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 essen