAC clones 890C4 and 936B3, flanking 928F9 . An increased copy number in the minimal region was detected in nine cell lines and in multiple primary tumors . This suggests the possibility that a single gene mapping to 928F9 is involved in breast cancer development or progression and may be deregulated by copy number increases in some tumors and by translocation in others . Four expressed sequence tags were mapped to YAC 928F9 and analyzed for rearrangements by Southern analysis and for abnormal expression by Northern analysis.

Science, 1998 May 8, 280(5365), 855 - 60
Biodemographic trajectories of longevity; Vaupel JW et al.; Old-age survival has increased substantially since 1950 . Death rates decelerate with age for insects, worms, and yeast, as well as humans . This evidence of extended postreproductive survival is puzzling . Three biodemographic insights--concerning the correlation of death rates across age, individual differences in survival chances, and induced alterations in age patterns of fertility and mortality--offer clues and suggest research on the failure of complicated systems, on new demographic equations for evolutionary theory, and on fertility-longevity interactions . Nongenetic changes account for increases in human life-spans to date . Explication of these causes and the genetic license for extended survival, as well as discovery of genes and other survival attributes affecting longevity, will lead to even longer lives.

FEBS Lett, 1998 Apr 17, 426(2), 229 - 32
HAP1-huntingtin interactions do not contribute to the molecular pathology in Huntington's disease transgenic mice; Bertaux F et al.; HAP1 (huntingtin associated protein) has previously been found to interact with huntingtin (htt) in a glutamine length dependent manner and has been proposed to play a role in the cell specific neurodegeneration observed in Huntington's disease (HD) . We have isolated mouse HAP1 (hap1) and have shown that expression is not enriched in areas specifically affected in HD . We have used the yeast two hybrid system to demonstrate that htt amino acids 171-230 are necessary for the hap1-htt binding and that hapl does not interact with the transgene exon 1 protein in a transgenic model of HD.

Genomics, 1998 Apr 15, 49(2), 314 - 6
The human TRIP6 gene encodes a LIM domain protein and maps to chromosome 7q22, a region associated with tumorigenesis; Yi J et al.; The thyroid receptor interacting protein-6 (TRIP6) was first identified as a ligand-dependent binding partner for the thyroid hormone receptor in a yeast two-hybrid screen . A partial TRIP6 cDNA clone that was isolated in the initial screen encodes two copies of the LIM domain . The LIM domain is a double zinc-finger structure that mediates protein-protein interactions . Here we report the complete amino acid sequence of human TRIP6 . The TRIP6 protein displays a proline-rich N-terminal region linked to three tandemly arrayed C-terminal LIM domains . The global molecular architecture and sequence of TRIP6 place it in the same family as the adhesion plaque protein, zyxin, and the lipoma preferred partner (LPP) . Zyxin and LPP are implicated in cellular signaling and tumorigenesis, respectively . By radiation hybrid mapping, the human TRIP6 gene was assigned to a segment of chromosome 7q22 that is commonly deleted in malignant myeloid diseases and uterine leiomyoma.

Genomics, 1998 Apr 15, 49(2), 310 - 3
DLG3, the gene encoding human neuroendocrine Dlg (NE-Dlg), is located within the 1.8-Mb dystonia-parkinsonism region at Xq13.1; Stathakis DG et al.; Neuroendocrine-Dlg (NE-Dlg) is a member of the discs-large-related (DLG) subfamily of the membrane-associated guanylate kinase-related protein family . Based on evidence from model systems, this protein appears to be critical for synaptogenesis, acting as a site-specific organizational center for integral membrane proteins and their downstream signaling molecules associated with the cytoskeleton . NE-Dlg also directly interacts with the colorectal tumor suppressor adenomatous polyposis coli, suggesting that it may play a role in regulating cell proliferation in epithelial cells . To explore the genetic control of NE-Dlg, we developed a physical map of the chromosome region containing DLG3, the locus encoding NE-Dlg . Using human-hamster radiation hybrid mapping panels, we mapped DLG3 to Xq13.1 and established a sequence-tagged site marker map of the surrounding region . We then developed a yeast artificial chromosome (YAC) contig for this region . Encompassing approximately 2.0 Mb contained within five overlapping YACs, this contig also includes the dystonia-parkinsonism syndrome (DYT3) locus . The close proximity of DLG3 to the DYT3 region suggests that the gene encoding NE-Dlg is a candidate locus for this neurological disorder.

Genomics, 1998 Apr 15, 49(2), 230 - 6
The gene encoding a cationic amino acid transporter (SLC7A4) maps to the region deleted in the velocardiofacial syndrome; Sperandeo MP et al.; By screening an expressed sequence tag database, we identified a novel human gene, SLC7A4, encoding a solute carrier family 7 {cationic amino acid (CAA) CAT-4 transporter, y+ system} member 4 . The SLC7A4 cDNA is 2325 nt long and includes 78, 1911, and 336 nt in the 5' noncoding, coding, and 3'-noncoding regions, respectively . SLC7A4 displays high homology with SLC7A1 and SLC7A2, two previously known CAA transporters . By chromosomal in situ hybridization and YAC identification, SLC7A4 was mapped to 22q11.2, the commonly deleted region of the velocardiofacial syndrome (VCFS, Shprintzen syndrome) . In a patient affected by VCFS, deletion of SLC7A4 was demonstrated by chromosomal FISH . By Northern analysis, an abundant transcript was detected in brain, testis, and placenta . Microinjection of SLC7A4 mRNA into Xenopus laevis oocytes demonstrates a significant stimulation of CAA transport.

Genomics, 1998 Apr 15, 49(2), 200 - 8
High-resolution YAC fragmentation map of 1q21; Lioumi M et al.; Chromosomal band 1q21 contains a number of genes, constituting the Epidermal differentiation complex (EDC), most of which are involved in the process of terminal differentiation of the human epidermis and implicated in several disorders of keratinization and cancer . The physical map of 1q21 has been refined by generating 400 YAC derivatives . These products have allowed us to localize EDC genes and additional ESTs precisely . The transcriptional map of the region has been extended by positioning 20 ESTs reported to map between D1S442 and D1S305 . Eight of the ESTs are localized in two distinct clusters, confirmed by isolating PACs and chromosome 1-specific cosmids . Two of the ESTs correspond to the genes for YL1 and selenium-binding protein, both of which have potential tumor suppressor activity . Through the use of fragmented YACs and bacterial clones, the order of markers and ESTs in the region has been established as follows: cen-A002O32-Bda44g03-Cda10d12-Bdab5d06, H60056, A005K39-D1S442-WI5663-WI7969-Cx40-Cda0g e12-Cda0kh05-A002D26- A008S07-Cda0ff08-D1S498-S100A10-WI7815( THH)-WI7217(FLG)-D1S1664-INV-SPRR2A- LOR-A001X21-D1S305-tel.

Genomics, 1998 Apr 15, 49(2), 180 - 7
Transposition of RhoA to the murine Y chromosome; Boettger-Tong HL et al.; In an effort to produce a more complete transcription map of the short (approximately 5 Mb) arm of the mouse Y chromosome, we have initiated exon trapping from Yp-derived YACs . Sequence analysis of the trapped products has identified exons of previously cloned mouse Y-located genes Zfy and SSty and potential exons homologous to the human Y-located Tspy gene family . In addition, a family of three Yp-located transcripts that show close homology to human RHOA (locus designation ARHA), a member of the Ras family of small GTPases, has been identified . To determine whether these Yp sequences had been transposed from an autosomal ancestor, we used this trapped product to isolate a full-length autosomal mouse RhoA cDNA that is 80% identical at the nucleotide level and 98% identical at the amino acid level to human RHOA and maps to mouse Chromosome 2 (locus designation ArhA) . Sequence analysis indicates that the Y-linked copies have diverged from the autosomal form, with small deletions precluding maintenance of a significant open reading frame in all Yp copies . Yet RT-PCR analysis indicates that two of these pseudogenes, RhoAy1 and 3, are expressed in a testis-specific manner, in sharp contrast to the nearly ubiquitous expression pattern of the autosomal ancestor . The data indicate that the Y copies of RhoA have been transposed from an autosome, followed by subsequent duplication, sequence divergence, and acquisition of a testis-specific promoter/enhancer.

J Am Diet Assoc, 1998 May, 98(5), 537 - 47
Dietary sources of nutrients among US adults, 1989 to 1991; Subar AF et al.; OBJECTIVE: To identify major food sources of 27 nutrients and dietary constituents for US adults . DESIGN: Single 24-hour dietary recalls were used to assess intakes . From 3,970 individual foods reported, 112 groups were created on the basis of similarities in nutrient content or use . Food mixtures were disaggregated using the US Department of Agriculture (USDA) food grouping system . SUBJECTS/SETTING: A nationally representative sample of adults aged 19 years or older (n = 10,638) from USDA's 1989-91 Continuing Survey of Food Intakes by Individuals . ANALYSES PERFORMED: For each of 27 dietary components, the contribution of each food group to intake was obtained by summing the amount provided by the food group for all respondents and dividing by total intake from all food groups for all respondents . RESULTS: This article updates previous work and is, to the authors' knowledge the first to provide such data for carotenes, vitamin B-12, magnesium, and copper . Beef, yeast bread, poultry, cheese, and milk were among the top 10 sources of energy, fat, and protein . The following other major sources also contributed more than 2% to energy intakes: carbohydrate: yeast bread, soft drinks/soda, cakes/cookies/ quick breads/doughnuts, sugars/syrups/jams, potatoes (white), ready-to-eat cereal, and pasta; protein: pasta; and fat: margarine, salad dressings/mayonnaise, and cakes/ cookies/quick breads/doughnuts . Ready-to-eat cereals, primarily because of fortification, were among the top 10 food sources for 18 of 27 nutrients . APPLICATIONS/CONCLUSIONS: These analyses are the most current regarding food sources of nutrients and, because of disaggregation of mixtures, provide a truer picture of contributions of each food group.

Exp Cell Res, 1998 May 1, 240(2), 274 - 81
Cellular localization and expression of template-activating factor I in different cell types; Nagata K et al.; Template-activating factors I (TAF-I) alpha and beta have been identified as chromatin remodeling factors from human HeLa cells . TAF-I beta corresponds to the protein encoded by the set gene, which was found in an acute undifferentiated leukemia as a fusion version with the can gene via chromosomal translocation . To determine the localization of TAF-I, we raised both polyclonal and monoclonal antibodies against TAF-I . The proteins that react to the antibodies are present not only in human cells but also in mouse, frog, insect, and yeast cells . The mouse TAF-I homologue is ubiquitous in a variety of tissue cells, including liver, kidney, spleen, lung, heart, and brain . It is of interest that the amounts of TAF-I alpha and beta vary among hemopoietic cells and some specific cell types do not contain TAF-I alpha . The level of the TAF-I proteins does not change significantly during the cell cycle progression in either HeLa cells synchronized with an excess concentration of thymidine or NIH 3T3 cells released from the serum-depleted state . TAF-I is predominantly located in nuclei, while TAF-I that is devoid of its acidic region, the region which is essential for the TAF-I activity, shows both nuclear and cytoplasmic localization . The localization of TAF-I in conjunction with the regulation of its activity is discussed.

Zhonghua Yi Xue Za Zhi, 1997 Mar, 77(3), 205 - 7
{Inhibitory effects of two oligosaccharides on murine melanoma experimental metastasis}; Zhou R et al.; OBJECTIVE: To observe the effects of a chemically synthesized tetrose and a natural yeast mannan on mouse melanoma experimental liver metastasis . METHODS: After treated with 4 mg tetrose (tetrose group) or 4 mg mannan (mannan group) for 30 minutes at 37 degrees C, 0.5 ml 1 x 10(6)B16-MBK melanoma cells were injected intraspleen . 55 days later, melanoma metastasis nodes in the surfaces of the liver and other organs as well as mouse survival time were observed . RESULTS: Of 6 mice in control (B16 cell+PBS), 4 died naturally within 55 days, 2 were dissected in the 55th day . All of the 6 mice had metastases in the livers, the total number of the melanoma nodes on each liver surface ranged from 2 to 30, with the largest one fused to the whole liver . One mouse had a neoplasm in the remnant site of injection, 3 had metastases in lungs, while of the 6 mice in the tetrose group, one died on the 50th day on injection . In mannan group, all of the 6 mice survived and no metastasis was seen except the largest diameter of < 1 mm of 2 liver nodes in one mouse . Neither tetrose nor mannan group had metastasis of the liver, and the weights of liver in the two groups were significantly lower than in the control . CONCLUSION: Both tetrose and mannan had the effects in blocking melanoma experimental liver metastasis, inhibiting transmigration of the liver, and prolonging the survival time of the mouse.

Science, 1998 May 8, 280(5365), 909 - 12
Replication checkpoint enforced by kinases Cds1 and Chk1; Boddy MN et al.; Cdc2, the kinase that induces mitosis, is regulated by checkpoints that couple mitosis to the completion of DNA replication and repair . The repair checkpoint kinase Chk1 regulates Cdc25, a phosphatase that activates Cdc2 . Effectors of the replication checkpoint evoked by hydroxyurea (HU) are unknown . Treatment of fission yeast with HU stimulated the kinase Cds1, which appears to phosphorylate the kinase Wee1, an inhibitor of Cdc2 . The protein kinase Cds1 was also required for a large HU-induced increase in the amount of Mik1, a second inhibitor of Cdc2 . HU-induced arrest of cell division was abolished in cds1 chk1 cells . Thus, Cds1 and Chk1 appear to jointly enforce the replication checkpoint.

J Cell Biol, 1998 Apr 20, 141(2), 309 - 19
Centromere protein B null mice are mitotically and meiotically normal but have lower body and testis weights; Hudson DF et al.; CENP-B is a constitutive centromere DNA-binding protein that is conserved in a number of mammalian species and in yeast . Despite this conservation, earlier cytological and indirect experimental studies have provided conflicting evidence concerning the role of this protein in mitosis . The requirement of this protein in meiosis has also not previously been described . To resolve these uncertainties, we used targeted disruption of the Cenpb gene in mouse to study the functional significance of this protein in mitosis and meiosis . Male and female Cenpb null mice have normal body weights at birth and at weaning, but these subsequently lag behind those of the heterozygous and wild-type animals . The weight and sperm content of the testes of Cenpb null mice are also significantly decreased . Otherwise, the animals appear developmentally and reproductively normal . Cytogenetic fluorescence-activated cell sorting and histological analyses of somatic and germline tissues revealed no abnormality . These results indicate that Cenpb is not essential for mitosis or meiosis, although the observed weight reduction raises the possibility that Cenpb deficiency may subtly affect some aspects of centromere assembly and function, and result in reduced rate of cell cycle progression, efficiency of microtubule capture, and/or chromosome movement . A model for a functional redundancy of this protein is presented.

Curr Genet, 1998 Mar, 33(3), 231 - 7
Production and secretion of biologically active human epidermal growth factor in Yarrowia lipolytica; Hamsa PV et al.; The gene encoding human epidermal growth factor (hEGF) was expressed as a fusion protein with the leader peptide and pro I region of alkaline extracellular protease in the yeast Yarrowia lipolytica . hEGF was purified from culture supernatant by reverse-phase chromatography and analysed by Western-blot hybridisations . The biologically active hEGF in the purified sample was assayed using the radioreceptor assay and estimated to be 100 microg/l . However, the level of expression was found to be substantially low compared to the levels of homologous protein, alkaline extracellular protease (AEP), possibly due to degradation by secreted acid protease(s) . A novel and sensitive bioassay was developed to determine the biological activity of hEGF produced at low levels and is based on the effect produced by hEGF in the regenerating tails of the wall lizard . Intramuscular injections of culture supernatant from the recombinant yeast and the standard hEGF led to a drastic reduction in tail regeneration confirming the biological activity of the recombinant hEGF.

Curr Genet, 1998 Mar, 33(3), 165 - 70
Delta-9 fatty acid desaturase gene containing a carboxyl-terminal cytochrome b5 domain from the red alga Cyanidioschyzon merolae; Itoh R et al.; A delta-9 fatty acid desaturase gene, homologous to animal and fungal acyl-coenzyme A (CoA) desaturases, was isolated from the red alga Cyanidioschyzon merolae using a degenerate PCR strategy . This gene, designated as CmFAD9, has no intron . The encoded delta-9 fatty acid desaturase (CmFad9p) consists of 476 amino acids and has an estimated molecular mass of 55.4 kDa . CmFad9p is a unique delta-9 fatty acid desaturase among plants, in that it is fused with the cytochrome b5 domain at its carboxyl terminus . This is characteristic of yeast acyl-CoA desaturase . Genomic Southern hybridization suggested that the C . merolae genome contains a single gene for delta-9 fatty acid desaturase of the animal and fungal type . Southern hybridization combined with pulsed-field gel electrophoresis revealed that CmFAD9 is probably located on chromosome XI of the 17 C . merolae chromosomes . A 1.6-kb product of this gene was transcribed throughout a light/dark synchronization culture . The discovery of CmFAD9 indicates the existence of a novel type of plant delta-9 fatty acid desaturase that may function in the endoplasmic reticulum, but not in the plastid.

J Dairy Sci, 1998 Apr, 81(4), 1089 - 94
Effect of selenium supplements on the distribution of selenium among serum proteins in cattle; Awadeh FT et al.; The objective of this study was to determine the effects of the amount and chemical form of dietary Se on the distribution of Se among serum proteins . Six growing calves were assigned in a completely randomized design to receive diets containing either adequate (0.41 microgram/g) or excess (0.73 microgram/g) dietary Se . Proteins in serum collected from the calves were separated into albumin, glutathione peroxidase, and selenoprotein P fractions, and the concentration of Se in each was determined . The concentration of Se within serum was elevated by dietary Se supplementation . The selenoprotein P fraction within serum contained the largest percentage of Se among the serum proteins . In a second study, 12 mature cows were assigned to receive one of four experimental salt mixes containing 20, 60, or 120 micrograms of Se as sodium selenite/g of salt mix; the fourth treatment was 60 micrograms of Se as selenized yeast/g of salt mix . Cows given salt with 120 micrograms of Se as selenite or 60 micrograms of Se as selenized yeast had the highest concentrations of Se in whole blood; however, concentrations of Se in serum did not differ among treatments . Concentrations of Se in the protein fractions within serum were not affected by treatment . Within serum, the highest concentration of Se was in the selenoprotein P fraction (31.6 ng/ml), the smallest concentration was in the glutathione peroxidase fraction (4.7 ng/ml), and an intermediate amount of Se was obtained from the albumin fraction (8.5 ng/ml) . In conclusion, selenized yeast and selenite as sources of Se for supplementation of cattle resulted in similar patterns of Se distribution among proteins in serum . The greatest concentration of Se was found in the selenoprotein P fraction, which may contribute to Se transportation or function as an antioxidant.

Zhonghua Nei Ke Za Zhi, 1996 Aug, 35(8), 517 - 9
{A study on red blood cell immune function in patients with Guillain-Barré syndrome and multiple sclerosis}; Qi X et al.; In order to understand whether there is red blood cell (RBC) immune dysfunction and the relationship between RBC immune abnormality and clinical state in patients with multiple sclerosis (MS) and Guillain-Barre syndrome (GBS), RBC immune function and circulation immune complex (CIC) level were evaluated in patient with these two diseases by using RBC immune adhesion test . It was found that the rate of formation of red blood cell-C3b receptor-yeast rosette was significantly lower in patients with these two diseases before and after treatment than that in a control group (P < 0.01) . The rate of formation of red blood cell-immune complex-yeast rosette and the CIC level in GBS group were notably higher than that in the control group (P < 0.05) . Moreover, we observed that the immune functions in patients with severe GBS and active MS were different from those in patients with mild GBS and stable MS and the change of each immune index in GBS and MS patients was related with the degree of recovery and the clinical state . These results suggested that decreased capability of RBC immune adhesion in patients with GBC and MS may be one of factors causing these diseases.

Plant Physiol, 1998 May, 117(1), 263 - 71
Isolation of the ornithine-delta-aminotransferase cDNA and effect of salt stress on its expression in Arabidopsis thaliana; Roosens NH et al.; To evaluate the relative importance of ornithine (Orn) as a precursor in proline (Pro) synthesis, we isolated and sequenced a cDNA encoding the Orn-delta-aminotransferase (delta-OAT) from Arabidopsis thaliana . The deduced amino acid sequence showed high homology with bacterial, yeast, mammalian, and plant sequences, and the N-terminal residues exhibited several common features with a mitochondrial transit peptide . Our results show that under both salt stress and normal conditions, delta-OAT activity and mRNA in young plantlets are slightly higher than in older plants . This appears to be related to the necessity to dispose of an easy recycling product, glutamate . Analysis of the expression of the gene revealed a close association with salt stress and Pro production . In young plantlets, free Pro content, Delta1-pyrroline-5-carboxylate synthase mRNA, delta-OAT activity, and delta-OAT mRNA were all increased by salt-stress treatment . These results suggest that for A . thaliana, the Orn pathway, together with the glutamate pathway, plays an important role in Pro accumulation during osmotic stress . Conversely, in 4-week-old A . thaliana plants, although free Pro level also increased under salt-stress conditions, the delta-OAT activity appeared to be unchanged and delta-OAT mRNA was not detectable . Delta1-pyrroline-5-carboxylate synthase mRNA was still induced at a similar level . Therefore, for the adult plants the free Pro increase seemed to be due to the activity of the enzymes of the glutamate pathway.

Pharmacol Ther, 1998 Apr, 78(1), 29 - 46
Molecular mechanisms of interferon resistance mediated by viral-directed inhibition of PKR, the interferon-induced protein kinase; Gale M Jr et al.; The interferon (IFN)-induced cellular antiviral response is the first line of defense against viral infection within an animal host . In order to establish a productive infection, eukaryotic viruses must first overcome the IFN-induced blocks imposed on viral replication . The double-stranded RNA-activated protein kinase (PKR) is a key component mediating the antiviral actions of IFN . This IFN-induced protein kinase can restrict viral replication through its ability to phosphorylate the protein synthesis initiation factor eukaryotic initiation factor-2 alpha-subunit and reduce levels of viral protein synthesis . Viruses, therefore, must block the function of PKR in order to avoid these deleterious antiviral effects associated with PKR activity . Indeed, many viruses have developed effective measures to repress PKR activity during infection . This review will focus primarily on an overview of the different molecular mechanisms employed by these viruses to meet a common goal: the inhibition of PKR function, uncompromised viral protein synthesis, and unrestricted virus replication . The past few years have seen exciting new advances in this area . Rather unexpectedly, this area of research has benefited from the use of the yeast system to study PKR . Other recent advances include studies on PKR regulation by the herpes simplex viruses and data from our laboratory on the medically important hepatitis C viruses . We speculate that IFN is ineffective as a therapeutic agent against hepatitis C virus because the virus can effectively repress PKR function . Finally, we will discuss briefly the future directions of this PKR field.

Antimicrob Agents Chemother, 1998 May, 42(5), 1133 - 8
CNI-H0294, a nuclear importation inhibitor of the human immunodeficiency virus type 1 genome, abrogates virus replication in infected activated peripheral blood mononuclear cells; Haffar OK et al.; Active nuclear importation of the human immunodeficiency virus (HIV) type 1 (HIV-1) preintegration complex (PIC) is required for the productive infection of nondividing cells, but it is believed to be dispensable for the infection of proliferating cells, such as activated T lymphocytes . To investigate this question, we exploited the properties of the small arylene bis (methyl ketone) compound CNI-H0294 . We have previously shown that this compound associated with the HIV-1 matrix protein nuclear localization sequence and blocked binding of the HIV-1 PIC to yeast karyopherin alpha . CNI-H0294 abrogated nuclear importation of the HIV-1 genome in macrophages and effectively inhibited infection of nondividing cells . In this study we demonstrate that CNI-H0294 inhibits binding of the HIV-1 PIC to human karyopherin alpha and reduces nuclear importation of the viral genome in primary peripheral blood mononuclear cells (PBMCs) . We also demonstrate that CNI-H0294 inhibits acute infection of PBMC cultures in vitro with a primary isolate of HIV-1 and reduces virus replication and virus load in cultures of endogenously infected PBMCs from seropositive individuals . Thus, as for infection of nondividing, terminally differentiated macrophages, HIV-1 uses active nuclear importation of the virus genome to infect activated CD4+ T cells . These results support nuclear importation as a novel target and CNI-H0294 and its derivatives as novel compounds for therapeutic intervention in HIV infection and AIDS.

Nat Biotechnol, 1998 May, 16(5), 431 - 9
Construction of YAC-based mammalian artificial chromosomes; Ikeno M et al.; To construct a mammalian artificial chromosome (MAC), telomere repeats and selectable markers were introduced into a 100 kb yeast artificial chromosome (YAC) containing human centromeric DNA . This YAC, which has a regular repeat structure of alpha-satellite DNA and centromere protein B (CENP-B) boxes, efficiently formed MACs that segregated accurately and bound CENP-B, CENP-C, and CENP-E . The MACs appear to be about 1-5 Mb in size and contain YAC multimers . Structural analyses suggest that the MACs have not acquired host sequences and were formed by a de novo mechanism . The accurate segregation of the MACs suggests they have potential as vectors for introducing genes into mammals.

Nucleic Acids Res, 1998 Jun 1, 26(11), 2638 - 43
Characterization of a DEAD box ATPase/RNA helicase protein of Arabidopsis thaliana; Okanami M et al.; We have isolated cDNAs encoding a novel member of the DEAD box RNA helicase family from Arabidopsis . The protein, named AtDRH1, is composed of 619 amino acids and the central portion has high similarity with the helicase core region of a prototypic RNA helicase, the human nuclear protein p68 . The N- and C-terminal regions are considerably diverged from the animal and yeast p68 homologs at the amino acid sequence level, but like the p68 subfamily members, an RGG box-like domain is present near the C-terminus . RNA blot analysis showed that the AtDRH1 transcript accumulates at a high level and almost equally in every part of the Arabidopsis plant . The purified, recombinant AtDRH1 was capable of unwinding double-stranded RNA in the presence of ATP or dATP and of hydrolyzing ATP . The ATPase activity was stimulated by some single-stranded RNAs and DNAs, including poly(A) and poly(dT), but not by poly(dA) . The ability of the polynucleotides to stimulate the ATPase activity was largely consistent with their affinity for AtDRH1 . These results show that AtDRH1 is a novel type of ATP/dATP-dependent RNA helicase and polynucleotide-dependent ATPase.

Nucleic Acids Res, 1998 Jun 1, 26(11), 2572 - 9
SCP2: a major protein component of the axial elements of synaptonemal complexes of the rat; Offenberg HH et al.; In the axial elements of synaptonemal complexes (SCs) of the rat, major protein components have been identified, with relative electrophoretic mobilities (M rs) of 30 000-33 000 and 190 000 . Using monoclonal anti-SC antibodies, we isolated cDNA fragments which encode the 190 000 M r component of rat SCs . The translation product predicted from the nucleotide sequence of the cDNA, called SCP2 (for synaptonemal complex protein 2), is a basic protein (pI = 8.0) with a molecular mass of 173 kDa . At the C-terminus, a stretch of approximately 50 amino acid residues is predicted to be capable of forming coiled-coil structures . SCP2 contains two clusters of S/T-P motifs, which are common in DNA-binding proteins . These clusters flank the central, most basic part of the protein (pI = 9.5) . Three of the S/T-P motifs are potential target sites for p34(cdc2) protein kinase . In addition, SCP2 has eight potential cAMP/cGMP-dependent protein kinase target sites . The gene encoding SCP2 is transcribed specifically in the testis, in meiotic prophase cells . At the amino acid sequence and secondary structural level, SCP2 shows some similarity to the Red1 protein, which is involved in meiotic recombination and the assembly of axial elements of SCs in yeast . We speculate that SCP2 is a DNA-binding protein involved in the structural organization of meiotic prophase chromosomes.

Nucleic Acids Res, 1998 Jun 1, 26(11), 2536 - 40
Dnmt2 is not required for de novo and maintenance methylation of viral DNA in embryonic stem cells; Okano M et al.; We have shown previously that de novo methylation activities persist in mouse embryonic stem (ES) cells homozygous for a null mutation of Dnmt1 that encodes the major DNA cytosine methyltransferase . In this study, we have cloned a putative mammalian DNA methyltransferase gene, termed Dnmt2 , that is homologous to pmt1 of fission yeast . Different from pmt1 in which the catalytic Pro-Pro-Cys (PPC) motif is 'mutated' to Pro-Ser-Cys, Dnmt2 contains all the conserved methyltransferase motifs, thus likely encoding a functional cytosine methyltransferase . However, baculovirus-expressed Dnmt2 protein failed to methylate DNA in vitro . To investigate whether Dnmt2 functions as a DNA methyltransferase in vivo , we inactivated the Dnmt2 gene by targeted deletion of the putative catalytic PPC motif in ES cells . We showed that endogenous virus was fully methylated in Dnmt2 -deficient mutant ES cells . Furthermore, newly integrated retrovirus DNA was methylated de novo in infected mutant ES cells as efficiently as in wild-type cells . These results indicate that Dnmt2 is not essential for global de novo or maintenance methylation of DNA in ES cells.

Genes Chromosomes Cancer, 1998 May, 22(1), 1 - 8
Identification of a 910-kb region of common allelic loss in chromosome bands 16q24.1-q24.2 in human lung cancer; Sato M et al.; To understand the molecular pathogenesis of human lung cancer, we analyzed allelic deletions on the long arm of chromosome 16 by PCR amplification of microsatellite markers . A total of 203 lung cancer specimens (78 squamous cell carcinomas and 125 adenocarcinomas) were analyzed . In both cell types, a common region of allelic loss was identified in 16q24.1-q24.2; it is flanked by the two markers D16S534 and D16S422 that spanned at most 910 kb . These results were confirmed by fluorescence in situ hybridization . There was no correlation between allelic loss and histopathologic diagnosis or clinical stage . These results suggest the existence of a tumor-suppressor gene that plays an important role in the course of carcinogenesis in both squamous cell carcinoma and adenocarcinoma of the lungs.

Biophys Chem, 1998 Mar 30, 71(1), 9 - 20
Effect of charge interactions on the carboxylate vibrational stretching frequency in c-type cytochromes investigated by continuum electrostatic calculations and FTIR spectroscopy; Laberge M et al.; The FTIR spectra of the asymmetric carboxylate absorption region of three c-type cytochromes--namely horse heart, yeast and bonito cytochromes c--as well as continuum electrostatic calculations performed on their respective protein matrices, show that these combined methods can target specific protein regions and yield pertinent protein charge information that correlates with the observed spectral data . Deconvolution of the IR carboxylate stretch frequency region (1525-1675 cm-1) in the three cytochromes yield different v(oco)a distributions . In the case of the bonito cytochrome c carboxylates, two v(oco)a populations are clearly distinguishable in the deconvoluted spectra--which is not the case for the more complex v(oco)a deconvolutions of the other two cytochromes . The frequency distributions of the calculated potentials are consistent with the experimental observations and we conclude that the IR carboxylate absorption in proteins can be modified by the electrostatic environment.

Lipids, 1998 Apr, 33(4), 393 - 9
Effect of selenium and vitamin E supplements on tissue lipids, peroxides, and fatty acid distribution in experimental diabetes; Douillet C et al.; The protective role of selenium (Se), given as a Se-rich yeast, selenomethionine or selenomethionine + vitamin E supplement, toward changes in lipid, peroxide, and fatty acid distribution in tissues of streptozotocin-induced diabetic rats, was investigated, after 24 wk of disease . Diabetes increased liver thiobarbituric acid-reactive substances and conjugated dienes; Se supplement completely corrected these changes . In kidney, as in heart, the peroxide levels were not significantly changed by diabetes . In diabetic rat liver, a significant drop in triglycerides and phospholipids (P < 0.05) was observed; this was modulated by Se + vitamin E supplementation . Se + vitamin E supplementation also inhibited the decrease in 18:2n-6 and the increase in 22:6n-3 observed in liver of diabetic rats, changes which reflect altered glycemic control . In kidney, heart, and aorta, diabetes produced some changes in lipid content and fatty acid distribution, especially an increase in heart triglycerides which was also corrected by the Se supplement . Se supplementation to diabetic rats also increased 18:0 ether-linked alcohol, 20:4 n-6, and 22:5 n-3 in cardiac lipids . In aorta, Se + vitamin E significantly increased 20:5 n-3 . These polyunsaturated fatty acids are precursors, in situ, of prostaglandin I2 (PGI2) and PGI3 which may protect against cardiovascular dysfunction . In kidney, conversely, Se decreased 20:4 n-6, the precursor of thromboxane A2 implicated in diabetic glomerular injury . Thus Se, and more efficiently Se + Vitamin E supplementation, in experimental diabetes could play a role in controlling oxidative status and altered lipid metabolism in liver, thereby maintaining favorable fatty acid distribution in the major tissues affected by diabetic complications.

Nat Genet, 1998 May, 19(1), 74 - 8
Human deltex is a conserved regulator of Notch signalling; Matsuno K et al.; A fundamental cell-fate control mechanism regulating multicellular development is defined by the Notch-signalling pathway . Developmental and genetic studies of wild type and activated Notch-receptor expression in diverse organisms suggest that Notch plays a general role in development by governing the ability of undifferentiated precursor cells to respond to specific signals . Notch signalling has been conserved throughout evolution and controls the differentiation of a broad spectrum of cell types during development . Genetic studies in Drosophila have led to the identification of several components of the Notch pathway . Two of the positive regulators of the pathway are encoded by the suppressor of hairless {Su(H)} and deltex (dx) genes . Drosophila dx encodes a ubiquitous, novel cytoplasmic protein of unknown biochemical function . We have cloned a human deltex homologue and characterized it in parallel with its Drosophila counterpart in biochemical assays to assess deltex function . Both human and Drosophila deltex bind to Notch across species and carry putative SH3-binding domains . Using the yeast interaction trap system, we find that Drosophila and human deltex bind to the human SH3-domain containing protein Grb2 (ref . 10) . Results from two different reporter assays allow us for the first time to associate deltex with Notch-dependent transcriptional events . We present evidence linking deltex to the modulation of basic helix-loop-helix (bHLH) transcription factor activity.

J Immunol, 1998 May 15, 160(10), 4881 - 8
Phosphorylation of FADD/MORT1 and Fas by kinases that associate with the membrane-proximal cytoplasmic domain of Fas; Kennedy NJ et al.; Fas (Apo-1, CD95), a member of the TNFR family, is expressed on a variety of cell types and transduces an apoptotic signal . Since Fas does not possess known enzymatic activities, proteins that interact with the cytoplasmic domain of Fas regulate the death signal . Several proteins have been identified, primarily using the yeast two-hybrid system, that associate with the death domain of Fas . One of these proteins, FADD/MORT1, can be phosphorylated, although the kinase that is responsible has not been identified . Furthermore, direct signaling connections between Fas and its known activation of sphingomyelinase or NF-kappaB have not been made, suggesting that other proteins may associate with Fas . In this study, a series of glutathione S-transferase fusion proteins was constructed that contained the cytoplasmic domain of murine Fas . These proteins were used to search for additional proteins that associate with Fas . Novel proteins, including kinases, were identified that associated specifically with the membrane-proximal, cytoplasmic tail of Fas but not with the death domain . One of these kinases phosphorylates FADD/MORT1 . Moreover, the membrane-proximal region of Fas itself was phosphorylated by one of the associating kinases . These findings suggest that, similar to the Fas-related p55 TNFR, the membrane-proximal region of Fas likely participates in signaling.

Cell, 1998 May 1, 93(3), 423 - 32
A function for the AP3 coat complex in synaptic vesicle formation from endosomes; Faundez V et al.; Synaptic vesicles can be coated in vitro in a reaction that is ARF-, ATP-, and temperature-dependent and requires synaptic vesicle membrane proteins . The coat is largely made up of the heterotetrameric complex, adaptor protein 3, recently implicated in Golgi-to-vacuole traffic in yeast . Depletion of AP3 from brain cytosol inhibits small vesicle formation from PC12 endosomes in vitro . Budding from washed membranes can be reconstituted with purified AP3 and recombinant ARF1 . We conclude that AP3 coating is involved in at least one pathway of small vesicle formation from endosomes.

Biochimie, 1998 Feb, 80(2), 137 - 50
The mitochondrial ADP/ATP carrier: structural, physiological and pathological aspects; Fiore C et al.; Under the conditions of oxidative phosphorylation, the mitochondrial ADP/ATP carrier catalyses the one to one exchange of cytosolic ADP against matrix ATP across the inner mitochondrial membrane . The ADP/ATP transport system can be blocked very specifically by two families of inhibitors: atractyloside (ATR) and carboxyatractyloside (CATR) on one hand, and bongkrekic acid (BA) and isobongkrekic acid (isoBA) on the other hand . It is well established that these inhibitors recognise two different conformations of the carrier protein, the CATR- and BA-conformations, which exhibit different chemical, immunochemical and enzymatic reactivities . The reversible transition of the ADP/ATP carrier between the two conformations was studied by fluorometric techniques . This transconversion, which is only triggered by transportable nucleotides, is probably the same as that which occurs during the functioning of ADP/ATP transport system . The fluorometric approach, using the tryptophanyl residues of the yeast carrier as intrinsic fluorescence probes, was combined to a mutagenesis approach to elucidate the ADP/ATP transport mechanism at the molecular level . Finally, recent reports that myopathies might result from defect in ADP/ATP transport led us to develop a method to quantify the carrier protein in muscular biopsies.

Am J Clin Nutr, 1998 May, 67(5 Suppl), 972S - 977S
Aceruloplasminemia: an inherited neurodegenerative disease with impairment of iron homeostasis; Harris ZL et al.; Aceruloplasminemia is an autosomal recessive disorder characterized by progressive neurodegeneration of the retina and basal ganglia associated with specific inherited mutations in the ceruloplasmin gene . Clinical and pathologic studies in patients with aceruloplasminemia revealed a marked accumulation of iron in affected parenchymal tissues, a finding consistent with early work identifying ceruloplasmin as a ferroxidase and with recent findings showing an essential role for a homologous copper oxidase in iron metabolism in yeast . The presence of neurologic symptoms in aceruloplasminemia is unique among the known inherited and acquired disorders of iron metabolism; recent studies revealed an essential role for astrocyte-specific expression of ceruloplasmin in iron metabolism and neuronal survival in the central nervous system . Recognition of aceruloplasminemia provides new insights into the genetic and environmental determinants of copper metabolism and has important implications for our understanding of the role of copper in human neurodegenerative diseases.

Am J Clin Nutr, 1998 May, 67(5 Suppl), 952S - 959S
Essentiality of copper in humans; Uauy R et al.; The biochemical basis for the essentiality of copper, the adequacy of the dietary copper supply, factors that condition deficiency, and the special conditions of copper nutriture in early infancy are reviewed . New biochemical and crystallographic evidence define copper as being necessary for structural and catalytic properties of cuproenzymes . Mechanisms responsible for the control of cuproprotein gene expression are not known in mammals; however, studies using yeast as a eukaryote model support the existence of a copper-dependent gene regulatory element . Diets in Western countries provide copper below or in the low range of the estimated safe and adequate daily dietary intake . Copper deficiency is usually the consequence of decreased copper stores at birth, inadequate dietary copper intake, poor absorption, elevated requirements induced by rapid growth, or increased copper losses . The most frequent clinical manifestations of copper deficiency are anemia, neutropenia, and bone abnormalities . Recommendations for dietary copper intake and total copper exposure, including that from potable water, should consider that copper is an essential nutrient with potential toxicity if the load exceeds tolerance . A range of safe intakes should be defined for the general population, including a lower safe intake and an upper safe intake, to prevent deficiency as well as toxicity for most of the population.

J Mol Med, 1998 Apr, 76(5), 295 - 302
Clinical and molecular aspects of nephropathic cystinosis; McDowell GA et al.; Nephropathic cystinosis, an autosomal recessively inherited lysosomal storage disease, results from impaired transport of the disulfide amino acid cystine out of cellular lysosomes . The consequent accumulation and crystallization of cystine destroys tissues, causing growth retardation in infancy, renal failure at 10 years of age, and a variety of other complications . Early oral therapy with the cystine-depleting agent cysteamine prevents renal deterioration and enhances growth . Although the lysosomal cystine carrier has been extensively studied, its molecular structure remains unknown . The lysosomal cystine transporter gene has been mapped by linkage analysis to human chromosome 17p between polymorphic microsatellite markers D17S1583 and D17S1584 . Pertinent recombination events and homozygosity by descent has verified that the cystinosis gene lies in the 3.6 cM genetic interval between these two markers . The cystinosis region has been substantially reduced in size by the observation of recombination events in cystinosis patients between markers D17S1828 and D17S2167 . According to radiation hybrid analysis, these two markers are separated by 10.2 cR8000 (centirad using 8000 rad radiation hybrids) . Estimates of the physical size of this interval range from 187 to 510 kb . Four yeast artificial chromosomes have been identified which form a contig covering the original cystinosis region . Two P1 clones together may span the new, smaller interval, meaning that the cystinosis gene would lie on one of them . Current efforts are being directed toward using these P1 clones to isolate candidate cDNAs by a variety of methods . The ultimate cloning of the cystinosis gene will reveal how functional lysosomal porters are synthesized, targeted, processed, and integrated into the lysosomal membrane.

Eur J Cell Biol, 1998 Mar, 75(3), 223 - 31
Syntaxin-16, a putative Golgi t-SNARE; Simonsen A et al.; Members of the syntaxin family of integral membrane proteins have recently been implicated as vesicle receptors on target membranes, coresponsible for the specificity of intracellular membrane traffic . So far, only a small number of different mammalian syntaxins have been identified . Here we report the cloning of three new human syntaxin cDNAs, presumably originating from alternative splicing of the same transcript . Syntaxin-16A and syntaxin-16B are identical, except that the latter contains an insertion of 21 amino acid residues . Syntaxin-16C is a truncated version of syntaxin-16A, lacking the C-terminal coiled-coil and hydrophobic regions characteristic for syntaxins . Database searches identified putative yeast, plant and nematode homologues of syntaxin-16, indicating that this protein is conserved through evolution, and syntaxin-16 belongs to a new subgroup of syntaxins . Epitope-tagged syntaxin-16A and syntaxin-16B were found to colocalize with the Golgi marker beta-COP, while syntaxin-16C was found in the cytosol . Syntaxin-16A associates posttranslationally with microsomes, and appears to be transported to the Golgi via the endoplasmic reticulum . The three syntaxin-16 forms may have differential roles in intracellular trafficking.

Genome Res, 1998 May, 8(5), 509 - 23
A map of 75 human ribosomal protein genes; Kenmochi N et al.; We mapped 75 genes that collectively encode >90% of the proteins found in human ribosomes . Because localization of ribosomal protein genes (rp genes) is complicated by the existence of processed pseudogenes, multiple strategies were devised to identify PCR-detectable sequence-tagged sites (STSs) at introns . In some cases we exploited specific, pre-existing information about the intron/exon structure of a given human rp gene or its homolog in another vertebrate . When such information was unavailable, selection of PCR primer pairs was guided by general insights gleaned from analysis of all mammalian rp genes whose intron/exon structures have been published . For many genes, PCR amplification of introns was facilitated by use of YAC pool DNAs rather than total human genomic DNA as templates . We then assigned the rp gene STSs to individual human chromosomes by typing human-rodent hybrid cell lines . The genes were placed more precisely on the physical map of the human genome by typing of radiation hybrids or screening YAC libraries . Fifty-one previously unmapped rp genes were localized, and 24 previously reported rp gene localizations were confirmed, refined, or corrected . Though functionally related and coordinately expressed, the 75 mapped genes are widely dispersed: Both sex chromosomes and at least 20 of the 22 autosomes carry one or more rp genes . Chromosome 19, known to have a high gene density, contains an unusually large number of rp genes (12) . This map provides a foundation for the study of the possible roles of ribosomal protein deficiencies in chromosomal and Mendelian disorders.

Curr Biol, 1998 Apr 23, 8(9), R305 - 7
Auxin signalling: protein stability as a versatile control target; Leyser O; Two components of an auxin signalling pathway in Arabidopsis have been found to be homologous to budding yeast enzymes that are known to be involved in regulating the stability of key cell-cycle regulatory proteins, such as the cyclin-dependent kinase inhibitor Sic1p.

Cancer Lett, 1998 Apr 24, 126(2), 119 - 26
Cloning of a breakpoint cluster region on chromosome 14 in uterine leiomyoma; Bhugra B et al.; A recurrent reciprocal chromosomal translocation, t(12;14)(q15;q24) is frequently observed in uterine leiomyoma . Chromosome 12 breakpoints have been shown to occur in a region of approximately 150 kb that contains the gene for a high mobility group protein (HMGI-C) . The breakpoint region on chromosome 14 has not been precisely defined . We have generated a contig of overlapping yeast artificial chromosome (YAC) clones approximately 3 Mb in size . Fluorescence in situ hybridization (FISH) analysis showed that this contig spanned the t(12;14) breakpoints in three uterine leiomyomas and that the breakpoints in these tumors occurred within a 1 Mb region . A 30 kb cosmid spanning one of the breakpoints was isolated to set the stage for identifying regions on chromosome 14 that may cause this region to be a preferential site for chromosomal translocation.

Eur J Gastroenterol Hepatol, 1998 Mar, 10(3), 235 - 7
Nutritional factors in inflammatory bowel disease; Hunter JO; During the past 20 years there has been growing interest in the importance of nutritional factors in the pathogenesis of inflammatory bowel disease . There are so far no definite links between ulcerative colitis and diet, but links with Crohn's disease have been studied by both epidemiologists and clinicians . Epidemiological studies, although retrospective, have suggested that patients with Crohn's disease eat more sugar and sweets that control individuals; however, when dietary sugar is restricted, there is little clinical benefit . The clinical approach to nutrition in Crohn's disease has been by the use of elemental diets, which will produce symptomatic and objective remission in up to 90% of compliant patients . Those who return to normal eating soon relapse but, in some studies, have enjoyed prolonged remission on exclusion diets . The foods excluded have been not sugar, but predominantly cereals, dairy products and yeast . Attention has now switched to the possible harmful role of fat in Crohn's disease . The efficacy of elemental feeds appears to depend not on the presentation of nitrogen but on the amount of long chain triglyceride present . Increases in recent years in the frequency of Crohn's disease in Japan have been correlated with increased dietary fat intake, and a recent study suggested that W-3 fatty acids, which are metabolized by immunomodulatory leukotrienes and prostaglandins, may have a beneficial role to play . The links between nutrition and Crohn's disease have now become strong and the role of fat may be the most exciting of all.

Acta Biochim Pol, 1997, 44(4), 791 - 4
New intron-containing human tRNA(Leu) genes; Karwowska U et al.; Three new human nuclear tRNA(Leu) genes have been isolated and sequenced using the PCR technique . Two of them represent genes containing a CAA anticodon and both contain introns of 22 nucleotides in length but differing in sequence . Intron-containing prolongated anticodon stems can be folded into a secondary structure similar to that of yeast pre-tRNA(Leu) . The evolutionary conserved secondary structure suggests the same role of intron sequences in the human and yeast pre-tRNA(Leu) maturation pathway.

Development, 1998 Jun, 125(12), 2193 - 202
Spatial and temporal targeting of gene expression in Drosophila by means of a tetracycline-dependent transactivator system; Bello B et al.; In order to evaluate the efficiency of the tetracycline-regulated gene expression system in Drosophila, we have generated transgenic lines expressing a tetracycline-controlled transactivator protein (tTA), with specific expression patterns during embryonic and larval development . These lines were used to direct expression of a tTA-responsive promoter fused to the coding region of either the beta-galactosidase or the homeotic protein Antennapedia (ANTP), under various conditions of tetracycline treatment . We found that expression of beta-galactosidase can be efficiently inhibited in embryos and larvae with tetracycline provided in the food, and that a simple removal of the larvae from tetracycline exposure results in the induction of the enzyme in a time- and concentration-dependent manner . Similar treatments can be used to prevent the lethality associated with the ectopic expression of ANTP in embryos and, subsequently, to control the timing of expression of the homeoprotein ANTP specifically in the antennal imaginal disc . Our results show that the expression of a gene placed under the control of a tetracycline-responsive promoter can be tightly controlled, both spatially by the regulatory sequences driving the expression of tTA and temporally by tetracycline . This provides the basis of a versatile binary system for controlling gene expression in Drosophila, with an additional level of regulation as compared to the general method using the yeast transcription factor GAL4.

J Exp Zool, 1998 Apr 15, 280(6), 384 - 94
alpha-Glucosidase from the hepatopancreas of the shrimp, Penaeus vannamei (Crustacea-Decapoda); Le Chevalier P et al.; Penaeus vannamei is an omnivorous species, and it can be assumed that a high level of carbohydrates is necessary for growth . Alpha-glucosidases are important enzymes necessary for the ultimate liberation of glucose residues from various carbohydrates . Using acarbose affinity chromatography, a glycosylated alpha-glucosidase with a molecular mass of approximately 105 kDa was isolated for the first time from the hepatopancreas of the shrimp . Exhibiting an optimal catalytic activity in the temperature range from 40 degrees C to 50 degrees C at pH 6, the purified enzyme hydrolyses alpha 1-4 bonds and liberates glucose from different oligo and polysaccharides . By contrast to other known glucosidases, no alpha 1-6 glucose link with hydrolysis has been observed . This could explain the different rates of growth in shrimp aquaculture with starches from various origins . The amino-acid composition, together with the partial sequence of a hydrolytic peptide, shows a high degree of similarity to the alpha-glucosidases reported for various organisms including yeast and fungi and may help determine the phylogeny of the family.

Oncogene, 1998 Apr 9, 16(14), 1779 - 88
The kinase activity of c-Abl but not v-Abl is potentiated by direct interaction with RFXI, a protein that binds the enhancers of several viruses and cell-cycle regulated genes; Agami R et al.; c-Abl, the non-receptor tyrosine kinase is associated with EP, a DNA element found in promoters/enhancers of different viruses and cell-cycle regulated genes . EP-DNA binds RFXI, a member of a novel family of DNA-binding proteins that is conserved through evolution and in yeast, it controls differentiation and exit from the mitotic cycle to G0 . EP-associated proteins are preferentially tyrosine phosphorylated and the associated c-Abl has strong tyrosine kinase activity . Here we investigated the molecular mechanism underlying this c-Abl kinase activity . We show that RFXI and c-Abl are in direct interaction, in vitro and in cell extracts, through the RFXI proline rich (PxxP) motif and the c-Abl SH3 domain . Remarkably, this interaction significantly potentiates c-Abl but not v-Abl auto-kinase activity . Collectively, we describe a novel mechanism of c-Abl recruitment to a defined DNA-cis element with its concomitant kinase activation . We propose that this mechanism may act to regulate cell-cycle control genes.

J Marmara Univ Dent Fac, 1993 Sep, 1(4), 307 - 14
Aetiology of denture stomatitis; Kulak Y et al.; The purpose of this study was to determine the etiologic factors of denture stomatitis . Fifteen subjects with clinical evidence of localized simple denture stomatitis, fifteen subjects without clinical signs of denture stomatitis, and forty-five subjects with clinical evidence of generalized simple denture stomatitis were investigated clinically and mycologically . Subjects were evaluated according to age, sex, duration of denture usage, smoking habits, frequency of denture brushing, overnight denture wearing, pH level of saliva and degree of candidal colonization and candidal formation . Salivary samples and swabs were taken from the palate and the mucosal surfaces of the dentures investigated mycologically in order to identify the yeast colonies . Smears were taken from the palate and investigated in order to identify candidal formation . No statistically significant relationship was found between denture stomatitis and age, sex, duration of denture usage, frequency of denture brushing, overnight denture wearing or pH level of saliva . There was however, a statistically significant relationship between denture stomatitis and denture hygiene, smoking habits, candidal colonization and candidal formation.

Proc Natl Acad Sci U S A, 1998 Apr 14, 95(8), 4463 - 8
The lethal mutation of the mouse wasted (wst) is a deletion that abolishes expression of a tissue-specific isoform of translation elongation factor 1alpha, encoded by the Eef1a2 gene; Chambers DM et al.; We have identified the mutation responsible for the autosomal recessive wasted (wst) mutation of the mouse . Wasted mice are characterized by wasting and neurological and immunological abnormalities starting at 21 days after birth; they die by 28 days . A deletion of 15.8 kb in wasted mice abolishes expression of a gene called Eef1a2, encoding a protein that is 92% identical at the amino acid level to the translation elongation factor EF1alpha (locus Eef1a) . We have found no evidence for the involvement of another gene in this deletion . Expression of Eef1a2 is reciprocal with that of Eef1a . Expression of Eef1a2 takes over from Eef1a in heart and muscle at precisely the time at which the wasted phenotype becomes manifest . These data suggest that there are tissue-specific forms of the translation elongation apparatus essential for postnatal survival in the mouse.

Proc Natl Acad Sci U S A, 1998 Apr 14, 95(8), 4152 - 7
The C-terminal SET domains of ALL-1 and TRITHORAX interact with the INI1 and SNR1 proteins, components of the SWI/SNF complex; Rozenblatt-Rosen O et al.; The ALL-1 gene was discovered by virtue of its involvement in human acute leukemia . Its Drosophila homolog trithorax (trx) is a member of the trx-Polycomb gene family, which maintains correct spatial expression of the Antennapedia and bithorax complexes during embryogenesis . The C-terminal SET domain of ALL-1 and TRITHORAX (TRX) is a 150-aa motif, highly conserved during evolution . We performed yeast two hybrid screening of Drosophila cDNA library and detected interaction between a TRX polypeptide spanning SET and the SNR1 protein . SNR1 is a product of snr1, which is classified as a trx group gene . We found parallel interaction in yeast between the SET domain of ALL-1 and the human homolog of SNR1, INI1 (hSNF5) . These results were confirmed by in vitro binding studies and by demonstrating coimmunoprecipitation of the proteins from cultured cells and/or transgenic flies . Epitope-tagged SNR1 was detected at discrete sites on larval salivary gland polytene chromosomes, and these sites colocalized with around one-half of TRX binding sites . Because SNR1 and INI1 are constituents of the SWI/SNF complex, which acts to remodel chromatin and consequently to activate transcription, the interactions we observed suggest a mechanism by which the SWI/SNF complex is recruited to ALL-1/trx targets through physical interactions between the C-terminal domains of ALL-1 and TRX and INI1/SNR1.

J Biol Chem, 1998 Apr 10, 273(15), 8835 - 41
A modular DNA carrier protein based on the structure of diphtheria toxin mediates target cell-specific gene delivery; Uherek C et al.; Modular fusion proteins that combine distinct functions required for cell type-specific uptake and intracellular delivery of DNA present an attractive approach for the development of self-assembling vectors for targeted gene delivery . Here, we describe a novel DNA carrier protein termed GD5 that mimics the structure of the bacterial diphtheria toxin (DT) and facilitates target cell-specific gene transfer via receptor-mediated endocytosis . GD5 carries at the N terminus the DNA-binding domain of the yeast transcription factor Gal4, which is connected to a C-terminal antibody fragment specific for the tumor-associated ErbB2 antigen via an internal DT translocation domain as an endosome escape activity . Bacterially expressed GD5 protein specifically bound to ErbB2-expressing cells and formed protein-DNA complexes with a luciferase reporter gene construct . These complexes, after compensation of excess negative charge with poly-L-lysine, served as a specific transfection vector for ErbB2-expressing cells . Inhibitors of endosomal acidification drastically reduced GD5-mediated transfection, indicating that the DT translocation domain of GD5, similar to the parental toxin, is strictly dependent on the transit through an acidic environment . Our results suggest that fusion proteins that employ the natural endosome escape mechanism of bacterial toxins might aid in the development of efficient nonviral vectors for applications in gene therapy.

Biochem Biophys Res Commun, 1998 Apr 7, 245(1), 53 - 8
Protonation of the neutral repeats of the RNA polymerase II CTD; Morris DP et al.; The CTD (carboxy-terminal repeat domain) of the largest subunit of RNA Polymerase II in most eukaryotes consists of from 26 to 52 seven amino acid repeats, the consensus sequence of which is YSPTSPS . Even though this consensus repeat does not contain residues that are normally protonated under the conditions used for positive ion electrospray mass spectrometry, we find that the CTD acquires about one proton per repeat when analyzed by this procedure . We have termed this phenomenon superprotonation . Superprotonation is apparently a property of the consensus sequence as the repeat peptide, (YSPTSPS)4, is superprotonated whereas other proteins and the repeat peptides (YSPTSPK)4, (YSPTSPR)4 and (YSPTAPR)4 are not . The highly conserved nature of the contiguous consensus repeats in organisms ranging from yeast to mammals implies that the functionally significant behavior of the domain is easily perturbed . We propose that CTD superprotonation is a manifestation of a unique biophysical property that will influence and could be the basis for consensus repeat function in vivo .

Biochem Biophys Res Commun, 1998 Apr 7, 245(1), 43 - 9
alpha-Mannosidase from Trichoderma reesei participates in the postsecretory deglycosylation of glycoproteins; Eneyskaya EV et al.; The 160 kDa alpha-mannosidase (E.C . 3.2.1.24) isolated from culture filtrate of Trichoderma reesei has wide aglycon specificity but cleaves the alpha1 --> 2 and alpha1 --> 3 mannosidic bonds with higher rate than alpha1 --> 6 bond and slowly hydrolyses yeast mannan and 1,6-alpha-mannan . The specific activity of the enzyme and rate constant in the reaction with p-nitrophenyl-alpha-D-mannopyranoside were 0.15 U/mg and 1.62 x 10(-4) microM/min/microg, respectively, at optimal pH 6.5 . We have found that in vitro enzyme is able to cleave off 30% of total alpha-mannopyranosyl residues from N- and O-linked glycans of secreted glycoproteins . The activity of the alpha-mannosidase toward glycoproteins in vivo was studied comparing the structures of O- and N-linked glycans of glycoproteins isolated from the cultures growing with and without 1-deoxymannojirimycin, an inhibitor of alpha-mannosidases . Difference in structures of these glycans may be explained by postsecretory deglycosylation catalysed by the alpha-mannosidase .

Biochem J, 1998 Apr 1, 331 ( Pt 1), 291 - 7
The two activation domains of the CCAAT-binding factor CBF interact with the dTAFII110 component of the Drosophila TFIID complex; Coustry F et al.; The CCAAT-binding factor CBF is a heterotrimeric transcription factor that specifically binds to CCAAT sequences in many eukaryotic genes . Previous studies have shown that CBF contains two transcription activation domains: a glutamine-rich, serine-threonine-rich domain present in the CBF-B subunit and a glutamine-rich domain in the CBF-C subunit . In this study, by using a series of deletion mutations of CBF-B and CBF-C in transcription assay in vitro, we further delineated smaller segments in these domains that were sufficient to support transcriptional activation by CBF . To test whether transcription activation by CBF requires co-activators, we examined the interaction between CBF and dTAF110, a component of the Drosophila TFIID complex . Recent work has demonstrated that glutamine-rich domains of the Sp1 transcription factor interact with dTAF110 and that this interaction has an important role in mediating transcription activation . Here we first demonstrate in a direct interaction assay in vitro that CBF binds dTAF110 . By using a yeast two-hybrid system we show that both of the transcription activation domains of CBF interact with dTAF110 . A deletion analysis suggests that a segment of CBF-B needed for transcription activation is also involved in interaction with dTAF110 . In CBF-C the C-terminal portion of the molecule seems to be needed for these two activities . Our results suggest that TAF110 might represent one of the co-activators that mediate transcriptional activation by CBF.

J Anim Sci, 1998 Apr, 76(4), 1204 - 15
Effect of level and source of dietary selenium on concentrations of thyroid hormones and immunoglobulins in beef cows and calves; Awadeh FT et al.; Our objective was to determine the effect of level and chemical form of dietary selenium on productivity of beef cows, concentrations of triiodothyronine (T3), and thyroxine (T4) in plasma, and immunoglobulins (IgG and IgM) in plasma and colostrum of cows . Pregnant cows (n = 60) were randomly allocated among four dietary treatments of 20, 60, or 120 ppm Se as selenite and 60 ppm as selenomethionine from selenized yeast (SeY) in salts offered free-choice . Treatments began 90 d prepartum and continued through the second parturition . Treatments did not affect the final body weights of cows or birth weights or weaning weights of calves . At parturition, cows given salt with 20 ppm Se as selenite had lower (P < . 05) concentrations of Se in blood than cows with access to higher-Se salts . Treatments affected (P < .01) the concentration of T3 and the ratio of T3:T4 in plasma of cows . The concentration of T3 in plasma of cows with access to salt with 20 ppm Se was 14% lower than that in cows supplemented with 60 ppm Se as selenite or SeY . Plasma IgG in cows and calves, colostrum, and Se concentrations in colostrum, casein, and whey were lowest (P < .01) for cows given salt with only 20 ppm Se . Thus, salts with concentrations of 60 and 120 ppm Se improved measures of Se status in cows and calves . Consideration should be given to the concentrations of T3 and IgG when determining the nutritional requirements for Se in cattle.

Cancer Res, 1998 May 1, 58(9), 1793 - 7
Selective sensitivity to radiation of cerebral glioblastomas harboring p53 mutations; Tada M et al.; Recent studies suggest that a balance may exist between the cell cycle arrest and apoptosis-inducing functions of the p53 tumor suppressor gene . Adenoviral p21 transduction attenuates apoptosis, whereas deletion of the p21 gene promotes it, and p21-null xenografts respond better than isogenic p21-wild type tumors to irradiation . Hence, the role of p53 in dictating the clinical response to radiotherapy and chemotherapy may be more complex than previously thought . We have analyzed survival and radiation response (regrowth-free period) of 42 patients with glioblastomas whose p53 status was determined by a sensitive yeast functional assay . Multivariate analysis revealed that p53 mutation is associated with longer survival (P < 0.02) . Among 36 radiation-treated patients, the regrowth-free period after treatment was significantly longer for tumors with p53 mutations (P < 0.0001), and p53 mutation was the sole independent factor predictive of radiotherapeutic response (P < 0.01) . Survival time after regrowth was independent of p53 status, suggesting that the difference in survival was related to the treatment rather than to the intrinsic aggressiveness of the tumor . Thus, in this Northern Japanese population, p53 mutation is a marker for better radiation response in glioblastomas, and this results in significantly longer survival.

Biochem J, 1997 Nov 1, 327 ( Pt 3), 773 - 9
Human and rat testis express two mRNA species encoding variants of NRD convertase, a metalloendopeptidase of the insulinase family; Hospital V et al.; Rat testis NRD convertase (EC 3.4.24.61) is a Zn2+-dependent endopeptidase that cleaves, in vitro, peptide substrates at the N-terminus of Arg residues in dibasic sites . This putative processing enzyme of the insulinase family of metallopeptidases exhibits a significant degree of similarity to insulinase and two yeast processing enzymes, Axl1 and Ste23 . We report the cloning of two human testis cDNA species encoding isoforms of NRD convertase, hNRD1 and hNRD2 . Whereas the hNRD1 transcript (3.7 kb) is equivalent to the previously characterized rat cDNA (rNRD1), hNRD2 and rNRD2 are 3.9 kb novel forms containing a nucleotide insertion encoding a 68-residue segment . This motif, which is inserted N-terminal of the Zn2+-binding site, HXXEH, is contained within the most conserved region among the insulinase family members . Analysis of the deduced primary sequences revealed 92% identity between rat and human orthologues . The human gene encoding NRD convertase was localized to chromosome 1p32.1-p32.2 . Whereas NRD convertase is mostly expressed in testis and in 24 cell lines, low mRNA levels were detected in most of the 27 other tissues tested.

Biochem J, 1997 Nov 1, 327 ( Pt 3), 765 - 71
14-3-3 proteins interact with the insulin-like growth factor receptor but not the insulin receptor; Furlanetto RW et al.; We have used a yeast two-hybrid system to identify proteins which bind to the cytosolic portion of the type 1 insulin-like growth factor (IGF) receptor (IGFIR) but not the insulin receptor (IR) . This analysis identified 14-3-3beta and zeta proteins . 14-3-3beta also binds to the IGFIR but not the IR in vitro and 14-3-3-IGFIR complexes are present in insect cells overexpressing the IGFIR cytoplasmic domain . 14-3-3 proteins are substrates of the IGFIR in the yeast system and in vitro . The interaction of 14-3-3 with the IGFIR requires receptor-kinase activity and maps to the C-terminus of the receptor, but does not depend on tyrosine residues in this or the juxtamembrane regions . Instead, the binding maps to serine residue 1283 and requires phosphorylation of this residue . 14-3-3 proteins are phosphoserine-binding proteins which have been shown to interact directly with components of the mitogenic and apoptotic signalling pathways, suggesting that they participate in growth regulation . Our findings suggest that 14-3-3 proteins may play a role in IGFIR signal transduction and may contribute to the differences in IGF and IR signalling capabilities.

Glycoconj J, 1998 Mar, 15(3), 251 - 63
Effect of shape, size, and valency of multivalent mannosides on their binding properties to phytohemagglutinins; Roy R et al.; Clusters of di-, tri-, and tetra-antennary alpha-D-mannopyranosides were synthesized in good yields based on the coupling of amine-bearing mono- or trisaccharide {Man alpha(1 --> 6){Man alpha(1 --> 3)}Man} haptens to poly-isocyanate or -isothiocyanate tethering cores . The relative binding properties of the resulting multivalent ligands were determined by turbidimetric and solid phase enzyme-linked lectin assays (ELLA) using plant lectins (phytohemagglutinins) Concanavalin A (Con A) and Pisum sativum (pea lectin) having four and two carbohydrate binding sites, respectively . Rapid and efficient cross-linking between tetravalent Con A and mannopyranosylated clusters were measured by a microtiter plate version of turbidimetric analyses . In inhibition of binding of the lectins to yeast mannan, the best tetravalent monosaccharide (30) and trisaccharide (31) inhibitors were shown to be 140 and 1155 times more potent inhibitors than monomeric methyl alpha-D-mannopyranoside against pea lectin and Con A, respectively . Compounds 30 and 31 were thus 35- and 289-fold more potent than the reference monosaccharide based on their hapten contents . As a general observation, the ligands bearing the Man alpha(1 --> 6){Man alpha(1 --> 3)}Man trimannoside structures were found to be more potent inhibitors for Con A than the ligands having single mannoside residues, whereas pea lectin could not discriminate between the two types of ligands.

Eur J Biochem, 1998 Apr 1, 253(1), 154 - 60
Processing of vertebrate box C/D small nucleolar RNAs in plant cells; Leader DJ et al.; The recent isolation of a number of plant box C/D small nucleolar (sno)RNAs demonstrates the conservation in plants of sequence and structural elements of processed box C/D snoRNAs . Boxes C and D, and terminal inverted repeats are known to be essential for accumulation and processing in vertebrates and yeast . Processing of vertebrate box C/D snoRNAs was examined by expression of various mouse hsc70 intron 5-U14 constructs in tobacco protoplasts . Full-length U14 and internally deleted U14 accumulated in the plant cells . Human U3 and U8 fragments, consistent with processing to internal box C/C' sequences, also accumulated in the plant cells . The similarity of processing behaviour of the vertebrate box C/D constructs in tobacco protoplasts and Xenopus oocytes suggests the mechanism of processing, involving recognition and association of proteins, is conserved in plants.

Biochem J, 1998 May 15, 332 ( Pt 1), 189 - 93
Enzymic, cysteine-specific ADP-ribosylation in bovine liver mitochondria; Jorcke D et al.; NAD+ glycohydrolase (NADase) and non-enzymic ADP-ribosylation have been thought to be involved in the regulation of mitochondrial Ca2+ fluxes . In this study it was found that several conditions (5 mM nicotinamide, 5 mM 3-aminobenzamide, 2 mM EDTA, 1 mM ATP, 10 mM dithiothreitol) known to strongly inhibit the NADase decreased ADP-ribosylation in bovine liver mitochondrial membranes with {32P}NAD+ as substrate to only a limited extent, if at all . The reaction led to the specific modification of two proteins with apparent molecular masses of approx . 26 and 53 kDa . An excess of added free ADP-ribose diminished the incorporation of label from {32P}NAD+ only slightly . Dithiothreitol inactivated the NADase, whereas ADP-ribosylation was unaffected . At low concentrations (25 microM) ADP-ribosylation was efficient with NAD+, but not ADP-ribose, as substrate . Under these conditions mitochondrial ADP-ribosylation seems to occur as an enzymic reaction rather than a non-enzymic transfer of ADP-ribose previously liberated from NAD+ by NAD+ glycohydrolase . The chemical stability of the protein-ADP-ribose bonds in the mitochondrial membranes indicated that cysteine residues are the predominant acceptors . Moreover, yeast aldehyde dehydrogenase, known to be a substrate for thiol-associated ADP-ribosylation, was efficiently ADP-ribosylated by using the mitochondrial activity and NAD+ as substrate . The modification of a cysteine residue in the aldehyde dehydrogenase was verified by the observation that pretreatment of this acceptor protein with N-ethylmaleimide substantially decreased its modification . It is therefore concluded that bovine liver mitochondria contain a cysteine-specific ADP-ribosyltransferase.

Biochem J, 1998 May 15, 332 ( Pt 1), 9 - 19
Reactions of nitric oxide with mitochondrial cytochrome c: a novel mechanism for the formation of nitroxyl anion and peroxynitrite; Sharpe MA et al.; The aerobic reactions of nitric oxide with cytochrome c were analysed . Nitric oxide (NO) reacts with ferrocytochrome c at a rate of 200 M-1 s-1 to form ferricytochrome c and nitroxyl anion (NO-) . Ferricytochrome c was detected by optical spectroscopy; NO- was detected by trapping with metmyoglobin (Mb3+) to form the EPR-detectable Mb-nitrosyl complex, and by the formation of dimers in yeast ferrocytochrome c via cross-linking of the free cysteine residue . The NO- formed subsequently reacted with oxygen to form peroxynitrite, as measured by the oxidation of dihydrorhodamine 123 . NO binds to ferricytochrome c to form the ferricytochrome c-NO complex . The on-rate for this reaction is 1.3+/-0.4x10(3) M-1.s-1, and the off-rate is 0.087+/-0.054 s-1 . The dissociation constant (Kd) of the complex is 22+/-7 microM . These reactions of NO with cytochrome c are likely to be relevant to mitochondrial metabolism of NO . Ferricytochrome c can act as a reversible sink for excess NO in the mitochondria . The reduction of NO to NO- by ferrocytochrome c may play a role in the irreversible inhibition of mitochondrial oxygen consumption by peroxynitrite . It is generally assumed that peroxynitrite would be formed in mitochondria via the reaction of NO with superoxide . The finding that NO- is formed from the reaction of NO and ferrocytochrome c provides a means of producing peroxynitrite in the absence of superoxide, via the reaction of NO- with oxygen.

Mech Dev, 1998 Mar, 72(1-2), 53 - 63
The suppressor of forked protein of Drosophila, a homologue of the human 77K protein required for mRNA 3'-end formation, accumulates in mitotically-active cells; Audibert A et al.; The suppressor of forked (Su(f)) protein of Drosophila melanogaster is highly homologous to two proteins involved in mRNA 3'-end formation, the yeast RNA14 protein and the 77K subunit of human cleavage stimulation factor (CstF) . This suggests a role for su(f) in mRNA 3'-end-processing, probably as part of Drosophila CstF . We have investigated the expression pattern of su(f) during Drosophila development and found that the su(f) gene product is not detected ubiquitously . The Su(f) protein accumulates in mitotically-active cells, but does not in non-dividing cells . This expression pattern corroborates earlier data suggesting that the phenotypes of su(f) mutants could result from a defect in cell proliferation . Our results suggest that, in Drosophila, Su(f) is involved in the regulatory function of CstF .

J Cell Sci, 1998 Mar, 111 ( Pt 6), 803 - 13
HiPER1, a phosphatase of the endoplasmic reticulum with a role in chondrocyte maturation; Romano PR et al.; We have previously identified and partially cloned Band 17, a gene expressed in growth plate chondrocytes transiting from proliferation to hypertrophy . We now rename this gene HiPER1, Histidine Phosphatase of the Endoplasmic Reticulum-1, based on the results reported here . HiPER1 encodes two proteins of 318 (HiPER1(318)) and 449 (HiPER1(449)) amino acids, which are 20-21% identical to a group of yeast acid phosphatases that are in the histidine phosphatase family . HiPER1(449) is significantly more abundant than HiPER1(318), correlating with the abundance of the alternatively spliced messages encoding HiPER449 and HiPER318 . Anti-HiPER1 antibodies detect two proteins of 53 and 55 kDa in growth plate chondrocytes that are absent in articular chondrocytes . We confirm that the 53 and 55 kDa proteins are HiPER1(449) by heterologous expression of the HiPER1(449) coding sequence in chick embryo fibroblasts . The 53 and 55 kDa proteins are glycosylated forms of HiPER1(449), as N-glycosidase F digestion reduces these proteins to 48 kDa, the predicted size of HiPER1(449) without the N-terminal signal sequence . Immunocytochemistry demonstrates that HiPER1(449) is found in chondrocytes maturing from proliferation to hypertrophy, but is not detectable in resting zone, deep hypertrophic zone or articular chondrocytes, a distribution that is consistent with the message distribution . HiPER1(449) was predicted to localize to the lumen of endoplasmic reticulum by an N-terminal signal sequence and by the C-terminal sequence Ala-Asp-Glu-Leu, which closely matches the consensus signal for ER retention, Lys-Asp-Glu-Leu . We confirm this prediction by demonstrating colocalization of HiPER1(449) with the ER protein HSP47 using dual-label immunofluorescence . PTHrP, a peptide that prevents hypertrophy in chondrocytes, suppressed HiPER1 and HiPER1(449) expression in vitro, an observation that further supports a role for HiPER1 in chondrocyte maturation . The yeast phosphatase homology, localization to the endoplasmic reticulum and pattern of expression suggest that HiPER1 represents a previously unrecognized intracellular pathway, involved in differentiation of chondrocytes.

J Basic Microbiol, 1998, 38(1), 61 - 9
Adaptation and reutilization of Candida guilliermondii cells for xylitol production in bagasse hydrolysate; Sene L et al.; The xylitol productivity increased by about 15% with the use of cells of Candida guilliermondii FTI 20037 previously recycled through four consecutive batch cultures and adapted to the sugar cane bagasse hemicellulosic hydrolysate . Furthermore, the more concentrated the hydrolysate, the more necessary was the adaptation of the cells, owing to the presence of toxic substances at high concentration which inhibited the xylose-xylitol conversion by the yeast.

Gene, 1998 Apr 14, 210(2), 239 - 45
The human RIL gene: mapping to human chromosome 5q31.1, genomic organization and alternative transcripts; Bashirova AA et al.; The ril gene encoding a LIM domain protein of an unknown function was previously identified by differential expression cloning as a candidate tumor suppressor gene in rat fibroblasts (Kiess, M., Scharm, B., Aguzzi, A., Hajnal, A., Klemenz, R., Schwarte-Waldhoff, I., Schafer, R., 1995 . Expression of ril, a novel LIM domain gene, is down-regulated in HRAS-transformed cells and restored in phenotypic revertants . Oncogene 10, 61-68) . Searching for novel genes on human chromosome 5q31.1 by the cDNA selection technique, we isolated a cDNA clone identical with the cDNA of the human RIL gene (GenBank Accession No . X93510) . The human 5q31.1 region is of interest because it contains the cytokine gene cluster and is frequently deleted in the malignant cells of patients with myelodysplasia and myeloid leukemia . Using Southern blot analysis and restriction mapping of genomic YAC (yeast artificial chromosome) and cosmid clones, we located the human RIL gene 240-260 kb telomeric to the IRF1 gene and characterized its genomic structure . PCR analysis indicated the presence of two alternative RIL transcripts in human fetal brain mRNA . The major transcript is identical with the RIL cDNA previously deposited in GenBank and contains seven exons distributed over 14.5 kb of genomic DNA with the two last 3'-exons coding a LIM domain . The minor transcript lacks the sixth exon compared with the major transcript, which leads to the loss of the LIM domain . We also identified two putative transcription start points (tsp) and sequenced the 5'-flanking region of RIL to reveal potential binding sites for transcriptional factors.

Arch Biochem Biophys, 1998 Apr 1, 352(1), 31 - 6
Molecular cloning and characterization of a novel nuclear protein kinase in mice; Zelko I et al.; We cloned cDNAs which encode a mouse liver nuclear protein with an apparent molecular mass of 51 kDa, using sequences derived from a purified protein as the basis for designing specific primers . The deduced amino acid sequences revealed that the 51-kDa protein contains characteristic subdomain structures of a protein kinase . The bacterially expressed recombinant 51-kDa protein catalyzed phosphorylation of general substrates such as casein and was autophosphorylated at serine residue(s) . This 51-kDa protein kinase, designated 51PK, is 40% identical to the 34-kDa protein kinase encoded by the vaccinia virus B1 gene and 25% identical to the casein kinase I isoforms, including yeast HRR25 . The 51PK mRNA was expressed as two splice variants and the 51PK protein was exclusively localized in nuclei . Northern hybridization showed that 51PK mRNA was expressed in various tissues, with highest levels in testis, spleen, lung, and liver . These results, therefore, indicate that 51PK is a nuclear serine/threonine kinase and a novel distinct member of the protein kinase superfamily .

Proc Natl Acad Sci U S A, 1998 Mar 31, 95(7), 3764 - 9
Rapid isolation of cDNA by hybridization; Hamaguchi M et al.; The isolation of genes from a given genomic region can be a rate-limiting step in the discovery of disease genes . We describe an approach to the isolation of cDNAs that have sequences in common with large genomic clones such as bacterial artificial chromosomes . We applied this method to loci both amplified and deleted in cancer, illustrating its usage in the identification of both oncogenes and tumor suppressor genes, respectively . The method, called rapid isolation of cDNAs by hybridization (RICH), depends on solution hybridization, enzymatic modification, and amplification/selection of sequences present in both cDNA populations and the genomic clones . The method should facilitate the development of transcription maps for large genomic clones, possibly even yeast artificial chromosomes.

Proc Natl Acad Sci U S A, 1998 Mar 31, 95(7), 3419 - 24
HIV-1 Vpr interacts with a human 34-kDa mov34 homologue, a cellular factor linked to the G2/M phase transition of the mammalian cell cycle; Mahalingam S et al.; Several important and possibly interrelated functions have been identified for the HIV-1 accessory gene product Vpr . These include import of the HIV reverse transcription complex into the nucleus of nondividing cells, cellular differentiation including cell cycle arrest at the G2/M phase border, immune suppression, and enhancement of virus replication . We have cloned a candidate Vpr ligand, termed human Vpr interacting protein (hVIP/MOV34), by using a yeast two-hybrid assay . This gene is homologous to a simultaneously identified 34-kDa human mov34 homologue . The MOV34 family includes proteins that function as transcriptional and proteolytic regulators of cell growth and differentiation . We demonstrate direct interactions between the putative ligand hVIP/MOV34 and Vpr in vitro and in vivo . hVIP/MOV34 localizes to the nucleus and appears to function as a component of the cell cycle cascade . We observe an association between the induction of cell cycle arrest at the G2/M phase border by Vpr and a change in the subcellular localization of hVIP/MOV34 from a nuclear to a perinuclear localization . This was further associated with the inhibition of maturation promoting factor-associated histone H1 kinase activity . We conclude that hVIP/MOV34 is involved in the regulation of the cell cycle and a likely cellular cofactor for HIV-1 Vpr.

Arch Virol, 1998, 143(3), 563 - 70
Telomeric sequences from human herpesvirus 6 do not mediate nuclear retention of episomal DNA in human cells; Bulboaca GH et al.; Telomeric repeat sequences (TRS) have been identified close to, but not at, the genome termini of several lymphotropic herpesviruses, including human herpesviruses 6 and 7 (HHV-6, HHV-7) . The functional significance of these motifs remains uncertain . Since telomeric sequences can mediate stable retention of episomal DNA in yeast, we have tested whether the TRS motifs from HHV-6 might mediate a similar function in human cells . Several candidate sequences were assessed for their ability to provide nuclear retention to an autonomously replicating vector in rapidly dividing human tissue culture cells, including HHV-6 TRS DNA, as well as telomeric DNA from human cells and sequences from Epstein-Barr virus (EBV) . However, only a vector carrying the EBV-derived retention mechanism showed a significant level of nuclear retention . Neither the HHV-6 TRS motifs, nor human telomeric sequences, mediated nuclear retention of episomal DNA in human cells.

Semin Cell Dev Biol, 1998 Feb, 9(1), 11 - 7
Subtilisin-related serine proteases in the mammalian constitutive secretory pathway; Gensberg K et al.; Proteolytic cleavage is a key step in the activation of many secreted proteins . Since the identification of the prototype yeast enzyme Kex2, seven subtilisin-related serine proteases, together with a number of isoforms, have been identified in mammalian cells, five of which act within the constitutive secretory pathway . Overlapping expression patterns and substrate specificities complicate analysis but individual roles are beginning to emerge for some members of the group.

Mol Cells, 1998 Feb 28, 8(1), 19 - 26
Cloning of a cDNA encoding phospholipase D from Pimpinella brachycarpa; Cha YR et al.; Phospholipase D (PLD, EC 3.1.4.4) has been known to be related to various cellular processes in plants . To gain an understanding of the property of the enzyme in Pimpinella brachycarpa, the cDNA of the enzyme was isolated by PCR with degenerate primers, cDNA library screening, and 5' RACE . The full-length PLD cDNA is 2859 bp long and contains an open reading frame of 2424 bp coding for a polypeptide of 808 amino acids . The deduced enzyme has a calculated molecular mass of 91.7 kDa and pI of 5.86 . The percent identity and similarity values of P . brachycarpa PLD with those of other PLDs in plants are 70 approximately 78 and 84 approximately 95, respectively . It was identified that PLD from P . brachycarpa has HQKIVVVD and HAKMMIVD sequences which were homologous with a duplicated HXKXXXXD motif that has been conserved in PLDs from plants, animals, and yeast . Based on the analysis of amino acid similarity, it is believed that PLD from P . brachycarpa is an alpha form which is distinct from PLD beta reported recently . The N-terminus is homologous to the C2 domain which is present in a number of different proteins involved in signal transduction and membrane trafficking in animals . Southern and northern blot analyses indicated that PLD was expressed from one copy of PLD gene in the genome of P . brachycarpa.

Methods, 1998 Feb, 14(2), 135 - 51
Physical mapping of the mouse genome; Herman GE; This review is intended to provide an overview of techniques and a source of reagents for physical mapping of the mouse genome . It focuses on those applications, methods, or resources unique to the mouse and on the generation of comparative physical maps . The reference list is not comprehensive; rather, recent reviews on each topic and selected representative examples are given.

Genomics, 1998 Apr 1, 49(1), 103 - 11
Identification, characterization, and genetic mapping of Rad51d, a new mouse and human RAD51/RecA-related gene; Pittman DL et al.; Homologous DNA recombination occurs in all organisms and is important for repair of DNA damage during mitosis . One of the critical genes for DNA repair and meiotic recombination in yeast is RAD51, and homologs of RAD51 have been identified in several species, including mouse and human . Here we describe a new RAD51-related mammalian gene, named Rad51d, identified by searching the EST database with the yeast RAD55 and human RAD51B/REC2 genes . A full-length 1.5-kb mouse cDNA clone that encodes a predicted 329-amino-acid protein was isolated . Rad51d mRNA was present in every mouse tissue examined . Four different transcript sizes were detected, one of which was specific to testis . Human cDNA clones that predicted 71% amino acid identity to the mouse protein were also isolated . Interestingly, the sequences of these human clones and of RT-PCR-derived products provided evidence for alternative splicing . These mRNAs are predicted to encode proteins that are truncated relative to the mouse and lack the ATP-binding motif characteristic of RecA-related proteins . Using an interspecific backcross mapping panel, Rad51d was mapped to mouse Chromosome 11, 48.5 cM from the centromere . By radiation hybrid mapping, the human ortholog RAD51D was mapped to chromosome 17q11, which is a region syntenic to mouse Chromosome 11 . Due to its expression pattern and sequence similarity to other RAD51 family members, it is likely that Rad51d is part of a complex of proteins required for DNA repair and meiotic recombination.

Genomics, 1998 Apr 1, 49(1), 1 - 13
Construction of a 2.5-Mb integrated physical and gene map of distal 21q22.3; Lapenta V et al.; The gene-rich telomeric region of 21q harbors several loci relevant to human diseases including autoimmune polyglandular disease type I, nonsyndromic deafness, Knobloch syndrome, holoprosencephaly, and bipolar affective disorder . A contig of genomic clones in this region would facilitate the isolation of these genes . However, distal 21q22.3 has yet been poorly mapped, presumably due to the presence of sequences that are underrepresented in yeast artificial chromosome (YAC) libraries . We generated a framework of YACs and used these clones as starting points for the isolation of a combination of bacterial artificial chromosome clones, P1-derived artificial chromosome clones, and cosmid clones by chromosome walking procedures . These studies resulted in the construction of a high-resolution contig map spanning the 2.5-Mb region from PFKL to the telomere, approximately 2 Mb of which are covered by ready-to-sequence contigs . Within this map we determined the location and relative distance of 21 markers . These include 9 established genetic markers, the order of which is cen-PFKL-D21S154-D21S170-D21S171-D21S1903- D21S1897- D21S112-D21S1446-D21S1575-tel . Moreover, we established the precise map position of 13 genes and 4 ESTs including the recently isolated genes C21ORF2, SMT3H1, RNA editing deaminase 1 (ADARB1), folate transporter (SLC19A1), COL18A1, lanosterol synthase (LSS-PEN), pericentrin (PCNT), and arginine methyltransferase (HRMT1L1) . This integrated map provides a useful resource for the mapping and isolation of disease genes and for the construction of a complete transcription map of distal 21q as well as for large-scale sequencing efforts.

Mamm Genome, 1998 May, 9(5), 355 - 60
Factors affecting ectopic gene conversion in mice; Cooper DM et al.; Duplicated genes and repetitive sequences are distributed throughout the genomes of complex organisms . The homology between related sequences can promote nonallelic (ectopic) recombination, including gene conversion and reciprocal exchange . Resolution of these events can result in translocations, deletions, or other harmful rearrangements . In yeast, ectopic recombination between sequences on nonhomologous chromosomes occurs at high frequency . Because the mammalian genome is replete with duplicated sequences and repetitive elements, high levels of ectopic exchange would cause aneuploidy and genome instability . To understand the factors regulating ectopic recombination in mice, we evaluated the effects of homology length on gene conversion between unlinked sequences in the male germline . Previously, we found high levels of gene conversion between lacZ transgenes containing 2557 bp of homology . We report here that genetic background can play a major role in ectopic recombination; frequency of gene conversion was reduced by more than an order of magnitude by transferring the transgenes from a CF1 strain background to C57BL/6J . Additionally, conversion rates decreased as the homology length decreased . Sequences sharing 1214 bp of sequence identity underwent ectopic conversion less frequently than a pair sharing 2557 bp of identity, while 624 bp was insufficient to catalyze gene conversion at significant levels . These results suggest that the germline recombination machinery in mammals has evolved in a way that prevents high levels of ectopic recombination between smaller classes of repetitive sequences, such as the Alu family . Additionally, genomic location appeared to influence the availability of sequences for ectopic recombination.

Oncogene, 1998 Mar 26, 16(12), 1579 - 86
The Ski oncoprotein interacts with Skip, the human homolog of Drosophila Bx42; Dahl R et al.; The v-Ski avian retroviral oncogene is postulated to act as a transcription factor . Since protein-protein interactions have been shown to play an important role in the transcription process, we attempted to identify Ski protein partners with the yeast two-hybrid system . Using v-Ski sequence as bait, the human gene skip (Ski Interacting Protein) was identified as encoding a protein which interacts with both the cellular and viral forms of Ski in the two-hybrid system . Skip is highly homologous to the Drosophila melanogaster protein Bx42 which is found associated with chromatin in transcriptionally active puffs of salivary glands . The Ski-Skip interaction is potentially important in Ski's transforming activity since Skip was demonstrated to interact with a highly conserved region of Ski required for transforming activity . Like Ski, Skip is expressed in multiple tissue types and is localized to the cell nucleus.

Mol Cell Biol, 1998 May, 18(5), 2431 - 43
Double-stranded RNA-independent dimerization of interferon-induced protein kinase PKR and inhibition of dimerization by the cellular P58IPK inhibitor; Tan SL et al.; The interferon (IFN)-induced, double-stranded RNA-activated protein kinase (PKR) mediates the antiviral and antiproliferative actions of IFN, in part, via its translational inhibitory properties . Previous studies have demonstrated that PKR forms dimers and that dimerization is likely to be required for activation and/or function . In the present study we used multiple approaches to examine the modulation of PKR dimerization . Deletion analysis with the lambda repressor fusion system identified a previously unrecognized site involved in PKR dimerization . This site comprised amino acids (aa) 244 to 296, which span part of the third basic region of PKR and the catalytic subdomains I and II . Using the yeast two-hybrid system and far-Western analysis, we verified the importance of this region for dimerization . Furthermore, coexpression of the 52-aa region alone inhibited the formation of full-length PKR dimers in the lambda repressor fusion and two-hybrid systems . Importantly, coexpression of aa 244 to 296 exerted a dominant-negative effect on wild-type kinase activity in a functional assay . Due to its role as a mediator of IFN-induced antiviral resistance, PKR is a target of viral and cellular inhibitors . Curiously, PKR aa 244 to 296 contain the binding site for a select group of specific inhibitors, including the cellular protein P58IPK . We demonstrated, utilizing both the yeast and lambda systems, that P58IPK, a member of the tetratricopeptide repeat protein family, can block kinase activity by preventing PKR dimerization . In contrast, a nonfunctional form of P58IPK lacking a TPR motif did not inhibit kinase activity or perturb PKR dimers . These results highlight a potential mechanism of PKR inhibition and define a novel class of PKR inhibitors . Finally, the data document the first known example of inhibition of protein kinase dimerization by a cellular protein inhibitor . On the basis of these results we propose a model for the regulation of PKR dimerization.

Mol Cell Biol, 1998 May, 18(5), 2867 - 75
Axil, a member of the Axin family, interacts with both glycogen synthase kinase 3beta and beta-catenin and inhibits axis formation of Xenopus embryos; Yamamoto H et al.; Using a yeast two-hybrid method, we identified a novel protein which interacts with glycogen synthase kinase 3beta (GSK-3beta) . This protein had 44% amino acid identity with Axin, a negative regulator of the Wnt signaling pathway.We designated this protein Axil for Axin like . Like Axin, Axil ventralized Xenopus embryos and inhibited Xwnt8-induced Xenopus axis duplication . Axil was phosphorylated by GSK-3beta . Axil bound not only to GSK-3beta but also to beta-catenin, and the GSK-3beta-binding site of Axil was distinct from the beta-catenin-binding site . Furthermore, Axil enhanced GSK-3beta-dependent phosphorylation of beta-catenin . These results indicate that Axil negatively regulates the Wnt signaling pathway by mediating GSK-3beta-dependent phosphorylation of beta-catenin, thereby inhibiting axis formation.

Mol Cell Biol, 1998 May, 18(5), 2825 - 34
The Drosophila esc and E(z) proteins are direct partners in polycomb group-mediated repression; Jones CA et al.; The extra sex combs (esc) and Enhancer of zeste {E(z)} proteins are members of the Drosophila Polycomb group (Pc-G) of transcriptional repressors . Here we present evidence for direct physical interaction between the esc and E(z) proteins using yeast two-hybrid and in vitro binding assays . In addition, coimmunoprecipitation from embryo extracts demonstrates association of esc and E(z) in vivo . We have delimited the esc-binding domain of E(z) to an N-terminal 33-amino-acid region . Furthermore, we demonstrate that site-directed mutations in the esc protein previously shown to impair esc function in vivo disrupt esc-E(z) interactions in vitro . We also show an in vitro interaction between the heed and EZH1 proteins, which are human homologs of esc and E(z), respectively . These results suggest that the esc-E(z) molecular partnership has been conserved in evolution . Previous studies suggested that esc is primarily involved in the early stages of Pc-G-mediated silencing during embryogenesis . However, E(z) is continuously required in order to maintain chromosome binding by other Pc-G proteins . In light of these earlier observations and the molecular data presented here, we discuss how esc-E(z) protein complexes may contribute to transcriptional silencing by the Pc-G.

Mol Cell Biol, 1998 May, 18(5), 2758 - 67
Human CDC6/Cdc18 associates with Orc1 and cyclin-cdk and is selectively eliminated from the nucleus at the onset of S phase; Saha P et al.; In a two-hybrid screen for proteins that interact with human PCNA, we identified and cloned a human protein (hCdc18) homologous to yeast CDC6/Cdc18 and human Orc1 . Unlike yeast, in which the rapid and total destruction of CDC6/Cdc18 protein in S phase is a central feature of DNA replication, the total level of the human protein is unchanged throughout the cell cycle . Epitope-tagged protein is nuclear in G1 and cytoplasmic in S-phase cells, suggesting that DNA replication may be regulated by either the translocation of this protein between the nucleus and the cytoplasm or the selective degradation of the protein in the nucleus . Mutation of the only nuclear localization signal of this protein does not alter its nuclear localization, implying that the protein is translocated to the nucleus through its association with other nuclear proteins . Rapid elimination of the nuclear pool of this protein after the onset of DNA replication and its association with human Orc1 protein and cyclin-cdks supports its identification as human CDC6/Cdc18 protein.

Mol Cell Biol, 1998 May, 18(5), 2748 - 57
Negative regulation of DNA replication by the retinoblastoma protein is mediated by its association with MCM7; Sterner JM et al.; A yeast two-hybrid screen was employed to identify human proteins that specifically bind the amino-terminal 400 amino acids of the retinoblastoma (Rb) protein . Two independent cDNAs resulting from this screen were found to encode the carboxy-terminal 137 amino acids of MCM7, a member of a family of proteins that comprise replication licensing factor . Full-length Rb and MCM7 form protein complexes in vitro, and the amino termini of two Rb-related proteins, p107 and p130, also bind MCM7 . Protein complexes between Rb and MCM7 were also detected in anti-Rb immunoprecipitates prepared from human cells . The amino-termini of Rb and p130 strongly inhibited DNA replication in an MCM7-dependent fashion in a Xenopus in vitro DNA replication assay system . These data provide the first evidence that Rb and Rb-related proteins can directly regulate DNA replication and that components of licensing factor are targets of the products of tumor suppressor genes.

Mol Cell Biol, 1998 May, 18(5), 2712 - 20
The Drosophila polycomb group protein Psc contacts ph and Pc through specific conserved domains; Kyba M et al.; The Polycomb group proteins are transcriptional repressors that are thought to act through multimeric nuclear complexes . We show that ph and Psc coprecipitate with Pc from nuclear extracts . We have analyzed the domains required for the association of Psc with ph and Pc by using the yeast two-hybrid system and an in vitro protein-binding assay . Psc and ph interact through regions of sequence conservation with mammalian homologs, i.e., the H1 domain of ph (amino acids 1297 to 1418) and the helix-turn-helix-containing region of Psc (amino acids 336 to 473) . Psc contacts Pc primarily at the helix-turn-helix-containing region of Psc (amino acids 336 to 473), but also at the ring finger (amino acids 250 to 335) . The Pc chromobox is not required for this interaction . We discuss the implication of these results for the nature of the complexes formed by Polycomb group proteins.

Mol Cell Biol, 1998 May, 18(5), 2608 - 16
The second catalytic domain of protein tyrosine phosphatase delta (PTP delta) binds to and inhibits the first catalytic domain of PTP sigma; Wallace MJ et al.; The LAR family protein tyrosine phosphatases (PTPs), including LAR, PTP delta, and PTP sigma, are transmembrane proteins composed of a cell adhesion molecule-like ectodomain and two cytoplasmic catalytic domains: active D1 and inactive D2 . We performed a yeast two-hybrid screen with the first catalytic domain of PTP sigma (PTP sigma-D1) as bait to identify interacting regulatory proteins . Using this screen, we identified the second catalytic domain of PTP delta (PTP delta-D2) as an interactor of PTP sigma-D1 . Both yeast two-hybrid binding assays and coprecipitation from mammalian cells revealed strong binding between PTP sigma-D1 and PTP delta-D2, an association which required the presence of the wedge sequence in PTP sigma-D1, a sequence recently shown to mediate D1-D1 homodimerization in the phosphatase RPTP alpha . This interaction was not reciprocal, as PTP delta-D1 did not bind PTP sigma-D2 . Addition of a glutathione S-transferase (GST)-PTP delta-D2 fusion protein (but not GST alone) to GST-PTP sigma-D1 led to approximately 50% inhibition of the catalytic activity of PTP sigma-D1, as determined by an in vitro phosphatase assay against p-nitrophenylphosphate . A similar inhibition of PTP sigma-D1 activity was obtained with coimmunoprecipitated PTP delta-D2 . Interestingly, the second catalytic domains of LAR (LAR-D2) and PTP sigma (PTP sigma-D2), very similar in sequence to PTP delta-D2, bound poorly to PTP sigma-D1 . PTP delta-D1 and LAR-D1 were also able to bind PTP delta-D2, but more weakly than PTP sigma-D1, with a binding hierarchy of PTP sigma-D1 >> PTP delta-D1 > LAR-D1 . These results suggest that association between PTP sigma-D1 and PTP delta-D2, possibly via receptor heterodimerization, provides a negative regulatory function and that the second catalytic domains of this and likely other receptor PTPs, which are often inactive, may function instead to regulate the activity of the first catalytic domains.

Mol Cell Biol, 1998 May, 18(5), 2444 - 54
Intrinsic transcriptional activation-inhibition domains of the polyomavirus enhancer binding protein 2/core binding factor alpha subunit revealed in the presence of the beta subunit; Kanno T et al.; A member of the polyomavirus enhancer binding protein 2/core binding factor (PEBP2/CBF) is composed of PEBP2 alphaB1/AML1 (as the alpha subunit) and a beta subunit . It plays an essential role in definitive hematopoiesis and is frequently involved in the chromosomal abnormalities associated with leukemia . In the present study, we report functionally separable modular structures in PEBP2 alphaB1 for DNA binding and for transcriptional activation . DNA binding through the Runt domain of PEBP2 alphaB1 was hindered by the adjacent carboxy-terminal region, and this inhibition was relieved by interaction with the beta subunit . Utilizing a reporter assay system in which both the alpha and beta subunits are required to achieve strong transactivation, we uncovered the presence of transcriptional activation and inhibitory domains in PEBP2 alphaB1 that were only apparent in the presence of the beta subunit . The inhibitory domain keeps the full transactivation potential of full-length PEBP2 alphaB1 below its maximum potential . Fusion of the transactivation domain of PEBP2 alphaB1 to the yeast GAL4 DNA-binding domain conferred transactivation potential, but further addition of the inhibitory domain diminished the activity . These results suggest that the activity of the alpha subunit as a transcriptional activator is regulated intramolecularly as well as by the beta subunit . PEBP2 alphaB1 and the beta subunit were targeted to the nuclear matrix via signals distinct from the nuclear localization signal . Moreover, the transactivation domain by itself was capable of associating with the nuclear matrix, which implies the existence of a relationship between transactivation and nuclear matrix attachment.

Mol Biochem Parasitol, 1998 Mar 15, 91(2), 207 - 20
Thioredoxin peroxidases from Brugia malayi; Ghosh I et al.; Parasite-derived antioxidant proteins have been implicated in playing an important role in protection against the oxygen radicals that are generated during aerobic metabolism and in defense against host immune cell attack . Here we report that filarial nematodes include the thioredoxin peroxidase/thiol-specific antioxidant (TPx/TSA) family of antioxidant proteins as part of their complex defense against radical-mediated damage . At the protein level, the TPx/TSA from Brugia malayi (Bm-TPx-1) was approximately 50% identical and approximately 60% similar to TPx/TSAs from mammals, amphibians and yeast . Bm-TPx-1 was also approximately 60% identical to putative TPx proteins from a related filarial nematode, Onchocerca volvulus, and from the free-living nematode Caenorhabditis elegans . That B . malayi may express multiple forms of molecules with TPx/TSA activity was indicated by the identification of a B . malayi gene encoding a second, distinct member of the TPx/TSA family (Bm-tpx-2) . Bm-tpx-1 was found to be transcribed in all stages of the parasite present in the mammalian host and the 25 kDa translation product was present in all of the developmental stages studied . The results of immunohistochemical, immunofluorescent and immunoprecipitation studies showed Bm-TPx-1 to be localized in the cells of the hypodermis/lateral chord in adult parasites and not to be present at the surface or in excretory/secretory products . The distribution in the parasite suggests that Bm-TPx-1 may play its major role in countering radicals produced within cells . A recombinant form of Bm-TPx-1 was biologically active and capable of protecting DNA from oxygen radical-mediated damage . Thioredoxin peroxidases may prove to be a critical component in the parasite's defense against injury caused by oxygen radicals derived from endogenous and exogenous sources.

Mem Inst Oswaldo Cruz, 1997 Nov-Dec, 92(6), 843 - 52
Towards the physical map of the Trypanosoma cruzi nuclear genome: construction of YAC and BAC libraries of the reference clone T . cruzi CL-Brener; Ferrari I et al.; Strategies to construct the physical map of the Trypanosoma cruzi nuclear genome have to capitalize on three main advantages of the parasite genome, namely (a) its small size, (b) the fact that all chromosomes can be defined, and many of them can be isolated by pulse field gel electrophoresis, and (c) the fact that simple Southern blots of electrophoretic karyotypes can be used to map sequence tagged sites and expressed sequence tags to chromosomal bands . A major drawback to cope with is the complexity of T . cruzi genetics, that hinders the construction of a comprehensive genetic map . As a first step towards physical mapping, we report the construction and partial characterization of a T . cruzi CL-Brener genomic library in yeast artificial chromosomes (YACs) that consists of 2,770 individual YACs with a mean insert size of 365 kb encompassing around 10 genomic equivalents . Two libraries in bacterial artificial chromosomes (BACs) have been constructed, BACI and BACII . Both libraries represent about three genome equivalents . A third BAC library (BAC III) is being constructed . YACs and BACs are invaluable tools for physical mapping . More generally, they have to be considered as a common resource for research in Chagas disease.

Mem Inst Oswaldo Cruz, 1997 Nov-Dec, 92(6), 829 - 34
Proceedings of the schistosome genome project; Tanaka M et al.; "The host-parasite relationship" is a vast and diverse research field which, despite huge human and financial input over many years, remains largely shrouded in mystery . Clearly, the adaptation of parasites to their different host species, and to the different environmental stresses that they represent, depends on interactions with, and responses to, various molecules of host and/or parasite origin . The schistosome genome project is a primary strategy to reach the goal; this systematic research project has successfully developed novel technologies for qualitative and quantitative characterization of schistosome genes and genome organization by extensive international collaboration between top quality laboratories . Schistosomes are a family of parasitic blood flukes (Phylum Platyhelminthes), which have seven pairs of autosomal chromosomes and one pair of sex chromosomes (ZZ for a male worm and ZW for a female), of a haploid genome size of 2.7 x 10(8) base pairs (Simpson et al . 1982) . Schistosomes are ideal model organisms for the development of genome mapping strategies since they have a small genome size comparable to that of well-characterized model organisms such as Caenorhabditis elegans (100 Mb) and Drosophila (165 Mb), and contain functional genes with a high level of homology to the host mammalian genes . Here we summarize the current progress in the schistosome genome project, the information of 3,047 transcribed genes (Expressed Sequence Tags; EST), complete sets of cDNA and genomic DNA libraries (including YAC and cosmid libraries) with a mapping technique to the well defined schistosome chromosomes . The schistosome genome project will further identify and characterize the key molecules that are responsible for host-parasite adaptation, i.e., successful growth, development, maturation and reproduction of the parasite within its host in the near future.

Int J Syst Bacteriol, 1998 Jan, 48 Pt 1, 31 - 8
Sulfur-inhibited Thermosphaera aggregans sp . nov., a new genus of hyperthermophilic archaea isolated after its prediction from environmentally derived 16S rRNA sequences; Huber R et al.; Recently, a new procedure was developed which allowed for the first time the isolation of a hyperthermophilic archaeum tracked by 165 rRNA analysis from a terrestrial hot solfataric spring ('Obsidian Pool', Yellowstone National Park, WY, USA) . This novel isolate is characterized here . Cells are round cocci with a diameter of 0.2-0.8 micron, occurring singly, in pairs, short chains and in grape-like aggregates . The aggregates exhibit a weak bluish-green fluorescence under UV radiation at 420 nm . The new isolate is an anaerobic obligate heterotroph, using preferentially yeast extract for growth . The metabolic products include CO2, H2, acetate and isovalerate . Growth is observed between 65 and 90 degrees C (optimum: 85 degrees C), from pH 5.0 to 7.0 (optimum: 6.5) and up to 0.7% NaCl . The apparent activation energy for growth is about 149 kJ mol-1 . Elemental sulfur or hydrogen inhibits growth . The core lipids consist mainly of acyclic and cyclic glycerol diphytanyl tetraethers . The cell envelope contains a cytoplasmic membrane covered by an amorphous layer of unknown composition; there is no evidence for a regularly arrayed surface-layer protein . The G + C content is 46 mol% . On the basis of 165 rRNA sequence comparisons in combination with morphological, physiological and biochemical properties, the isolate represents a new genus within the Desulfurococcaceae, which has been named Thermosphaera . The type species is Thermosphaera aggregans, the type strain is isolate M11TLT (= DSM 11486T).

EMBO J, 1998 Mar 16, 17(6), 1728 - 39
Cooperative interaction of ets-1 with USF-1 required for HIV-1 enhancer activity in T cells; Sieweke MH et al.; The distal enhancer region of the human immunodeficiency virus 1 (HIV-1) long terminal repeat (LTR) is known to be essential for HIV replication and to contain immediately adjacent E-box and Ets binding sites . Based on a yeast one-hybrid screen we have identified the E-box binding protein USF-1 as a direct interaction partner of Ets-1 and found that the complex acts on this enhancer element . The binding surfaces of USF-1 and Ets-1 map to their DNA-binding domains and although these domains are highly conserved, the interaction is very selective within the respective protein family . USF-1 and Ets-1 synergize in specific DNA binding as well as in the transactivation of reporter constructs containing the enhancer element, and mutations of the individual binding sites dramatically reduce reporter activity in T cells . In addition, a dominant negative Ets-1 mutant inhibits both USF-1-mediated transactivation and the activity of the HIV-1 LTR in T cells . The inhibition is independent of Ets DNA-binding sites but requires the Ets binding surface on USF-1, highlighting the importance of the direct protein-protein interaction . Together these results indicate that the interaction between Ets-1 and USF-1 is required for full transcriptional activity of the HIV-1 LTR in T cells.

Mol Gen Genet, 1998 Mar, 257(5), 568 - 75
Isolation of lambda and YAC clones from defined regions of the rye genome; Langridge P et al.; A dispersed, rye-specific element has been used to isolate clones of rye origin from wheat plants containing only a single rye chromosome arm or segment . In this way a set of 23 YAC clones has been isolated from the short arm of rye chromosome 1 (1RS) . This technique was extended to isolate clones from a small region of 1RS that contains a large number of agronomically important genes . The targeted cloning method allowed the isolation of 26 classes of lambda clones representing about 5% of the region . Ten of the lambda clones could be mapped to segments within this region . A third example of the application of this technique involved the isolation of clones from a very small but fully functional rye chromosome, the midget chromosome . These clones have allowed the confirmation of the origin of the midget from 1RL, and may provide a tool for the isolation of structural elements of cereal chromosomes . This technique allows the identification of clone libraries for any rye chromosome or chromosome arm, since substitution, addition and translocation lines are available for all rye chromosomes . Furthermore, the technique allows isolation of clones derived from segments of the rye genome recombined into wheat . The method is technically simple and both lambda and YAC libraries can be constructed . Synteny between the genomes of the cereals allows region-specific libraries from rye to be used to target regions of the wheat and barley genomes.

Biol Chem, 1998 Mar, 379(3), 275 - 82
The role of molecular chaperones in protein transport into the mammalian endoplasmic reticulum; Zimmermann R; Molecular chaperones belonging to the Hsp70 protein family play various roles in posttranslational protein transport into the yeast endoplasmic reticulum (ER): (i) Cytosolic Hsp70 (Hsc70) together with the J-domain containing Hsp40 preserves the transport competent state of presecretory proteins; (ii) ER-lumenal Hsp70 (Kar2p or Lhs1p) triggers the initial insertion of precursor proteins into the core of the protein translocase in the ER-membrane and cooperates with a J-domain containing membrane protein (Sec63p); (iii) the ER-lumenal Hsp70 and its membrane receptor also are involved in completion of translocation . Here a working model is presented for the putative roles of molecular chaperones in post- as well as cotranslational protein transport into the mammalian ER.

FEBS Lett, 1998 Apr 3, 425(3), 431 - 5
hUBC9 associates with MEKK1 and type I TNF-alpha receptor and stimulates NFkappaB activity; Saltzman A et al.; hUBC9, an E2 ubiquitin conjugating enzyme, was identified by yeast two-hybrid screening and coprecipitation studies to interact with MEKK1 and the type I TNF-alpha receptor, respectively . Because both of these proteins regulate NFkappaB activity, the role of hUBC9 in modulating NFkappaB activity was investigated . Overexpression of hUBC9 in HeLa cells stimulated the activity of NFkappaB as determined by NFkappaB reporter and IL-6 secretion assays . hUBC9 also synergized with MEKK1 to activate NFkappaB reporter activity . Thus, hUBC9 modulates NFkappaB activity which, at least in part, can be attributed to its interaction with MEKK1 and the type I TNF-alpha receptor.

FEBS Lett, 1998 Apr 3, 425(3), 401 - 6
Polypyrimidine tract-binding protein interacts with HnRNP L; Hahm B et al.; Polypyrimidine tract-binding protein (PTB) is involved in pre-mRNA splicing and internal ribosomal entry site (IRES)-dependent translation . In order to identify cellular protein(s) interacting with PTB, we performed a yeast two-hybrid screening . Heterogeneous nuclear ribonucleoprotein L (hnRNP L) was identified as a PTB-binding protein . The interaction between PTB and hnRNP L was confirmed in an in vitro binding assay . Both PTB and hnRNP L were found to localize in the nucleoplasm, excepting the nucleoli, in HeLa cells by the green fluorescent protein (GFP)-fused protein detection method . The N-terminal half of PTB (aa 1-329) and most of hnRNP L (aa 141-558) is required for the interaction between PTB and hnRNP L.

Virus Genes, 1998, 16(1), 119 - 31
Origin, adaptation and evolutionary pathways of fungal viruses; Ghabrial SA; Fungal viruses or mycoviruses are widespread in fungi and are believed to be of ancient origin . They have evolved in concert with their hosts and are usually associated with symptomless infections . Mycoviruses are transmitted intracellularly during cell division, sporogenesis and cell fusion, and they lack an extracellular phase to their life cycles . Their natural host ranges are limited to individuals within the same or closely related vegetative compatibility groups . Typically, fungal viruses are isometric particles 25-50 nm in diameter, and possess dsRNA genomes . The best characterized of these belong to the family Totiviridae whose members have simple undivided dsRNA genomes comprised of a coat protein (CP) gene and an RNA dependent RNA polymerase (RDRP) gene . A recently characterized totivirus infecting a filamentous fungus was found to be more closely related to protozoan totiviruses than to yeast totiviruses suggesting these viruses existed prior to the divergence of fungi and protozoa . Although the dsRNA viruses at large are polyphyletic, based on RDRP sequence comparisons, the totiviruses are monophyletic . The theory of a cellular self-replicating mRNA as the origin of totiviruses is attractive because of their apparent ancient origin, the close relationships among their RDRPs, genome simplicity and the ability to use host proteins efficiently . Mycoviruses with bipartite genomes (partitiviruses), like the totiviruses, have simple genomes, but the CP and RDRP genes are on separate dsRNA segments . Because of RDRP sequence similarity, the partitiviruses are probably derived from a totivirus ancestor . The mycoviruses with unencapsidated dsRNA-like genomes (hypoviruses) and those with bacilliform (+) strand RNA genomes (barnaviruses) have more complex genomes and appear to have common ancestry with plant (+) strand RNA viruses in supergroup 1 with potyvirus and sobemovirus lineages, respectively . The La France isometric virus (LIV), an unclassified virus with multipartite dsRNA genome, is associated with a severe die-back disease of the cultivated mushroom . LIV appears to be of recent origin since it differs from its host in codon usage.

Mol Cell Biochem, 1998 Apr, 181(1-2), 97 - 106
Characterization of multiple transcripts of the hamster dolichol-P-dependent N-acetylglucosamine-1-P transferase suggests functionally complex expression; Huang GT et al.; The evolutionarily conserved dolichol-P-dependent N-acetylglucosamine-1-P transferase gene, ALG7, functions by initiating the dolichol pathway of protein N-glycosylation . In yeast, ALG7 has a complex expression pattern and plays a critical role in diverse cellular functions, including proliferation and morphological response . In Chinese hamster ovary cells (CHO), ALG7 gives rise to three mRNAs of 1.5, 1.9 and 2.2 kb . We report results of RNA blotting assays, ribonuclease protection, PCR-amplification and sequencing of the CHO ALG7 transcripts 5' and 3' ends which suggest that the 1.5 and 1.9 kb transcripts are produced as a consequence of initiation at 2 distinct start sites, 350-379 bp apart . The transcriptional start site for the 1.5 kb mRNA is positioned between the first two in frame ATGs, while that of the 1.9 kb species is located upstream of these two in-frame ATGs . In order to test the translational competence of the 1.5 and 1.9 kb mRNAs, we constructed DNA templates specifying these transcripts and used them for in vitro transcription/translation . Our data show that the 1.9 kb mRNA served in the synthesis of 36 and 24 kDa species, as well as a low-abundance 32 kDa protein . The 1.5 kb transcript gave rise to a translation product of 32 kDa . The latter is synthesized in CHO cells and hamster submandibular glands . These results suggest the possibility that the 1.5 and 1.9 kb transcripts give rise to related protein isoforms with different lengths of their NH2-terminal regions.

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