Cell Motil Cytoskeleton, 1997, 38(4), 373 - 84 Relationship of actin, microtubules, and crosswall synthesis during septation in Aspergillus nidulans; Momany M et al.; Studies of cytokinesis in animal cells demonstrate that microtubules play an important role in signaling the position of the actin-containing contractile ring and subsequent formation of the cleavage furrow . Septation in several fungi closely resembles animal cell cytokinesis in that a circumferential ring of actin is visible at the incipient division site . However, this does not necessarily mean that division is contractile since actin may also serve to localize septal wall synthesis . In addition, several studies in fission yeast have suggested that microtubules are dispensable for actin ring formation . We have used synchronized cells and fluorescence microscopy to follow actin structures, nuclear division and septal wall synthesis during septation in Aspergillus nidulans . Our data suggest that actin first appears at the septum site as a circumferential ring and that it later broadens and invaginates, forming an hourglass-shaped structure coincident with septal cell wall synthesis . Depolymerization of microtubules early in septation prevents circumferential actin ring formation . Depolymerization of microtubules after circumferential actin ring formation blocks both the progression to invaginating bands and septal wall synthesis . In contrast to studies in yeast cells, our data suggest that microtubules are required for both the initiation and progression of septation in A . nidulans. Eur Cytokine Netw, 1997 Dec, 8(4), 359 - 65 Interleukin-6 receptor-interleukin-6 fusion proteins with enhanced interleukin-6 type pleiotropic activities; Chebath J et al.; An sIL-6R/IL-6 chimera, directly fusing the natural forms of soluble IL-6 receptor and IL-6, as found in human body fluids, was produced in transfected human cells . The secreted p85 glycoprotein was active at a concentration of 120 pM to produce growth-arrest and spindleoid differentiation of murine melanoma F10.9 cells, which do not respond to IL-6 alone . This fusion protein was as active as the yeast-produced p56 fusion protein containing a shortened sIL-6R, linked through a flexible peptide chain to IL-6 (Hyper IL-6) . The concentration of Hyper IL-6 needed to arrest the growth of F10.9 cells was much lower than that needed of a combination of IL-6 and sIL-6R, added separately . Hyper IL-6 was also more active than IL-6 in stimulating growth of murine plasmacytoma T1165 cells, the half maximal stimulation being obtained at 2 pM Hyper IL-6 versus 23 pM for IL-6 . In order to evaluate the effect of the fused sIL-6R/IL-6 proteins on human hematopoietic primitive progenitor cells, they were added to suspension cultures of CD34+ cells from human cord blood in addition to both flt3/flk2 ligand (FL) and stem cell factor (SCF) . Fused sIL-6R/IL-6 produced a marked stimulation of cell expansion and a marked increase in the number of colony forming units when subsequently plated in semi-solid medium with IL-3, GM-CSF, SCF and erythropoietin . Ex-vivo maintenance and expansion of early progenitor cells in bone marrow transplantation protocols may be a potential application for the sIL-6R/IL-6 chimeric glycoproteins. Biochim Biophys Acta, 1998 Jan 2, 1401(1), 93 - 103 Evolutionary analysis of G-proteins in early metazoans: cloning of alpha- and beta-subunits from the sponge Geodia cydonium; Seack J et al.; G-protein-coupled (seven-transmembrane segment)-receptors represent a major group of metazoan receptors, involved in transduction of extracellular signals . The G-proteins, which are made up of Galpha/beta/gamma-subunits, link the receptors to the effector system(s) . To analyze the phylogenetic relationships among the metazoan alpha-subunits of G-proteins, cDNAs of alpha-subunits were isolated from Geodia cydonium, a marine sponge belonging to the lowest metazoan phylum, Porifera . One encodes a putative isotype of a stimulator of the adenylyl cyclase (Galpha s), another one a putative inhibitor of the adenylyl cyclase (Galpha i/o) and the third one a putative activator of phospholipase C (Galpha q) . In addition one putative beta-subunit was cloned from the same species . The deduced amino acid sequences of the sponge Galpha s -(putative Mr 44749), the Galpha i/o -(Mr 41064) and the Galpha q subunits (Mr 41363) were found to display high similarity with the corresponding sequences from higher Metazoa, and are only distantly related to those of slime mold, yeast or plants . Of lower similarity are the sequences of the beta-subunits among animals and plants, thus not allowing robust grouping . These data demonstrate that the phylogenetic relationships, obtained from analyses of the alpha subunits from metazoan G-proteins, support the conclusion that all metazoan phyla, including the Porifera are of monophyletic origin. Biochim Biophys Acta, 1998 Jan 2, 1401(1), 21 - 37 YAC transgenesis: a study of conditions to protect YAC DNA from breakage and a protocol for transfection; Bauchwitz R et al.; Yeast artificial chromosomes (YACs) are providing a great boon to transgene technology by allowing the easy mutagenesis and study of very large DNAs . The large insert sizes of these vectors permit more accurate analysis of the regulation of transgene expression than smaller, more artificially assembled constructs . Transfection of mammalian cells by YACs can be accomplished by a number of methods; the most prevalent, using gel-purified DNA, is dependent upon compaction by salts to protect the large YAC DNA from breakage . We show that the common reliance on NaCl to compact YAC DNA sufficiently to protect it from breakage is not well-founded . Even the use of mixtures of polyamines and NaCl allows substantial damage to purified YACs . The use of polyamines alone in low salt buffers to compact YAC DNA provides the best protection from breakage and allows very effective transfection of murine embryonic stem (ES) cells . We provide a detailed method for ES cell transfection by YACs utilizing the DOTAP lipofection reagent that optimizes transfection efficiency and recovery of intact YACs. J Infect, 1997 Nov, 35(3), 231 - 5 A pilot phase II study of the safety and immunogenicity of HIV p17/p24:VLP (p24-VLP) in asymptomatic HIV seropositive subjects; Peters BS et al.; The aim of this phase II study was to evaluate the safety, immunogenicity and tolerability of the yeast-derived virus-like particle immunogen, Ty.p24.VLP (p24-VLP), in HIV-antibody-positive asymptomatic volunteers . Fifteen informed and consented volunteers, with p24 Antibody titres >1/100, p24 Antigen <20 pg/l, and CD4>350 x 10(9)/l were enrolled . Five were immunized with aluminium hydroxide placebo, five with 25 microg, and five with 100 microg p24-VLP in Alum adjuvant at weeks 0 and 4 by the intramuscular route . Patients were followed for 16 weeks post vaccination and the main outcome assessments were CD4 and CD8 lymphocyte counts, p24 antigen and antibody, Ty antibody and quantitative viral cultures . No serious adverse events were observed in any of the groups . There were increases in CD4 counts in the treated groups but not in the controls, although these changes were not statistically significant . There were no significant intrasubject or intergroup changes in the other parameters, such as p24 antigen and antibody . No pattern of change in plasma viraemia was detected, and most cultures were negative . Therefore we conclude that p24-VLP immunizations of 25 microg and 100 microg are well tolerated, and the CD4 changes are encouraging, but higher doses and larger numbers are required to see if there are significant humoral or cellular responses, and extended phase II studies are now in progress. Support Care Cancer, 1998 Jan, 6(1), 31 - 8 Progress in fighting systemic fungal infections in haematological neoplasia; De Pauw BE et al.; Considering the limited data available, there is clearly a need for thorough, well-designed clinical research on the epidemiology, diagnosis, treatment and prevention of invasive fungal infection in patients who are treated for cancer . Our knowledge has increased, but the information obtained so far is patchy and not generally applicable, as it is influenced by local problems and circumstances . New diagnostic tools have become available, but they are still insufficient in many cases . Until the value of the presently available chemoprophylaxis has been established beyond doubt, the strategy should be one of wait-and-see for patients with a low or moderate risk of developing infection . In bone marrow transplant recipients fluconazole has shown favourable results in eliminating yeast infections, but in patients at high risk of mould infections early initiation of intravenous treatment with amphotericin B at a therapeutic dose remains the best approach . The question of the optimal time point to start empirical antifungal treatment remains and has even been extended by the dispute about what antifungal drugs should be used for this purpose . Amphotericin B is still the drug of choice for the treatment of disseminated fungal infection, but its lipid formulations seem to offer a safer, though far more expensive, alternative . Head-to-head comparisons between the different formulations are required before a final conclusion on their respective efficacies and toxicities can be drawn, and it is questionable whether a higher dose will produce better results . Fluconazole appears very useful against the majority of Candida infections, whereas itraconazole is effective against both yeast and moulds, providing that adequate resorption can be ensured . The results of the first clinical trial of voriconazole in pulmonary aspergillosis have proved very promising. J Exp Med, 1997 Dec 15, 186(12), 1975 - 83 The E1B 19K/Bcl-2-binding protein Nip3 is a dimeric mitochondrial protein that activates apoptosis; Chen G et al.; Nip3 (nineteen kD interacting protein-3) is an E1B 19K and Bcl-2 binding protein of unknown function . Nip3 is detected as both a 60- and 30-kD protein in vivo and in vitro and exhibits strong homologous interaction in a yeast two-hybrid system indicating that it can homodimerize . Nip3 is expressed in mitochondria and a mutant (Nip3(163)) lacking the putative transmembrane domain and COOH terminus does not dimerize or localize to mitochondria . Transient transfection of epitope-tagged Nip3 in Rat-1 fibroblasts and MCF-7 breast carcinoma induces apoptosis within 12 h while cells transfected with the Nip3(163) mutant have a normal phenotype, suggesting that mitochondrial localization is necessary for induction of cell death . Nip3 overexpression increases the sensitivity to apoptosis induced by granzyme B and topoisomerase I and II inhibitors . After transfection, both Nip3 and Nip3(163) protein levels decrease steadily over 48 h indicating that the protein is rapidly degraded and this occurs in the absence of cell death . Bcl-2 overexpression initially delays the onset of apoptosis induced by Nip3 but the resistance is completely overcome in longer periods of incubation . Nip3 protein levels are much higher and persist longer in Bcl-2 expressing cells . In conclusion, Nip3 is an apoptosis-inducing dimeric mitochondrial protein that can overcome Bcl-2 suppression. J Biol Chem, 1997 Dec 12, 272(50), 31738 - 46 Cig30, a mouse member of a novel membrane protein gene family, is involved in the recruitment of brown adipose tissue; Tvrdik P et al.; We have identified a previously uncharacterized gene that is implicated in the thermogenic function of brown adipose tissue of mice . This gene, termed Cig30, is the first mammalian member of a novel gene family comprising several nematode and yeast genes, such as SUR4 and FEN1, mutation of which is associated with highly pleiotropic phenotypes . It codes for a 30-kDa plasma membrane glycoprotein with five putative transmembrane domains . The Cig30 mRNA was readily detected only in brown fat and liver . When animals were exposed to a 3-day cold stress, the Cig30 expression was selectively elevated in brown fat more than 200-fold . Similar increases were brought about in two other conditions of brown fat recruitment, namely during perinatal development and after cafeteria diet . The magnitude of Cig30 mRNA induction in the cold could be mimicked by chronic norepinephrine treatment in vivo . However, in primary cultures of brown adipocytes, a synergistic action of norepinephrine and dexamethasone was required for full expression of the gene, indicating that both catecholamines and glucocorticoids are required for the induction of Cig30 . We propose that the CIG30 protein is involved in a pathway connected with brown fat hyperplasia. J Biol Chem, 1997 Dec 12, 272(50), 31636 - 40 Hsp110 protects heat-denatured proteins and confers cellular thermoresistance; Oh HJ et al.; The 110-kDa heat shock protein (hsp110) has long been recognized as one of the primary heat shock proteins in mammalian cells . It belongs to a recently described protein family that is a significantly diverged subgroup of the hsp70 family and has been found in organisms as diverse as yeast and mammals . We describe here the first analysis of the ability of hsp110 to protect cellular and molecular targets from heat damage . It was observed that the overexpression in vivo of hsp110 conferred substantial heat resistance to both Rat-1 and HeLa cells . In vitro heat denaturation and refolding assays demonstrate that hsp110 is highly efficient in selectively recognizing denatured proteins and maintaining them in a soluble, folding-competent state and is significantly more efficient in performing this function than is hsc70 . hsp110-bound proteins can then be refolded by the addition of rabbit reticulocyte lysate or hsc70 and Hdj-1, whereas Hdj-1 does not itself function as a co-chaperone in folding with hsp110 . hsp110 is one of the principal molecular chaperones of mammalian cells and represents a newly identified component of the primary protection/repair pathway for denatured proteins and thermotolerance expression in vivo. J Biol Chem, 1997 Dec 12, 272(50), 31420 - 6 The molecular chaperone function of the secretory vesicle cysteine string proteins; Chamberlain LH et al.; The "J" domains of eukaryotic DnaJ-like proteins specify interaction with various Hsp70s . The conserved tripeptide, HPD, present in all J domains has been shown to be important for the interaction between yeast and bacterial DnaJ/Hsp70 protein pairs . We have characterized mutations in the HPD motif of the synaptic vesicle protein cysteine-string protein (Csp) . Mutation of the histidine (H43Q) or aspartic acid (D45A) residues of this motif reduced the ability of Csp to stimulate the ATPase activity of mammalian Hsc70 . The H43Q and D45A mutant proteins were not able to stimulate the ATPase activity of Hsc70 to any significant extent . The mutant proteins were characterized by competition assays, tryptic digestion analysis, and direct binding analysis from which it was seen that these proteins were defective in binding to Hsc70 . Thus, the HPD motif of Csp is required for binding to Hsc70 . We also analyzed the interaction between Csp and a model substrate protein, denatured firefly luciferase . Both Csp1 and the C-terminally truncated isoform Csp2 were able to prevent aggregation of heat-denatured luciferase, and they also cooperated with Hsc70 to prevent aggregation . In addition, complexes of Csp1 or Csp2 with Hsc70 and luciferase were isolated, confirming that these proteins interact and that Csps can bind directly to denatured proteins . Csp1 and Csp2 isoforms must differ in some aspect other than interaction with Hsc70 and substrate protein . These results show that both Csp1 and Csp2 can bind a partially unfolded protein and act as chaperones . This suggests that Csps may have a general chaperone function in regulated exocytosis. J Biol Chem, 1997 Dec 12, 272(50), 31230 - 4 An Eps homology (EH) domain protein that binds to the Ral-GTPase target, RalBP1; Yamaguchi A et al.; Ral proteins constitute a family of small GTPases that can be activated by Ras in cells . In the GTP-bound state, Ral proteins bind to RalBP1, a GTPase-activating protein for CDC42 and Rac GTPases . We have used the two-hybrid system in yeast to clone a cDNA for a novel approximately 85-kDa protein that can bind to an additional site on RalBP1 . This newly identified protein contains an Eps homology (EH) domain, which was first detected in the epidermal growth factor (EGF) receptor substrate Eps15 . Recently, the EH domain of Eps15 has been shown to bind to proteins containing an asparagine-proline-phenylalanine motif . Moreover, EH domains have been found in proteins involved in endocytosis and/or actin cytoskeleton regulation . The RalBP1 associated Eps-homology domain protein, Reps1, is tyrosine-phosphorylated in response to EGF stimulation of cells . In addition, Reps1 has the capacity to form a complex with the SH3 domains of the adapter proteins Crk and Grb2, which may link Reps1 to an EGF-responsive tyrosine kinase . Thus, Reps1 may coordinate the cellular actions of activated EGF receptors and Ral-GTPases. Proc Natl Acad Sci U S A, 1997 Dec 9, 94(25), 13738 - 42 Evolutionary specialization of the nuclear targeting apparatus; Malik HS et al.; The alpha- and beta-karyopherins (Kaps), also called importins, mediate the nuclear transport of proteins . All alpha-Kaps contain a central domain composed of eight approximately 40 amino acid, tandemly arranged, armadillo-like (Arm) repeats . The number and order of these repeats have not changed since the common origin of fungi, plants, and mammals . Phylogenetic analysis suggests that the various alpha-Kaps fall into two groups, alpha1 and alpha2 . Whereas animals encode both types, the yeast genome encodes only an alpha1-Kap . The beta-Kaps are characterized by 14-15 tandemly arranged HEAT motifs . We show that the Arm repeats of alpha-Kaps and the HEAT motifs of beta-Kaps are similar, suggesting that the alpha-Kaps and beta-Kaps (and for that matter, all Arm and HEAT repeat-containing proteins) are members of the same protein superfamily . Phylogenetic analysis indicates that there are at least three major groups of beta-Kaps, consistent with their proposed cargo specificities . We present a model in which an alpha-independent beta-Kap progenitor gave rise to the alpha-dependent beta-Kaps and the alpha-Kaps. Proc Natl Acad Sci U S A, 1997 Dec 9, 94(25), 13624 - 9 The glyoxysomal and plastid molecular chaperones (70-kDa heat shock protein) of watermelon cotyledons are encoded by a single gene; Wimmer B et al.; The monoclonal a-70-kDa heat shock protein (hsp70) antibody recognizes in crude extracts from watermelon (Citrullus vulgaris) cotyledons two hsps with molecular masses of 70 and 72 kDa . Immunocytochemistry on watermelon cotyledon tissue and on isolated glyoxysomes identified hsp70s in the matrix of glyoxysomes and plastids . Affinity purification and partial amino acid determination revealed the 70-kDa protein to share high sequence identity with cytosolic hsp70s from a number of plant species, while the 72 kDa protein was very similar to plastid hsp70s from pea and cucumber . A full-length cDNA clone encoding the 72-kDa hsp70 was isolated and identified two start methionines in frame within the N-terminal presequence leading either to an N-terminal extension of 67 amino acids or to a shorter one of 47 amino acids . The longer presequence was necessary and sufficient to target a reporter protein into watermelon proplastids in vitro . The shorter extension starting from the second methionine within the long version harbored a consensus peroxisomal targeting signal (RT-X5-KL) that directed in vivo a reporter protein into peroxisomes of the yeast Hansenula polymorpha . Peroxisomal targeting was however prevented, when the 67-residue presequence was fused to the reporter protein, indicating that the peroxisomal targeting signal 2 information is hidden in this context . We propose that the 72-kDa hsp70 is encoded by a single gene, but targeted alternatively into two organelles by the modulated use of its presequence. Proc Natl Acad Sci U S A, 1997 Dec 9, 94(25), 13504 - 9 Glucocorticoid-mediated repression of nuclear factor-kappaB-dependent transcription involves direct interference with transactivation; De Bosscher K et al.; Glucocorticoids exert multiple anti-inflammatory activities, one of which is the inhibition of transcription dependent on the nuclear factor (NF)-kappaB . It has been suggested that the effect of dexamethasone (DEX), a glucocorticoid analog, is attributed to an increased production of the inhibitory IkappaB molecule, which in turn would bind and remove activated, DNA-bound NF-kappaB complexes in the cell nucleus . Upon investigating DEX-mediated repression of interleukin-6 expression induced by tumor necrosis factor, DEX treatment was found to act directly on NF-kappaB-dependent transcription, without changing the expression level of IkappaB . Neither the mRNA of IkappaB nor the protein was significantly elevated by a combined treatment with tumor necrosis factor and DEX of murine endothelial or fibroblast cells . The DNA-binding activity of induced NF-kappaB also remained unchanged after stimulation of cells with DEX . Evidence for a direct nuclear mechanism of action was obtained by analysis of cell lines stably expressing a fusion protein between the DNA-binding domain of the yeast Gal4 protein and the transactivating p65 subunit of NF-kappaB . Expression of a Gal4-dependent luciferase reporter gene activated by this nuclear fusion protein was also strongly repressed after addition of DEX . Because the DNA-binding activity of the Gal4 fusion protein was not affected by DEX, it can be concluded that the reduction of gene activation was caused by interference of the activated glucocorticoid receptor with the transactivation potential of the NF-kappaB p65 subunit. Chem Biol, 1995 Jul, 2(7), 471 - 81 Structure-activity studies of rapamycin analogs: evidence that the C-7 methoxy group is part of the effector domain and positioned at the FKBP12-FRAP interface; Luengo JI et al.; BACKGROUND: Rapamycin is an immunosuppressant natural product, which blocks T-cell mitogenesis and yeast proliferation . In the cytoplasm, rapamycin binds to the immunophilin FKBP12 and the complex of these two molecules binds to a recently discovered protein, FRAP . The rapamycin molecule has two functional domains, defined by their interaction with FKBP12 (binding domain) or with FRAP (effector domain) . We previously showed that the allylic methoxy group at C-7 of rapamycin could be replaced by a variety of different substituents . We set out to examine the effects of such substitutions on FKBP12 binding and on biological activity . RESULTS: Rapamycin C-7-modified analogs of both R and S configurations were shown to have high affinities for FKBP12, yet these congeners displayed a wide range of potencies in splenocyte and yeast proliferation assays . The X-ray crystal structures of four rapamycin analogs in complexes with FKBP12 were determined and revealed that protein and ligand backbone conformations were essentially the same as those observed for the parent rapamycin-FKBP12 complex and that the C-7 group remained exposed to solvent . We then prepared a rapamycin analog with a photoreactive functionality as part of the C-7 substituent . This compound specifically labeled, in an FKBP12-dependent manner, a protein of approximately 250 kDa, which comigrates with recombinant FRAP . CONCLUSIONS: We conclude that the C-7 methoxy group of rapamycin is part of the effector domain . In the ternary complex, this group is situated in close proximity to FRAP, at the interface between FRAP and FKBP12. Plant J, 1997 Oct, 12(4), 921 - 30 Developmental and environmental regulation of tissue- and cell-specific expression for a pea protein farnesyltransferase gene in transgenic plants; Zhou D et al.; Farnesylation mediates membrane targeting and in vivo activities of several key regulatory proteins such as Ras and Ras-related GTPases and protein kinases in yeast and mammals, and is implicated in cell cycle control and abscisic acid (ABA) signaling in plants . In this study, the developmental expression of a pea protein farnesyltransferase (FTase) gene was examined using transgenic expression of the beta-glucuronidase (GUS) gene fused to a 3.2 kb 5' upstream sequence of the gene encoding the pea FTase beta subunit . Coordinate expression of the GUS transgene and endogenous tobacco FTase beta subunit gene in tobacco cell lines suggests that the 3.2 kb region contains the key FTase promoter elements . In transgenic tobacco plants, GUS expression is most prominent in meristematic tissues such as root tips, lateral root primordia and the shoot apex, supporting a role for FTase in the control of the cell cycle in plants . GUS activity was also detected in mature embryos and imbibed embryos, in accordance with a role for FTase in ABA signaling that modulates seed dormancy and germination . In addition, GUS activity was detected in regions that border two organs, e.g . junctions between stems and leaf petioles, cotyledons and hypocotyls, roots and hypocotyls, and primary and secondary roots . GUS is expressed in phloem complexes that are adjacent to actively growing tissues such as young leaves, roots of light-grown seedlings, and hypocotyls of dark-grown seedlings . Both light and sugar (e.g . sucrose) treatments repressed GUS expression in dark-grown seedlings . These expression patterns suggest a potential involvement of FTase in the regulation of nutrient allocation into actively growing tissues. Plant J, 1997 Oct, 12(4), 875 - 84 Regulation of expression of a cDNA from barley roots encoding a high affinity sulphate transporter; Smith FW et al.; A cDNA encoding a high-affinity sulphate transporter has been isolated from barley by complementation of a yeast mutant . The cDNA, designated HVST1, encodes a polypeptide of 660 amino acids (M(r) = 72,550), which is predicted to have 12 membrane-spanning domains and has extensive sequence homology with other identified eukaryotic sulphate transporters . The K(m) for sulphate was 6.9 microM when the HVST1 cDNA was expressed in a yeast mutant deficient in the gene encoding for the yeast SUL1 sulphate transporter . The strong pH-dependency of sulphate uptake when HVST1 was expressed heterologously in yeast suggests that the HVST1 polypeptide is a proton/sulphate co-transporter . The gene encoding HVST1 is expressed specifically in root tissues and the abundance of the mRNA is strongly influenced by sulphur nutrition . During sulphur-starvation of barley, the abundance of mRNA corresponding to HVST1, and the capacity of the roots to take up sulphate, both increase . Upon re-supply of sulphate, the abundance of the mRNA corresponding to HVST1, and the capacity of the roots to take up sulphate, decrease rapidly, concomitant with rises in tissue sulphate, cysteine and glutathione contents . Addition of the cysteine precursor, O-acetylserine, to plants grown with adequate sulphur supply, leads to increases in sulphate transporter mRNA, sulphate uptake rates and tissue contents of glutathione and cysteine . It is suggested, that whilst sulphate, cysteine and glutathione may be candidates for negative metabolic regulators of sulphate transporter gene expression, this regulation may be overridden by O-acetylserine acting as a positive regulator. J Mol Biol, 1997 Nov 7, 273(4), 814 - 25 Possible mechanisms for the regulation of telomere length; Kowald A; Since DNA polymerases can only synthesise a new DNA strand in the 5'-3' direction and need a primer that provides a free 3' OH end, the cellular replication machinery is unable to duplicate the 3' ends of linear chromosomes unless special mechanisms are operative . While the telomeres seem to shorten continuously in human somatic cells because of the "end replication" problem, it appears that telomere length is maintained in cancer cells, the germ line and unicellular organisms like yeast and Tetrahymena by a mechanism involving the enzyme telomerase, which elongates the 3' ends of telomeres . However, telomerase must be part of a more complicated mechanism to ensure that there is no net gain or loss of telomeric ends . Here we describe a simple theoretical model that can explain several experimental findings . The simulations show that (i) the proposed mechanism is able to maintain telomeres at a constant length, (ii) this length constancy is independent of the initial telomere length, (iii) mutations of the telomeric sequence lead to an elongation of telomeres, (iv) inhibition of telomerase causes telomeric shortening, and (v) it reproduces and explains the experimental result that the addition of oligonucleotides to the culture medium leads to an increase of telomere length. Genomics, 1997 Nov 1, 45(3), 479 - 86 High-resolution physical map of the immunoglobulin lambda variant gene cluster assembled by quantitative DNA fiber mapping; Duell T et al.; Quantitative DNA fiber mapping (QDFM) allows rapid construction of near-kilobase-resolution physical maps by hybridizing specific probes to individual stretched DNA molecules . We evaluated the utility of QDFM for the large-scale physical mapping of a rather unstable, repeat-rich 850-kb region encompassing the immunoglobulin lambda variant (IGLV) gene segments . We mapped a minimal tiling path composed of 32 cosmid clones to three partially overlapping yeast artificial chromosome (YAC) clones and determined the physical size of each clone, the extent of overlap between clones, and contig orientation, as well as the sizes of gaps between adjacent contigs . Regions of germline DNA for which we had no YAC coverage were characterized by cosmid to cosmid hybridizations . Compared to other methods commonly used for physical map assembly, QDFM is a rapid, versatile technique delivering unambiguous data necessary for map closure and preparation of sequence-ready minimal tiling paths . Biochim Biophys Acta, 1997 Sep 12, 1353(3), 224 - 30 Molecular cloning of a novel alternatively spliced nuclear protein; Hartmann AM et al.; Using the yeast two hybrid system, we isolated a rat cDNA (E3-3) coding for a new protein with no homology to any other protein in the database . E3-3 is ubiquitously expressed . Variants that most likely arise through alternative splicing encode truncated forms of the protein . Testis is the only tissue that predominantly expresses the longest protein variant . When this variant is tagged with enhanced green fluorescent protein, the protein is located in the nucleus. Biochim Biophys Acta, 1997 Sep 12, 1353(3), 199 - 202 An Arabidopsis gene encoding a putative 14-3-3-interacting protein, caffeic acid/5-hydroxyferulic acid O-methyltransferase; Zhang H et al.; AFT1, a 14-3-3 protein from Arabidopsis thaliana, was used as a 'bait' in the two-hybrid system to identify its interacting proteins . A caffeic acid/5-hydroxyferulic acid O-methyltransferase, OMT1, was identified as one of the several proteins that specifically interacts with AFT1 in yeast cells . The physical interaction between AFT1 and a partial OMT1 polypeptide can be demonstrated in vitro by using bacterially expressed proteins . A single copy gene was found to encode OMT1 in Arabidopsis, and its expression is both spatially and temporally regulated. Mol Gen Genet, 1997 Sep, 256(2), 121 - 6 Map-based cloning of the tomato genomic region that spans the Sw-5 tospovirus resistance gene in tomato; Brommonschenkel SH et al.; Two yeast artificial chromosomes (YACs) containing genomic DNA from tomato have been isolated using CT220, an RFLP marker which is tightly linked to the tomato spotted wilt virus resistance gene, Sw-5 . High-resolution mapping of the YAC ends and internal YAC probes demonstrated that one of the YAC clones, TY257 (400 kb), spans Sw-5 . By chromosome walking in a cosmid library, the position of Sw-5 has been delimited within the YAC to a maximal chromosomal segment of 100 kb, spanned by nine overlapping cosmid clones. Oncogene, 1997 Oct 2, 15(14), 1635 - 42 Rapamycin and p53 act on different pathways to induce G1 arrest in mammalian cells; Metcalfe SM et al.; Certain growth regulatory kinases contain a common domain related to the phospho-inositol 3 (PI-3) kinase catalytic site . These include the ATM gene product, DNA-PKcs, and the target of rapamycin (TOR in yeast; and FRAP in mammalian cells) . Rapamycin inhibits growth factor signalling and induces G1 arrest in many cell types . Some growth regulatory PI-3 kinases appear functionally linked to p53 and we have explored potential links between cellular effects induced by rapamycin and p53 . In p53 null cells rapamycin inhibited cell cycling but did not induce G1 arrest . In cells which showed selective G1 arrest in response to rapamycin, rapamycin had no effect on basal levels of p53 protein . Similarly p21(WAF1) protein was not induced by rapamycin . The kinetics of the cellular p53/p21(WAF1) response to ionising radiation was unaffected by rapamycin; and the ability of growth factor to protect against p53-mediated apoptosis in response to DNA damage was also unaffected by rapamycin . The ATM gene is mutated in the cancer susceptibility syndrome ataxia telangiectasia (AT) but such mutant cells showed a similar sensitivity to rapamycin compared to their normal counterparts . RKO cell lines of common genetic background, but with different levels of functional p53 protein, also responded similarly to rapamycin . Thus, although rapamycin and p53 are each able to induce G1 arrest, they appear to act through independent growth regulatory pathways. Oncogene, 1997 Oct 2, 15(14), 1625 - 34 Effect of Bcr sequences on the cellular function of the Bcr-Abl oncoprotein; McWhirter JR et al.; In Philadelphia chromosome (Ph1)-positive human leukemia, the c-Abl tyrosine kinase is activated by fusion to sequences encoded by the breakpoint cluster region (bcr) gene . Two major types of Bcr-Abl fusion proteins have been found in human leukemia . Fusion of the N-terminal 426 amino acids of Bcr generates p190(Bcr-Abl) which is mostly found in acute lymphocytic leukemia (ALL), whereas fusion of the N-terminal 902 or 927 amino acids of Bcr generates p210(Bcr-Abl) mostly found with chronic myelogenous leukemia (CML) . Previous studies have demonstrated that both the Bcr and the Abl functional domains contribute to the oncogenic activity of Bcr-Abl proteins . Present in both p190 and p210 is the N-terminal coiled-coil of Bcr (aa 1-63), which is shown here to be functionally replaceable with the leucine zipper of the yeast transcription factor GCN4 . The ZIP-Bcr-Abl protein transforms Rat-1/myc cells, is autophosphorylated on tyrosine and localized predominantly to actin filaments . Thus, formation of homo-oligomers through either Bcr or GCN4 coiled-coil can activate the tyrosine kinase and F-actin binding functions of Abl . We also found that a Bcr-Abl fusion containing only Bcr amino acids (1-191) can efficiently transform Rat-1/myc cells . Fusion of additional Bcr sequences (aa 192-923) did not affect the transformation of Rat-1/myc cells but progressively reduced the disruptive effect on the actin cytoskeleton . In particular, the Dbl homology domain present in p210(Bcr-Abl) but not in p190(Bcr-Abl) contributes to the stabilization of actin fibers . The modulatory effect of Bcr sequences on actin structure may underlie the apparent pathogenic variations between the different Bcr-Abl fusion proteins. Cell, 1998 Jan 23, 92(2), 265 - 77 MIX-1: an essential component of the C . elegans mitotic machinery executes X chromosome dosage compensation; Lieb JD et al.; We show that a functional component of the C . elegans mitotic machinery regulates X chromosome gene expression . This protein, MIX-1, is a member of the dosage compensation complex that associates specifically with hermaphrodite X chromosomes to reduce their gene expression during interphase . MIX-1 also associates with all mitotic chromosomes to ensure their proper segregation . Both dosage compensation and mitosis are severely disrupted by mix-1 mutations . MIX-1 belongs to the SMC protein family required for mitotic chromosome condensation and segregation in yeast and frogs . Thus, an essential, conserved component of mitotic chromosomes has been recruited to the dosage compensation process . Rather than dosage compensation and mitosis being achieved by two separate sets of related genes, these two processes share an identical component, indicating a common mechanism for establishing higher order chromosome structure and proper X chromosome gene expression. DNA Res, 1997 Oct 31, 4(5), 341 - 3 Chromosomal mapping of genes in the RBCS family in Arabidopsis thaliana; Niwa Y et al.; In Arabidopsis thaliana, four genes have been identified in the RBCS gene family, one being assigned to subfamily RBCS-A and the other three to subfamily RBCS-B (1B, 2B and 3B) . To determine the chromosomal location of these genes, hybridization analysis with CIC YAC high-density filters was carried out for the RBCS-A gene, and CAPS analysis for the three RBCS-B genes, based on the finding that restriction fragment length polymorphism is present in the upstream region of the gene RBCS-3B . The RBCS-A gene was mapped at 100.8 cM from the top of chromosome 1 and the three RBCS-B genes at 62.70 cM from the top of chromosome 5. Virology, 1998 Jan 20, 240(2), 175 - 82 Identification and characterization of a protein kinase-interacting protein encoded by the Autographa californica nuclear polyhedrosis virus; Fan X et al.; We have used a yeast two-hybrid system to identify a baculoviral gene encoding a protein kinase-interacting protein (PKIP) . The pkip gene is located at 15.8-16.2 m.u (map units) and represents ORF 24 in the sequence of Ayres et al . (1994) of the Autographa californica nuclear polyhedrosis virus . PKIP is a 19.2-kDa protein and appears to stimulate the activity of the viral protein kinase-1 in vitro . Northern blotting analysis revealed that pkip is a late gene . Attempts to plaque purify a pkip- virus were unsuccessful. J Pharm Sci, 1998 Jan, 87(1), 76 - 80 Characterization of recombinant hepatitis B surface antigen using surface plasmon resonance; Tung JS et al.; Two commercial monoclonal antibodies were shown to interact with distinct epitopes within the major antigenic a determinant region of yeast recombinant hepatitis B surface antigen (rHBsAg) particles, contained in the hepatitis B vaccine, by using the surface plasmon resonance phenomenon on a BIAcore system . Apparent avidity for these antibodies were compared among different rHBsAg batches, showing the consistency of these epitopes . Furthermore, the effect of the alteration of these epitopes, achieved by chemical modifications, was readily reflected by changes in the apparent avidity for these antibodies . The procedures used in the present study provide a novel and efficient way to characterize rHBsAg particles present in different hepatitis B vaccine products. Eur J Biochem, 1997 Nov 15, 250(1), 150 - 7 Metabolism of tentoxin by hepatic cytochrome P-450 3A isozymes; Delaforge M et al.; The interaction between rat and human liver cytochrome P-450 with tentoxin, a natural phytotoxic cyclotetrapeptide having chlorotic properties, was studied by difference ultraviolet visible spectroscopy . Tentoxin interacted with rat liver microsomes and the difference spectrum was characteristic of binding to a protein site close to the heme . The intensity of this spectrum was clearly dependent on the amounts of P-450 3A in the microsomes and was optimal in dexamethasone-treated rat microsomes . Tentoxin exhibited a high affinity for P-450 3A (Ks approximately 10 microM) . Similar results were observed with human P-450 isozymes expressed in yeast . Only P-450 3A4 and 3A5 were able to give spectral interactions with tentoxin . Liver microsomes from rats pretreated with dexamethasone, a specific inducer of P-450 3A, were found to be particularly active for the oxidation of tentoxin, which occurs mainly on its Ala(Me) function leading to demethylation . Yeast-expressed P-450 3A also exhibited high activity to metabolize tentoxin . The metabolites were identified by their ultraviolet and mass spectra in fast atom bombardment and collision-activated dissociation modes . In addition to the major N-demethylated metabolite, other hydroxylated metabolites were formed . Preliminary analysis showed that as tentoxin, some metabolites were still efficient chloroplast ATPase inhibitors, while at least one of them exhibited even at low concentration stimulatory effects. J Neurosci, 1998 Jan 15, 18(2), 720 - 30 Splice variant-specific interaction of the NMDA receptor subunit NR1 with neuronal intermediate filaments; Ehlers MD et al.; NMDA receptors are excitatory neurotransmitter receptors critical for synaptic plasticity and neuronal development in the mammalian brain . These receptors are found highly concentrated in the postsynaptic membrane of glutamatergic synapses . To investigate the molecular mechanisms underlying NMDA receptor localization, we used the yeast two-hybrid system to identify proteins expressed in the brain that interact with the NMDA receptor subunit NR1 . Here we report that the 68 kDa neurofilament subunit NF-L directly interacts with the NR1 subunit . This interaction occurs between the cytoplasmic C-terminal domain of NR1 and the rod domain of NF-L . However, NR1 splice variants lacking the first C-terminal exon cassette (C1) failed to associate with NF-L . Immunogold electron microscopy revealed a preferential localization of NR1 at the ends of in vitro-assembled neurofilaments . Overexpression of C1 cassette-containing NR1 constructs in fibroblast cells disrupted the assembly of recombinant neurofilaments . In addition, NR1 and NF-L cofractionated in detergent-treated rat brain synaptic plasma membranes . Furthermore, NR1 and NF-L colocalize in the dendrites and growth cones of cultured hippocampal neurons . These results demonstrate the splice variant-specific association of NR1 with neurofilaments and suggest a possible mechanism for anchoring or localizing NMDA receptors in the neuronal plasma membrane. J Biol Chem, 1998 Jan 2, 273(1), 584 - 91 Activation and processing of non-anchored yapsin 1 (Yap3p); Cawley NX et al.; A C-terminally truncated form of yapsin 1 (yeast aspartic protease 3), the first member of the novel sub-class of aspartic proteases with specificity for basic residues (designated the Yapsins), was overexpressed and purified to apparent homogeneity, yielding approximately 1 microg of yapsin 1/g of wet yeast . N-terminal amino acid analysis of the purified protein confirmed that the propeptide was absent and that the mature enzyme began at Ala68 . The mature enzyme was shown to be composed of approximately equimolar amounts of two subunits, designated alpha and beta, that were associated to each other by a disulfide bond . C-terminally truncated proyapsin 1 was also expressed in the baculovirus/Sf9 insect cell expression system and secreted as a zymogen that could be activated upon incubation at an acidic pH with an optimum at approximately 4.0 . When expressed without its pro-region, it was localized intracellularly and lacked activity, indicating that the pro-region was required for the correct folding of the enzyme . The activation of proyapsin 1 in vitro exhibited linear kinetics and generated an intermediate form of yapsin 1 or pseudo-yapsin 1. J Biol Chem, 1998 Jan 2, 273(1), 484 - 94 Negative regulation of beta enolase gene transcription in embryonic muscle is dependent upon a zinc finger factor that binds to the G-rich box within the muscle-specific enhancer; Passantino R et al.; We have previously identified a muscle-specific enhancer within the first intron of the human beta enolase gene . Present in this enhancer are an A/T-rich box that binds MEF-2 protein(s) and a G-rich box (AGTGGGGGAGGGGGCTGCG) that interacts with ubiquitously expressed factors . Both elements are required for tissue-specific expression of the gene in skeletal muscle cells . Here, we report the identification and characterization of a Kruppel-like zinc finger protein, termed beta enolase repressor factor 1, that binds in a sequence-specific manner to the G-rich box and functions as a repressor of the beta enolase gene transcription in transient transfection assays . Using fusion polypeptides of beta enolase repressor factor 1 and the yeast GAL4 DNA-binding domain, we have identified an amino-terminal region responsible for the transcriptional repression activity, whereas a carboxyl-terminal region was shown to contain a potential transcriptional activation domain . The expression of this protein decreases in developing skeletal muscles, correlating with lack of binding activity in nuclear extract from adult skeletal tissue, in which novel binding activities have been detected . These results suggest that in addition to the identified factor, which functionally acts as a negative regulator and is enriched in embryonic muscle, the G-rich box binds other factors, presumably exerting a positive control on transcription . The interplay between factors that repress or activate transcription may constitute a developmentally regulated mechanism that modulates beta enolase gene expression in skeletal muscle. J Biol Chem, 1998 Jan 2, 273(1), 262 - 70 Directly energized uptake of beta-estradiol 17-(beta-D-glucuronide) in plant vacuoles is strongly stimulated by glutathione conjugates; Klein M et al.; A directly energized vacuolar pump for glutathione (GS) conjugates has been described for several plant species . Since glucuronate conjugates also occur in plants, we addressed the question whether plant vacuoles take up the abiotic glucuronate conjugate estradiol 17-(beta-glucuronide) (E217G) via a GS conjugate pump, which in some cases has been reported to accept various organic anions as substrates, or via a distinct glucuronate transporter . Uptake studies into vacuoles from rye and barley were performed with E217G and metolachlor-GS (MOC-GS), a substrate of the GS conjugate ATPase, to compare glucuronate conjugate transport into vacuoles containing endogenous flavone glucuronides with those lacking specific glucuronate conjugates, respectively . Our results indicate that E217G and MOC-GS are taken up into vacuoles of both plants via a directly energized mechanism since transport was (i) strictly ATP-dependent; (ii) inhibited by vanadate but not by bafilomycin A1, azide, verapamil, nor by dissipation of the vacuolar DeltapH or DeltaPsi; (iii) E217G uptake into rye vacuoles was partially driven by other nucleotides in the following order of efficiency: ATP > GTP > UTP congruent with CTP, whereas the non-hydrolyzable ATP analogue 5'-adenylyl-beta,gamma-imidodiphosphate, ADP, or PPi did not energize uptake . E217G transport into rye vacuoles was saturable (Km approximately 0.2 mM) . The rye-specific luteolin glucuronides decreased uptake rates of E217G and MOC-GS into rye and barley vacuoles to comparable degrees with the mono- and diglucuronidated derivatives (40-60% inhibition) being more effective than the triglucuronide . Inhibition of E217G uptake by luteolin 7-O-diglucuronide was competitive (Ki = 120 microM) . Taurocholate had no effect on E217G transport, and uptake of MOC-GS was not inhibited by E217G . Although GS conjugates and oxidized GS decreased MOC-GS transport, E217G uptake into rye and barley vacuoles was stimulated up to 7-fold in a concentration-dependent manner by these substances, with dinitrobenzene-GS being most effective . The stimulation of the GS conjugates was not due to detergent or redox effects and was specific for the E217G pump . GS conjugate stimulation of glucuronate uptake was unique for plants as E217G uptake into yeast microsomal vesicles was not affected . By comparison with a DeltaYCF1 yeast mutant, defective in vacuolar transport of GS conjugates mediated by YCF1, it was shown that E217G was taken up into yeast vesicles via a YCF1-independent directly energized pump . These results indicate that E217G as a glucuronate conjugate is transported across the vacuolar membranes of plants and yeast by a carrier distinct from the GS conjugate ATPase. J Biol Chem, 1998 Jan 2, 273(1), 110 - 7 A novel ets-related transcription factor, ERT/ESX/ESE-1, regulates expression of the transforming growth factor-beta type II receptor; Choi SG et al.; A 2.5-kilobase cDNA clone that encodes a 371-amino acid novel transcription factor was isolated from a human placenta cDNA library using a yeast one-hybrid system . The novel ets-related transcription factor (ERT) showed a homology with the ETS DNA-binding domain . Using constructs of the transforming growth factor-beta (TGF-beta) type II receptor (RII) promoter linked to the luciferase gene, we have demonstrated that ERT activates transcription of the TGF-beta RII gene through the 5'-TTTCCTGTTTCC-3' response element spanning nucleotides +13 to +24 and multiple additional ETS binding sites between -1816 and -82 of the TGF-beta RII promoter . A specific interaction between ERT and the ETS binding sites was also demonstrated using an electrophoretic mobility shift assay . Deletion mapping of ERT protein suggests that the transactivation domain resides in the amino terminus while the DNA-binding domain is localized to the carboxyl-terminal region . Our results suggest that ERT might be a major transcription factor involved in the transcriptional regulation of the TGF-beta RII gene. J Biol Chem, 1998 Jan 2, 273(1), 23 - 7 ARNO is a guanine nucleotide exchange factor for ADP-ribosylation factor 6; Frank S et al.; ADP-ribosylation factors (ARFs) constitute a family of small monomeric GTPases . ARFs 1 and 3 function in the recruitment of coat proteins to membranes of the Golgi apparatus, whereas ARF6 is localized to the plasma membrane, where it appears to modulate both the assembly of the actin cytoskeleton and endocytosis . Like other GTPases, ARF activation is facilitated by specific guanine nucleotide exchange factors (GEFs) . ARNO (ARF nucleotide-binding site opener) is a member of a growing family of ARF-GEFs that share a common, tripartite structure consisting of an N-terminal coiled-coil domain, a central domain with homology to the yeast protein Sec7p, and a C-terminal pleckstrin homology domain . Recently, ARNO and its close homologue cytohesin-1 were found to catalyze in vitro nucleotide exchange on ARF1 and ARF3, respectively, raising the possibility that these GEFs function in the Golgi . However, the actual function of these proteins may be determined in part by their ability to interact with specific ARFs and in part by their subcellular localization . We report here that in vitro ARNO can stimulate nucleotide exchange on both ARF1 and ARF6 . Furthermore, based on subcellular fractionation and immunolocalization experiments, we find that ARNO is localized to the plasma membrane in mammalian cells rather than the Golgi . It is therefore likely that ARNO functions in plasma membrane events by modulating the activity of ARF6 in vivo . These findings are consistent with the previous observation that cytohesin-1 regulates the adhesiveness of alphaLbeta2 integrins at the plasma membrane of lymphocytes. Blood, 1998 Jan 1, 91(1), 231 - 7 Molecular delineation of 13q deletion boundaries in 20 patients with myeloid malignancies; La Starza R et al.; Fluorescent in situ hybridization (FISH) analysis with a panel of DNA probes for 13q13.1-q14.3 was performed on 20 cases of myeloid malignancies, of which 17 showed a del(13)(q) and three had translocations affecting 13q . By chromosome morphology, deletions consistently involved bands q14 and q21 . In addition to confirming the chromosome data, FISH allowed us to delineate a commonly deleted region that was flanked by YAC 833A2 and YAC 854D4 . Three cases with 13q translocations unexpectedly showed accompanying cryptic microdeletions of 13q, and in one case the commonly deleted region could be narrowed to a genomic segment, which includes YAC 937C7, RB1, and YAC 745E3 . Homozygous deletions were not detected . This region overlaps with the smallest deleted region of 13q14 in chronic lymphocytic leukemia. J Cell Biol, 1997 Dec 29, 139(7), 1719 - 33 Cytoskeletal protein ABP-280 directs the intracellular trafficking of furin and modulates proprotein processing in the endocytic pathway; Liu G et al.; Furin catalyzes the proteolytic maturation of many proproteins within the trans-Golgi network (TGN)/endosomal system . Furin's cytosolic domain (cd) directs both the compartmentalization to and transit between its manifold processing compartments (i.e., TGN/biosynthetic pathway, cell surface, and endosomes) . Here we report the identification of the first furin cd sorting protein, ABP-280 (nonmuscle filamin), an actin gelation protein . The furin cd was used as bait in a yeast two-hybrid screen to identify ABP-280 as a furin-binding protein . Binding analyses in vitro and coimmunoprecipitation studies in vivo showed that furin and ABP-280 interact directly and that ABP-280 tethers furin molecules to the cell surface . Quantitative analysis of both ABP-280-deficient and genetically replete cells showed that ABP-280 modulates the rate of internalization of furin but not of the transferrin receptor, a cycling receptor . However, although ABP-280 directs the rate of furin internalization, the efficiency of sorting of the endoprotease from the cell surface to early endosomes is independent of expression of ABP-280 . By contrast, efficient sorting of furin from early endosomes to the TGN requires expression of ABP-280 . In addition, ABP-280 is also required for the correct localization of late endosomes (dextran bead uptake) and lysosomes (LAMP-1 staining), demonstrating a pleiotropic role for this actin binding protein in the organization of cellular compartments and directing protein traffic . Finally, and consistent with the trafficking studies on furin, we showed that ABP-280 modulates the processing of furin substrates in the endocytic but not the biosynthetic pathways . The novel roles of ABP-280 and the cytoskeleton in the sorting of furin in the TGN/ endosomal system and the formation of proprotein processing compartments are discussed. J Cell Biol, 1997 Dec 29, 139(7), 1621 - 34 Evidence for covalent modification of the nuclear dot-associated proteins PML and Sp100 by PIC1/SUMO-1; Sternsdorf T et al.; PML and Sp100 proteins are associated with nuclear domains, known as nuclear dots (NDs) . They were discovered in the context of leukemic transformation and as an autoantigen in primary biliary cirrhosis, respectively . Both proteins are expressed in the form of many COOH-terminally spliced variants, and their expression is enhanced by interferons (IFN) . The recent finding that PIC1/SUMO-1, a small ubiquitin-like protein, is covalently linked to the RanGAP1 protein of the nuclear pore complex and also binds PML in yeast cells led us to determine whether PML is covalently modified by PIC1/SUMO-1 and whether the same is true for Sp100 . We found an immune reaction of PML and Sp100 proteins with a PIC1/SUMO-1-specific monoclonal antibody by immunoblotting when using cell extracts prepared from stably transfected cells inducibly expressing one isoform of each protein as well as from nontransfected cells . In contrast, both proteins did not react when synthesized in vitro . Immunofluorescence staining showed that PIC1/SUMO-1 colocalized with Sp100 and PML in NDs except in mitotic cells, in which PML and Sp100 are dissociated . Cell fractionation and immunoblotting demonstrated that PIC1/SUMO-1 immunoreactive Sp100 in IFN-treated and untreated cells was exclusively nuclear, whereas nonmodified Sp100 was also found in the cytoplasm . Taken together, these data strongly suggest covalent modification of specific nuclear isoforms of Sp100 and PML by PIC1/SUMO-1 . This modification may play a regulatory role in ND structure, composition, and function. Proc Natl Acad Sci U S A, 1997 Dec 23, 94(26), 14954 - 9 Differentially regulated NADPH:cytochrome P450 oxidoreductases in parsley; Koopmann E et al.; Two NADPH:cytochrome P450 oxidoreductases (CPRs) from parsley (Petroselinum crispum) were cloned, and the complete proteins were expressed and functionally identified in yeast . The two enzymes, designated CPR1 and CPR2, are 80% identical in amino acid sequence with one another and about 75% identical with CPRs from several other plant species . The mRNA accumulation patterns for CPR1 and CPR2 in fungal elicitor-treated or UV-irradiated cultured parsley cells and in developing or infected parsley plants were compared with those for cinnamate 4-hydroxylase (C4H), one of the most abundant CPR-dependent P450 enzymes in plants . All treatments strongly induced the mRNAs for C4H and CPR1 but not for CPR2, suggesting distinct metabolic roles of CPR1 and CPR2 and a functional relationship between CPR1 and C4H. Proc Natl Acad Sci U S A, 1997 Dec 23, 94(26), 14815 - 20 Interactive cloning with the SH3 domain of N-src identifies a new brain specific ion channel protein, with homology to eag and cyclic nucleotide-gated channels; Santoro B et al.; We have isolated a novel cDNA, that appears to represent a new class of ion channels, by using the yeast two-hybrid system and the SH3 domain of the neural form of Src (N-src) as a bait . The encoded polypeptide, BCNG-1, is distantly related to cyclic nucleotide-gated channels and the voltage-gated channels, Eag and H-erg . BCNG-1 is expressed exclusively in the brain, as a glycosylated protein of approximately 132 kDa . Immunohistochemical analysis indicates that BCNG-1 is preferentially expressed in specific subsets of neurons in the neocortex, hippocampus, and cerebellum, in particular pyramidal neurons and basket cells . Within individual neurons, the BCNG-1 protein is localized to either the dendrites or the axon terminals depending on the cell type . Southern blot analysis shows that several other BCNG-related sequences are present in the mouse genome, indicating the emergence of an entire subfamily of ion channel coding genes . These findings suggest the existence of a new type of ion channel, which is potentially able to modulate membrane excitability in the brain and could respond to regulation by cyclic nucleotides. Proc Natl Acad Sci U S A, 1997 Dec 23, 94(26), 14671 - 6 WIP, a protein associated with wiskott-aldrich syndrome protein, induces actin polymerization and redistribution in lymphoid cells; Ramesh N et al.; Wiskott-Aldrich syndrome (WAS) is an X-linked immunodeficiency caused by mutations that affect the WAS protein (WASP) and characterized by cytoskeletal abnormalities in hematopoietic cells . By using the yeast two-hybrid system we have identified a proline-rich WASP-interacting protein (WIP), which coimmunoprecipitated with WASP from lymphocytes . WIP binds to WASP at a site distinct from the Cdc42 binding site and has actin as well as profilin binding motifs . Expression of WIP in human B cells, but not of a WIP truncation mutant that lacks the actin binding motif, increased polymerized actin content and induced the appearance of actin-containing cerebriform projections on the cell surface . These results suggest that WIP plays a role in cortical actin assembly that may be important for lymphocyte function. Proc Natl Acad Sci U S A, 1997 Dec 23, 94(26), 14524 - 9 A multimeric complex and the nuclear targeting of the Drosophila Rel protein Dorsal; Yang J et al.; The intracellular part of the Rel signal transduction pathway in Drosophila is encoded by Toll, tube, pelle, dorsal, and cactus, and it functions to form the dorsal-ventral axis in the Drosophila embryo . Upon activation of the transmembrane receptor Toll, Dorsal dissociates from its cytoplasmic inhibitor Cactus and enters the nucleus . Tube and Pelle are required to relay the signal from Toll to the Dorsal-Cactus complex . In a yeast two-hybrid assay, we found that both Tube and Pelle interact with Dorsal . We confirmed these interactions in an in vitro binding assay . Tube interacts with Dorsal via its C-terminal domain, whereas full-length Pelle is required for Dorsal binding . Tube and Pelle bind Dorsal in the N-terminal domain 1 of the Dorsal Rel homology region rather than at the Cactus binding site . Domain 1 has been found to be necessary for Dorsal nuclear targeting . Genetic experiments indicate that Tube-Dorsal interaction is necessary for normal signal transduction . We propose a model in which Tube, Pelle, Cactus, and Dorsal form a multimeric complex that represents an essential aspect of signal transduction. Proc Natl Acad Sci U S A, 1997 Dec 23, 94(26), 14495 - 9 Chemotaxis in a lymphocyte cell line transfected with C-C chemokine receptor 2B: evidence that directed migration is mediated by betagamma dimers released by activation of Galphai-coupled receptors; Arai H et al.; Chemotaxis is mediated by activation of seven-transmembrane domain, G protein-coupled receptors, but the signal transduction pathways leading to chemotaxis are poorly understood . To identify G proteins that signal the directed migration of cells, we stably transfected a lymphocyte cell line (300-19) with G protein-coupled receptors that couple exclusively to Galphaq (the m3 muscarinic receptor), Galphai (the kappa-opioid receptor), and Galphas (the beta-adrenergic receptor), as well as the human thrombin receptor (PAR-1) and the C-C chemokine receptor 2B . Cells expressing receptors that coupled to Galphai, but not to Galphaq or Galphas, migrated in response to a concentration gradient of the appropriate agonist . Overexpression of Galpha transducin, which binds to and inactivates free Gbetagamma dimers, completely blocked chemotaxis although having little or no effect on intracellular calcium mobilization or other measures of cell signaling . The identification of Gbetagamma dimers as a crucial intermediate in the chemotaxis signaling pathway provides further evidence that chemotaxis of mammalian cells has important similarities to polarized responses in yeast . We conclude that chemotaxis is dependent on activation of Galphai and the release of Gbetagamma dimers, and that Galphai-coupled receptors not traditionally associated with chemotaxis can mediate directed migration when they are expressed in hematopoietic cells. Proc Natl Acad Sci U S A, 1997 Dec 23, 94(26), 14438 - 43 Subunit structure of the mammalian exocyst complex; Kee Y et al.; The exocyst is a protein complex required for the late stages of secretion in yeast . Unlike the SNAREs (SNAP receptors), important secretory proteins that are broadly distributed on the target membrane, the exocyst is specifically located at sites of vesicle fusion . We have isolated cDNAs encoding the rexo70, rsec5, and rsec15 subunits of the mammalian complex . The amino acid sequences encoded by these genes are between 21% and 24% identical to their yeast homologs . All three genes are broadly expressed and multiple transcripts are observed for rexo70 and rsec15 . Characterization of cDNAs encoding the 84-kDa subunit of the mammalian complex revealed a novel protein . mAbs were generated to the mammalian rsec6 subunit of the exocyst complex . rsec6 immunoreactivity is found in a punctate distribution at terminals of PC12 cell processes at or near sites of granule exocytosis. Eur J Hum Genet, 1997 Nov-Dec, 5(6), 397 - 405 Refined mapping of a gene for autosomal dominant progressive sensorineural hearing loss (DFNA5) to a 2-cM region, and exclusion of a candidate gene that is expressed in the cochlea; Van Laer L et al.; A gene for an autosomal dominant form of progressive sensorineural hearing loss (DFNA5) was previously assigned by us to a 15-cM region on chromosome 7p15 . In this study, the DFNA5 candidate region was refined to less than 2 cM, and completely cloned in a YAC contig . The HOXA1 gene located in 7p15 was considered to be a good candidate gene for DFNA5 as it harbours mutations leading to developmental defects of the inner ear in mice . However, the refinement of the candidate region of DFNA5 excludes the HOXA1 gene as a candidate for DFNA5 . We cloned a novel candidate gene (CG1, candidate gene 1), which is expressed in human fetal cochlea, from the DFNA5 candidate region . The complete cDNA sequence of CG1, encoding a 423 amino acid protein of unknown function, was determined . Mutation analysis of the CG1 gene in DFNA5 patients, however, could not reveal a disease-causing mutation. J Appl Microbiol, 1997 Dec, 83(6), 771 - 8 Chrysosporium species, potential spoilage organisms of chocolate; Kinderlerer JL; Standard methods of analysing foods for the presence of moulds are inadequate for the detection of genera such as Chrysosporium which do not grow at the high water activities of most mycological media . The use of malt, yeast, 50% glucose agar (MY50G) in sealed containers as an enrichment medium allowed time for germination and growth of heat-stressed spores . Three Chrysosporium spp., C . xerophilum Pitt, C . inops (Carmichael) and C . farinicola (Burnside) Skou, were isolated from commercial chocolate bars with a water activity (aw) of approximately 0.28 . Chrysosporium inops was isolated from commercial milk crumb and a new Chrysosporium sp . was isolated from Ghanaian cocoa beans . In chocolates made by coating MY50G agar (aw = 0.89) with chocolate (aw = 0.27) containing C . inops arthroconidia, two types of deterioration were seen after storage . The first was fat bloom due to recrystallization of the cocoa butter on the outer and inner chocolate surface . The second was growth of C . inops which occurred on the inside chocolate surface adjacent to the MY50G agar filling and on the outside surface after holding at 92% equilibrium relative humidity (erh) for 12 d . There was some evidence that C . inops could grow on the outside of chocolates held at 5.7% erh after 4 months' storage at 25 degrees C . The appearance of the white fungal growth was not unlike fat bloom to the naked eye but was clearly different with the electron microscope. Folia Microbiol (Praha), 1996, 41(3), 223 - 7 Isolation and characterization of a new dsRNA virus from Wickerhamia fluorescens; Pospisek M et al.; Virus-like particles (VLPs) were isolated from the yeast Wickerhamia fluorescens strain CCY61-1-1 . The VLPs are approximately 42 nm in diameter and contain only one species of dsRNA molecule . The apparent length of the dsRNA determined by native agarose gel electrophoresis was 4.6 kbp . Analysis of protein content of the VLPs showed them to contain one major capsid protein with an apparent molar mass of 74.5 kDa. Antimicrob Agents Chemother, 1998 Jan, 42(1), 151 - 3 Treatment of histoplasmosis with MK-991 (L-743,872); Graybill JR et al.; BALB/c nu/+ immunocompetent and athymic (nu/nu) mice were infected intravenously with yeast cells of Histoplasma capsulatum . Mice were either given water (controls) intraperitoneally (i.p.) or given MK-991 i.p . once daily or twice daily . Protection was measured as prolonged survival or reduction in tissue counts . MK-991 was protective in immunocompetent mice, prolonging survival and reducing counts in spleen and livers at a dose as low as 0.05 mg/kg of body weight/day . MK-991 was modestly effective in athymic mice at a higher dose, 5 mg/kg/day . These studies suggest that MK-991 may be appropriate for clinical development in histoplasmosis. Virology, 1998 Jan 5, 240(1), 64 - 75 Autographa californica nuclear polyhedrosis virus: subcellular localization and protein trafficking of BV/ODV-E26 to intranuclear membranes and viral envelopes; Beniya H et al.; The Autographa californica nuclear polyhedrosis virus da26 gene codes for an envelope protein of both budded virus (BV) and occlusion derived virus (ODV) . Western blot and temporal analysis of infected cell extracts detected a protein of 26 kDa by 4 h postinfection (p.i.) . The amount of protein increased by 16 h p.i . and remained at high levels throughout infection . By 36 h p.i . several additional immunoreactive proteins were detected which migrated at approximately 18 kDa and remained through 96 h p.i . Western blot analysis of purified virus envelope and nucleocapsid preparations revealed that both the 26- and 18-kDa proteins are structural proteins of the envelope of BV and ODV . Immunoelectron microscopy performed at a time when only the 26-kDa species of the protein was present confirmed that the protein located to ODV envelope . The protein was named BV/ODV-E26 to designate incorporation into viral progeny, envelope location, and apparent molecular weight . Studies designed to follow localization of BV/ODV-E26 demonstrated that early in infection, the protein was incorporated into cytoplasmic vesicles and by 16 h p.i., BV/ODV-E26 was detected in the nucleus associated with virus-induced intranuclear microvesicles and ODV envelope . Coimmunoprecipitation and yeast two-hybrid assays showed that BV/ODV-E26 and FP25K were capable of interacting with each other to form a complex and coimmunoprecipitation assays indicated that cellular actin was a third component of this complex . Together, these data suggest that FP25K and cellular actin may participate in the regulation, or movement through the cell, of baculovirus proteins and/or virus nucleocapsids. Trends Genet, 1998 Jan, 14(1), 14 - 20 Molecular genetics of life span in C . elegans: how much does it teach us? Hekimi S, Lakowski B, Barnes TM, Ewbank JJ. Several loci have been identified in the nematode worm Caenorhabditis elegans that, when mutated, can increase life span . Three of these genes, age-1, daf-2 and clk-1, have now been cloned . Mutations in these three genes are highly pleiotropic and affect many aspects of worm development and behaviour, age-1 and daf-2 act in the same genetic pathway and have similar effects on the worm, age-1 encodes a homologue of the p110 subunit of phosphatidylinositol 3-kinase and daf-2 encodes an insulin receptor family member, clk-1 encodes a protein of unknown biochemical function similar to the yeast metabolic regulator Cat5p/Coq7p . The implications of these findings for our understanding of organismal ageing are discussed. Mol Cell Biol, 1998 Feb, 18(2), 859 - 71 Regulation of interferon-induced protein kinase PKR: modulation of P58IPK inhibitory function by a novel protein, P52rIPK; Gale M Jr et al.; The cellular response to environmental signals is largely dependent upon the induction of responsive protein kinase signaling pathways . Within these pathways, distinct protein-protein interactions play a role in determining the specificity of the response through regulation of kinase function . The interferon-induced serine/threonine protein kinase, PKR, is activated in response to various environmental stimuli . Like many protein kinases, PKR is regulated through direct interactions with activator and inhibitory molecules, including P58IPK, a cellular PKR inhibitor . P58IPK functions to represses PKR-mediated phosphorylation of the eukaryotic initiation factor 2alpha subunit (eIF-2alpha) through a direct interaction, thereby relieving the PKR-imposed block on mRNA translation and cell growth . To further define the molecular mechanism underlying regulation of PKR, we have utilized an interaction cloning strategy to identify a novel cDNA encoding a P58IPK-interacting protein . This protein, designated P52rIPK, possesses limited homology to the charged domain of Hsp90 and is expressed in a wide range of cell lines . P52rIPK and P58IPK interacted in a yeast two-hybrid assay and were recovered as a complex from mammalian cell extracts . When coexpressed with PKR in yeast, P58IPK repressed PKR-mediated eIF-2alpha phosphorylation, inhibiting the normally toxic and growth-suppressive effects associated with PKR function . Conversely, introduction of P52rIPK into these strains resulted in restoration of both PKR activity and eIF-2alpha phosphorylation, concomitant with growth suppression due to inhibition of P58IPK function . Furthermore, P52rIPK inhibited P58IPK function in a reconstituted in vitro PKR-regulatory assay . Our results demonstrate that P58IPK is inhibited through a direct interaction with P52rIPK which, in turn, results in upregulation of PKR activity . Taken together, our data describe a novel protein kinase-regulatory system which encompasses an intersection of interferon-, stress-, and growth-regulatory pathways. Mol Cell Biol, 1998 Feb, 18(2), 1029 - 41 POU transcription factors Brn-3a and Brn-3b interact with the estrogen receptor and differentially regulate transcriptional activity via an estrogen response element; Budhram-Mahadeo V et al.; The estrogen receptor (ER) modulates transcription by forming complexes with other proteins and then binding to the estrogen response element (ERE) . We have identified a novel interaction of this receptor with the POU transcription factors Brn-3a and Brn-3b which was independent of ligand binding . By pull-down assays and the yeast two-hybrid system, the POU domain of Brn-3a and Brn-3b was shown to interact with the DNA-binding domain of the ER . Brn-3-ER interactions also affect transcriptional activity of an ERE-containing promoter, such that in estradiol-stimulated cells, Brn-3b strongly activated the promoter via the ERE, while Brn-3a had a mild inhibitory effect . The POU domain of Brn-3b which interacts with the ER was sufficient to confer this activation potential, and the change of a single amino acid in the first helix of the POU homeodomain of Brn-3a to its equivalent in Brn-3b can change the mild repressive effect of Brn-3a to a stimulatory Brn-3b-like effect . These observations and their implications for transcriptional regulation by the ER are discussed. Mol Cell Biol, 1998 Feb, 18(2), 872 - 9 A novel, multifuntional c-Cbl binding protein in insulin receptor signaling in 3T3-L1 adipocytes; Ribon V et al.; The protein product of the c-Cbl proto-oncogene is prominently tyrosine phosphorylated in response to insulin in 3T3-L1 adipocytes and not in 3T3-L1 fibroblasts . After insulin-dependent tyrosine phosphorylation, c-Cbl specifically associates with endogenous c-Crk and Fyn . These results suggest a role for tyrosine-phosphorylated c-Cbl in 3T3-L1 adipocyte activation by insulin . A yeast two-hybrid cDNA library prepared from fully differentiated 3T3-L1 adipocytes was screened with full-length c-Cbl as the target protein in an attempt to identify adipose-specific signaling proteins that interact with c-Cbl and potentially are involved in its tyrosine phosphorylation in 3T3-L1 adipocytes . Here we describe the isolation and the characterization of a novel protein that we termed CAP for c-Cbl-associated protein . CAP contains a unique structure with three adjacent Src homology 3 (SH3) domains in the C terminus and a region showing significant sequence similarity with the peptide hormone sorbin . Both CAP mRNA and proteins are expressed predominately in 3T3-L1 adipocytes and not in 3T3-L1 fibroblasts . CAP associates with c-Cbl in 3T3-L1 adipocytes independently of insulin stimulation in vivo and in vitro in an SH3-domain-mediated manner . Furthermore, we detected the association of CAP with the insulin receptor . Insulin stimulation resulted in the dissociation of CAP from the insulin receptor . Taken together, these data suggest that CAP represents a novel c-Cbl binding protein in 3T3-L1 adipocytes likely to participate in insulin signaling. Acta Paediatr Jpn, 1997 Dec, 39(6), 647 - 52 Linkage analysis and identification of deletion in Alagille syndrome gene; Yuan ZR et al.; Alagille syndrome (AGS) is a genetic disease and the responsible gene has already been mapped at 20p12 . To more accurately detect the region of the AGS gene on the linkage map of chromosome 20p, 14 yeast artificial chromosome (YAC) clones were screened to construct a YAC contig in the candidate region and 13 locus markers and 2 sequence-tagged sites (STS) were ordered . Combining all of the analyses, a 1.3 Mb critical region from D20S507 to D20S61 for the AGS gene was identified . As the human Jagged 1 gene (JAG1) lies just in this region and is responsible for the AGS disease, the genomic DNA in an AGS family without a visible deletion were analyzed by single-strand conformational polymorphism (SSCP) and direct DNA sequencing, and a 2-bp (CT) deletion mutation at exon 26 of the JAG1 was identified. Biochem Biophys Res Commun, 1998 Jan 14, 242(2), 457 - 60 Phosphorylation of mitochondrial telomere binding protein of Candida parapsilosis by camp-dependent protein kinase; Tomaska L; Mitochondrial telomere-binding protein (mtTBP) of Candida parapsilosis binds with high affinity to 5' single-stranded overhang of the linear mitochondrial DNA of this yeast (Tomaska, L'., Nosek, J., and Fukuhara, H . (1997) J . Biol . Chem . 272, 3049-3056) . Here it is reported that mtTBP is phosphorylated by catalytic subunit of cAMP-dependent protein kinase in vitro . Phosphorylated mtTBP has dramatically reduced ability to bind telomeric oligonucleotide in the gel-mobility retardation assay without affecting the oligomerization of mtTBP in vitro . MtTBP is one of the few mitochondrial proteins and the first mitochondrial single-strand DNA binding proteins that was demonstrated to serve as a substrate for cAMP-dependent protein kinase. J Biol Chem, 1997 Dec 26, 272(52), 33167 - 74 Genetic and biochemical evidence for a novel avermectin-sensitive chloride channel in Caenorhabditis elegans . Isolation and characterization; Vassilatis DK et al.; Avermectins are a class of macrocyclic lactones that is widely used in crop protection and to treat helminth infections in man and animals . Two complementary DNAs (GluClalpha and GluClbeta) encoding chloride channels that are gated by avermectin and glutamate, respectively, were isolated from Caenorhabditis elegans . To study the role of these subunits in conferring avermectin sensitivity we isolated a mutant C . elegans strain with a Tc1 transposable element insertion that functionally inactivated the GluClalpha gene (GluClalpha::Tc1) . GluClalpha::Tc1 animals exhibit a normal phenotype including typical avermectin sensitivity . Xenopus oocytes expressing GluClalpha::Tc1 strain mRNA elicited reduced amplitude avermectin and glutamate-dependent chloride currents . Avermectin binding assays in GluClalpha::Tc1 strain membranes showed the presence of high affinity binding sites, with a reduced Bmax . These experiments suggest that GluClalpha is a target for avermectin and that additional glutamate-gated and avermectin-sensitive chloride channel subunits exist in C . elegans . We isolated a cDNA (GluClalpha2) encoding a chloride channel that shares 75% amino acid identity with GluClalpha . This subunit forms homomeric channels that are gated irreversibly by avermectin and reversibly by glutamate . GluClalpha2 coassembles with GluClbeta to form heteromeric channels that are gated by both ligands . The presence of subunits related to GluClalpha may explain the low level and rarity of target site involvement in resistance to the avermectin class of compounds. J Biol Chem, 1997 Dec 26, 272(52), 32750 - 8 Analogous F-actin binding by cofilin and gelsolin segment 2 substantiates their structural relationship; Van Troys M et al.; Cofilin is representative for a family of low molecular weight actin filament binding and depolymerizing proteins . Recently the three-dimensional structure of yeast cofilin and of the cofilin homologs destrin and actophorin were resolved, and a striking similarity to segments of gelsolin and related proteins was observed (Hatanaka, H., Ogura, K., Moriyama, K., Ichikawa, S., Yahara, I., and Inagaka, F . (1996) Cell 85, 1047-1055; Fedorov, A . A., Lappalainen, P., Fedorov, E . V., Drubin, D . G., and Almo, S . C . (1997) Nat . Struct . Biol . 4, 366-369; Leonard, S . A., Gittis, A . G., Petrella, E . C., Pollard, T . D., and Lattman, E . E . (1997) Nat . Struct . Biol . 4, 369-373) . Using peptide mimetics, we show that the actin binding site stretches over the entire cofilin alpha-helix 112-128 . In addition, we demonstrate that cofilin and its actin binding peptide compete with gelsolin segments 2-3 for binding to actin filaments . Based on these competition data, we propose that cofilin and segment 2 of gelsolin use a common structural topology to bind to actin and probably share a similar target site on the filament . This adds a functional dimension to their reported structural homology, and this F-actin binding mode provides a basis to further enlighten the effect of members of the cofilin family on actin filament dynamics. Biochemistry, 1997 Dec 16, 36(50), 15992 - 8 Role of beta87 Thr in the beta6 Val acceptor site during deoxy Hb S polymerization; Reddy LR et al.; Three new Hb S variants containing beta87 Leu, Trp, or Asp instead of Thr were expressed in yeast in order to further define the role of the beta87 position in stability and polymerization of deoxy Hb S . Previous studies showed that hydrophobicity at beta85 Phe and beta88 Leu is critical for stabilization of hemoglobin . Results with the three Hb S beta87 variants, however, showed minimal differences in stability, suggesting that beta87 amino acid hydrophobicity is not critical for stabilization of hemoglobin . Polymerization properties of the variants in the deoxy form, however, were affected by the beta87 amino acid . Polymerization of Hb S beta87 Thr --> Leu and Hb S beta87 Thr --> Trp was preceded by a delay time like Hb S, while Hb S beta87 Thr --> Asp did not show a delay time . In addition, changes in time required for half polymer formation (T1/2) as a function of hemoglobin concentration for Hb S beta87 Thr --> Asp were similar to that for beta87 Thr --> Gln . Hb S beta87 Thr --> Leu polymerized at a lower hemoglobin concentration than Hb S while beta87 Thr --> Trp and Hb S beta87 Thr --> Asp required much higher hemoglobin concentrations for polymer formation . Critical concentration required for deoxy Hb S beta87 Thr --> Asp polymerization was 6- and 2.3-fold greater than that for Hb S beta85 Phe --> Glu and Hb S beta88 Leu --> Glu, respectively . These results suggest that even though beta87 Thr is not a direct interaction site for beta6 Val in deoxy Hb S polymers, it does play a critical role in formation of the hydrophobic acceptor pocket which then promotes protein-protein interactions facilitating formation of stable nuclei and polymers of deoxy Hb S. Biochemistry, 1997 Dec 16, 36(50), 15975 - 82 Interaction of the cytoplasmic tail of CTLA-4 (CD152) with a clathrin-associated protein is negatively regulated by tyrosine phosphorylation; Bradshaw JD et al.; CTLA-4 (CD152), high-avidity receptor for CD80 and CD86, is a powerful regulator of T cell activation . While CTLA-4 functions at the cell surface, it is primarily localized in intracellular vesicles and cycles to the cell surface . The CTLA-4 cytoplasmic domain contains sequences that direct its intracellular localization and regulate its signaling . Here we demonstrate that effector molecules involved in receptor trafficking and signaling interact with distinct, but overlapping, sequences in the CTLA-4 cytoplasmic domain . Using the yeast two-hybrid method, we demonstrate association of the mu2 subunit of AP-2, the clathrin-associated complex found in plasma membrane-associated coated pits, with the cytoplasmic tail of CTLA-4, but not CD28 . The mu1 subunit of AP-1, found in Golgi-associated coated pits, associated with neither CTLA-4 nor CD28 . Sequences required for interaction of mu2 and CTLA-4 were localized to residues, 161TTGVY in CTLA-4; this sequence is N-terminal to, but overlaps with, a previously identified SH2 binding motif, 165YVKM, involved in CTLA-4 signaling . Mu2 interacted preferentially with CTLA-4 when residue 165Y was nonphosphorylated, whereas a PI3 kinase SH2 domain interacted preferentially when 165Y was phosphorylated . In co-transfection experiments, both tyrosine residues in the cytoplasmic tail of CTLA-4 (165Y and 182Y) were phosphorylated by the T lymphocyte-associated tyrosine kinase, p56lck . Thus, phosphorylation of CTLA-4 residue 165Y may reciprocally regulate signaling and trafficking of CTLA-4 by determining which effector molecules bind to its cytoplasmic tail. J Virol, 1998 Feb, 72(2), 1297 - 307 A protein linkage map of the P2 nonstructural proteins of poliovirus; Cuconati A et al.; The yeast two-hybrid system was used to catalog all detectable interactions among the P2 nonstructural cleavage products of poliovirus type 1 (Mahoney) . Evidence has been obtained for specific associations among 2A(pro), 2BC, 2C, and 2B . Specifically, 2A(pro) can interact with itself and 2BC and its cleavage products (2B and 2C) interact in all possible combinations, with the exception of 2C/2C . Detected interactions were confirmed in vitro by a glutathione S-transferase pulldown assay, which allowed us to detect 2C/2C association . transdominant-negative mutants of 2B (K . Johnson and P . J . Sarnow, J . Virol . 65:4341-4349, 1991) were examined and were found to retain interaction with wild-type 2B, perhaps reflecting a need for 2B multimerization in viral RNA replication . The multimerization of 2B was examined further by screening a mutagenized library for 2B variants that have lost the ability to bind wild-type 2B . The screen identified two nonconservative missense mutations within a central hydrophobic region, as well as truncations and frameshifts that implicate the C terminus in homointeraction . Introduction of the missense mutations into the genome of the virus conferred a quasi-infectious phenotype, an observation strongly suggesting that the 2B/2B interaction is required for replication of the viral genome. Proteins, 1998 Jan, 30(1), 49 - 60 Feature-extraction from endopeptidase cleavage sites in mitochondrial targeting peptides; Schneider G et al.; Cleavage sites in nuclear-encoded mitochondrial protein targeting peptides (mTPs) from mammals, yeast, and plants have been analysed for characteristic physicochemical features using statistical methods, perceptrons, multilayer neural networks, and self-organizing feature maps . Three different sequence motifs were found, revealing loosely defined arginine motifs with Arg in positions -10, -3, and -2 . A self-organizing feature map was able to cluster these three types of endopeptidase target sites but did not identify any species-specific characteristics in mTPs . Neural networks were used to define local sequence features around precursor cleavage sites. J Med Virol, 1998 Jan, 54(1), 1 - 6 A study of the antigenicity and immunogenicity of a new hepatitis B vaccine using a panel of monoclonal antibodies; Waters JA et al.; The successful prevention of infection with hepatitis B virus (HBV) has been achieved by vaccination with purified hepatitis B surface antigen (HBsAg) . The ability of a novel synthetic HBV envelope antigen vaccine (Hep B-3, Hepagene; Medeva), which contains part of the pre-S1 and the complete pre-S2 regions and the whole of the S region and was produced in a mammalian cell line, to induce antibodies required for a protective immune response is of importance . In this study, the use of a panel of monoclonal antibodies known to bind to epitopes within the common "a" determinant has demonstrated that the epitopes present on this new vaccine are comparable to those found with plasma-derived HBsAg . In addition, the epitope specificity of the antibodies induced by this vaccine was examined and shown to accord well with previous results obtained using both a plasma-derived vaccine and a recombinant vaccine prepared in yeast. J Cell Biochem, 1998 Feb 1, 68(2), 174 - 85 Transcription of metallothionein isoform promoters is differentially regulated in cadmium-sensitive and -resistant CHO cells; Yu CW et al.; Transcription regulation of metallothionein (MT) isoform promoters was investigated in Chinese hamster ovary (CHO) K1 and MT gene amplified, cadmium-resistant (CdR) cells . The transfected promoter of Chinese hamster MTI and MTII genes can be activated in both cell lines by stimulation with Cd or Zn ions, although no MT mRNA can be detected in CHO K1 cells after challenge with metal ions . Neither MT promoter used in this study can be activated by induction with dexamethasone, regardless of whether a sequence homologous to glucocorticoid responsive element is present . During induction by metal ions, differential promoter activities of the MT genes occurs in both CHO K1 and CdR cells where MTII promoter has a stronger activity than that of MTI . As indicated by a time course study in both cell lines, the relative induction ratios of both MTI and MTII promoters are similar at each time interval . This result is consistent with a differential transcriptional factor-promoter interaction for the two MT promoters . By using the CHO K1 and CdR cells as a model system, the occurrence of autoregulation for yeast CUP1 (MT) gene was examined in mammalian cells . Both MT promoters consistently show a lower basal activity but a higher induction ratio in CHO K1 than CdR cells; a result different from that of yeast CUP1 gene . When MTF-1 mRNA was examined, no difference in relative quantity was observed in CHO K1 and in CdR cells treated with metal ions or with metal ions absent. Genes Chromosomes Cancer, 1998 Jan, 21(1), 70 - 3 Characterization of a t(8;13)(p11;q11-12) in an atypical myeloproliferative disorder; Smedley D et al.; Fourteen cases of an atypical myeloproliferative disorder associated with consistent translocations involving 8p11-12 have previously been described . A t(8;13)(p11;q11-12) was the most common, but variant t(8;9)(p11-12;q32-34) and t(6;8)(q27;p12) were also reported . Here we have used a series of yeast artificial chromosomes (YACs) derived from the 8p11 and 13q11-12 regions to analyse one of the t(8;13) cases by fluorescence in situ hybridization (FISH) . YACs flanking the 13q11-12 breakpoint and spanning the 8p11 breakpoint have been isolated . These YACs will facilitate characterization of the genes involved in this rearrangement. Genes Chromosomes Cancer, 1998 Jan, 21(1), 39 - 48 Minimal deletion of 3p13-->14.2 associated with immortalization of human uroepithelial cells; Vieten L et al.; Immortalization and tumorigenic transformation of many human cell types, including human uroepithelial cells (HUCs), are frequently associated with loss of genetic material from the short arm of chromosome 3 (3p) . In addition, losses of 3p have been observed in many human cancers including renal cell carcinoma, lung cancer, breast cancer, and bladder cancer . Genetic studies suggest that there are at least two regions on 3p in which tumor suppressor genes might be located, but the precise location of these genes is not known . We studied chromosome 3 losses that were specifically associated with immortalization of five independent human papilloma virus 16 (HPV16) E6- or E7-transformed HUCs . Cytogenetic analysis showed that the smallest common region of deletion was 3p14.1-->14.2 . Fluorescence in situ hybridization using a 3p13-->14-specific yeast artificial chromosome (YAC) contig showed the precise localization of the breakpoints to be in 3p13 and 3p14.2, thus defining the smallest common overlap of 3p deletions in HPV16 E6- or E7-immortalized HUCs . These results suggest the presence in this region of genes involved in the control of senescence in vitro and possibly tumorigenesis in vivo. J Biol Chem, 1998 Jan 16, 273(3), 1832 - 7 p38 kinase activity is essential for osmotic induction of mRNAs for HSP70 and transporter for organic solute betaine in Madin-Darby canine kidney cells; Sheikh-Hamad D et al.; In renal cells, hypertonicity induces genes for heat shock proteins (HSP70, alpha B-crystallin), as well as enzymes and transporters directly involved in the metabolism and transport of protective organic osmolytes . While heat shock proteins are induced by many stresses including osmotic stress, the induction of the osmolytes genes appears to be specific to osmotic stress . These two adaptive mechanisms allow kidney cells to survive and function in the hypertonic environment that exists on routine basis in kidney medulla . In mammalian cells, hypertonicity induces three mitogen-activated protein kinase pathways: ERK (extracellular regulated kinase), JNK (Jun N-terminal kinase), and p38 . ERK activation by osmotic stress is a consistent finding in many cells, but it is not essential for transcriptional regulation of mRNA for transporter of organic osmolyte betaine . While the growth of yeast cells on NaCl-supplemented medium is dependent on HOG1 pathway, it is still unclear which pathway mediates the adaptation to osmotic stress in mammalian cells . Here, we show that inhibition of p38 kinase activity, using the specific inhibitor SB203580 (4-(fluorophenyl)-2-(4-methylsulfonyl-phenyl)-5-(4-pyridyl) imidazole), abolishes the hypertonicity-mediated induction of mRNAs for HSP70 and betaine transporter in Madin-Darby canine kidney cells . The inhibition is dose-dependent and correlates with the in situ activity of native p38 kinase, determined as MAPKAPK-2 activity in cell extracts . As reported previously, the activities of ERK-1 and -2 were not affected by SB203580, but surprisingly, inhibition of native p38 kinase activity correlates with up-regulation of native JNK-1 activity in osmotically stressed cells . p38 mRNA is induced by hypertonic stress and is attenuated with p38 kinase inhibition . We also find that thermal induction of HSP70 mRNA is not affected by p38 kinase inhibition . Such findings suggest that p38 kinase activity is essential for the induction of genes involved in the adaptation of mammalian cells to osmotic stress and that the increased activity of JNK-1 during p38 kinase inhibition is consistent with regulation of JNK-1 by p38 kinase in osmotically stressed cells . In addition, the transduction pathways mediating HSP70 mRNA induction by different stresses appear to be divergent; osmotic induction of HSP70 is p38 kinase-dependent, while thermal induction is not. J Biol Chem, 1998 Jan 23, 273(4), 2379 - 83 Rack1, a receptor for activated protein kinase C, interacts with integrin beta subunit; Liliental J et al.; The integrin beta subunit cytoplasmic domains are important for activation-dependent cell adhesion and adhesion-dependent signaling events . We report an interaction between integrin beta subunit cytoplasmic domain and Rack1, a Trp-Asp (WD) repeat protein that has been shown to bind activated protein kinase C . The Rack1-binding site on integrin beta 2 subunit resides within a conserved, membrane-proximal region . In the yeast two-hybrid assay, WD repeats five to seven of Rack1 (Rack1-WD5/7) interact with integrin beta 1, beta 2, and beta 5 cytoplasmic domain . In eukaryotic cells, Rack1 co-immunoprecipitates with at least two different beta integrins, beta 1 integrins in 293T cells and beta 2 integrins in JY lymphoblastoid cells . Whereas Rack1-WD5/7 binds integrins constitutively, the association of full-length Rack1 to integrins in vivo requires a treatment with phorbol esters, which promotes cell spreading and adhesion . These findings suggest that Rack1 may link protein kinase C directly to integrins and participate in the regulation of integrin functions. J Biol Chem, 1998 Jan 23, 273(4), 2296 - 305 Interaction between the retinoid X receptor and transcription factor IIB is ligand-dependent in vivo; Leong GM et al.; The retinoid X receptor (RXR) influences gene activation through heterodimeric and homodimeric association with DNA and associates with TATA binding protein, TAF110, and cAMP response element-binding protein-binding protein; yet the molecular mechanisms responsible for gene activation by RXRs remain incompletely defined . Since the general transcription factor IIB (TFIIB) is a common target of sequence-specific transcriptional activators, we suspected that RXR might regulate target genes via an interaction with TFIIB . Coimmunoprecipitation, far Western analysis, and glutathione S-transferase binding studies indicated that murine RXR beta (mRXR beta) was capable of binding to human TFIIB in vitro . Functional analysis with a dual-hybrid yeast system and cotransfection assays revealed the interaction of mRXR beta with TFIIB to be ligand-dependent in vivo . Truncation experiments mapped the essential binding regions to the carboxyl region of mRXR beta (amino acids (aa) 254-389) and two regions in the carboxyl region of TFIIB (aa 178-201 and aa 238-271) . Furthermore, the delta 390-410 mRXR beta mutant bound to TFIIB in vitro but was not active in the dual-hybrid yeast system, suggesting that the extreme carboxyl region of RXR was required for in vivo interaction with TFIIB . These data indicate that interaction of mRXR beta with TFIIB is specific, direct, and ligand-dependent in vivo and suggest that gene activation by RXR involves TFIIB. J Biol Chem, 1998 Jan 23, 273(4), 1970 - 81 Multiubiquitin chain binding and protein degradation are mediated by distinct domains within the 26 S proteasome subunit Mcb1; Fu H et al.; The 26 S proteasome is a multisubunit proteolytic complex responsible for degrading eukaryotic proteins targeted by ubiquitin modification . Substrate recognition by the complex is presumed to be mediated by one or more common receptor(s) with affinity for multiubiquitin chains, especially those internally linked through lysine 48 . We have identified previously a candidate for one such receptor from diverse species, designated here as Mcb1 for Multiubiquitin chain-binding protein, based on its ability to bind Lys48-linked multiubiquitin chains and its location within the 26 S proteasome complex . Even though Mcb1 is likely not the only receptor in yeast, it is necessary for conferring resistance to amino acid analogs and for degrading a subset of ubiquitin pathway substrates such as ubiquitin-Pro-beta-galactosidase (Ub-Pro-beta-gal) (van Nocker, S., Sadis, S., Rubin, D.M., Glickman, M., Fu, H., Coux, O., Wefes, I., Finley, D., and Vierstra, R . D . (1996) Mol . Cell . Biol . 16, 6020-28) . To further define the role of Mcb1 in substrate recognition by the 26 S proteasome, a structure/function analysis of various deletion and site-directed mutants of yeast and Arabidopsis Mcb1 was performed . From these studies, we identified a single stretch of conserved hydrophobic amino acids (LAM/LALRL/V (ScMcb1 228-234 and At-Mcb1 226-232)) within the C-terminal half of each polypeptide that is necessary for interaction with Lys48-linked multiubiquitin chains . Unexpectedly, this domain was not essential for either Ub-Pro-beta-gal degradation or conferring resistance to amino acid analogs . The domain responsible for these two activities was mapped to a conserved region near the N terminus . Yeast and Arabidopsis Mcb1 derivatives containing an intact multiubiquitin-binding site but missing the N-terminal region failed to promote Ub-Pro-beta-gal degradation and even accentuated the sensitivity of the yeast delta mcb1 strain to amino acid analogs . This hypersensitivity was not caused by a gross defect in 26 S proteasome assembly as mutants missing either the N-terminal domain or the multiubiquitin chain-binding site could still associate with 26 S proteasome and generate a complex indistinguishable in size from that present in wild-type yeast . Together, these data indicate that residues near the N terminus, and not the multiubiquitin chain-binding site, are most critical for Mcb1 function in vivo. J Biol Chem, 1998 Jan 23, 273(4), 1859 - 62 Regulation of GRP1-catalyzed ADP ribosylation factor guanine nucleotide exchange by phosphatidylinositol 3,4,5-trisphosphate; Klarlund JK et al.; Cellular levels of phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) are rapidly elevated in response to activation of growth factor receptor tyrosine kinases . This polyphosphoinositide binds the pleckstrin homology (PH) domain of GRP1, a protein that also contains 200 residues with high sequence similarity to a segment of the yeast Sec7 protein that functions as an ADP ribosylation exchange factor (ARF) (Klarlund, J., Guilherme, A., Holik, J . J., Virbasius, J . V., Chawla, A., and Czech, M . P . (1997) Science 275, 1927-1930) . Here we show that dioctanoyl PtdIns(3,4,5)P3 binds the PH domain of GRP1 with a Kd = 0.5 microM, an affinity 2 orders of magnitude greater than dioctanoyl-PtdIns(4,5)P2 . Further, the Sec7 domain of GRP1 is found to catalyze guanine nucleotide exchange of ARF1 and -5 but not ARF6 . Importantly, PtdIns(3,4,5)P3, but not PtdIns(4,5)P2, markedly enhances the ARF exchange activity of GRP1 in a reaction mixture containing dimyristoylphosphatidylcholine micelles, 3-{(3-cholamidopropyl)dimethylammonio}-1-propanesulfonic acid, and a low concentration of sodium cholate . PtdIns(3,4,5)P3-mediated ARF nucleotide exchange through GRP1 is selectively blocked by 100 microM inositol 1,3,4,5-tetrakisphosphate, which also binds the PH domain of GRP1 . Taken together, these data are consistent with the hypothesis that selective recruitment of GRP1 to PtdIns(3,4,5)P3 in membranes activates ARF1 and -5, known regulators of intracellular membrane trafficking. Methods, 1997 Nov, 13(3), 271 - 80 Replication initiation point mapping; Gerbi SA et al.; Replication in eukaryotes is bidirectional and semi-discontinuous . This asymmetry provides the basis for mapping the origin of bidirectional replication (OBR), which is the transition point from discontinuous to continuous synthesis . The regions of each DNA strand complementary to the leading strand or lagging strand can be measured by the methods of imbalanced DNA synthesis or Okazaki fragment distribution, respectively . The resolution of both of these hybridization-based procedures is a few hundred base pairs . Nucleotide resolution was previously achieved for viral origins by mapping the initiation sites of Okazaki fragments on sequencing gels . To overcome the background caused by nicked DNA, all DNA ends were phosphorylated, RNA primers were removed from the Okazaki fragments by NaOH hydrolysis, and the hydroxyl ends thus created were phosphorylated with 32P . Unfortunately, this method was not sensitive enough to map eukaryotic cellular origins . A new method, replication initiation point (RIP) mapping, that is 1000-fold more sensitive and has been applied to yeast ARS1 where the OBR is mapped to and 18-bp region from within element B1 toward B2 is described here . RIP mapping utilizes Vent (exo-) polymerase to extend from a labeled primer to the DNA/RNA junctions of nascent strand template in an asynchronous population of replicating molecules . The DNA is digested with lambda-exonuclease prior to primer extension to remove nicked contaminating DNA. Methods, 1997 Nov, 13(3), 247 - 57 Mapping replication origins, pause sites, and termini by neutral/alkaline two-dimensional gel electrophoresis; Huberman JA; Neutral/alkaline two-dimensional gel electrophoresis is a robust, easily interpretable, sensitive technique that has yielded insights about the in vivo replication of many types of DNA, from multicopy yeast plasmids to single-copy chromosome regions in unsynchronized mammalian cells . It can provide information about directions of replication fork movement and locations of replication origins, termini, and pause sites . Especially when combined with its partner technique, neutral/neutral two-dimensional gel electrophoresis, it is a method of choice for investigation of unknown situations. Genomics, 1997 Dec 15, 46(3), 520 - 4 Identification of a human LMX1 (LMX1.1)-related gene, LMX1.2: tissue-specific expression and linkage mapping on chromosome 9; Iannotti CA et al.; LMX1 is a LIM-homeodomain (LIM-HD)-containing protein expressed selectively in insulin-producing beta-cell lines, and it it has been shown to activate insulin gene transcription . The human LMX1 gene was mapped by fluorescence in situ hybridization to chromosome region 1q22-q23, yet Church et al . (1994, Nat . Genet . 6: 98-105) identified two exon-trapping products from human chromosome 9 that were highly homologous to hamster LMX1 . In the current study, we demonstrate tissue-specific expression of an LMX1 (now known as LMX1.1)-related gene, named LMX1.2 . The chicken C-LMX1 gene, recently cloned using the hamster LMX1.1 sequence and shown to specify dorsal cell fate during vertebrate limb development (9), is actually more related to human LMX1.2 than LMX1.1 . We have identified a unique simple sequence repeat polymorphic marker (hLMX1.2CA1) in a P1 genomic clone containing the human LMX1.2 gene and genetically mapped the marker on chromosome 9 between markers D9S1825 and D9S290 with odds of at least 1000:1 . In addition, we localized the human LMX1.1 gene to three CEPH "B" yeast artificial chromosome clones (907A11, 935B12, and 947B2), along with two nearby polymorphic markers (D1S426 and D1S194)) . Identification of this new LIM-HD-related gene may provide the opportunity to elucidate further the function of LIM class homeobox genes . Nearby polymorphic markers will be useful in testing the hypothesis that mutations in these LIM-HD genes result in genetic diseases such as non-insulin-dependent diabetes mellitus. Genomics, 1997 Dec 15, 46(3), 491 - 4 Subregional localization of 21 chromosome 7-specific expressed sequence tags (ESTs) by FISH using newly identified YACs and P1s; Morton SM et al.; Twenty-one putative chromosome 7-derived expressed sequence tags (ESTs) identified 33 yeast artificial chromosomes (YACs) or P1 clones, which were then used as reagents for physical mapping . FISH mapping established that the ESTs contained within these clones were distributed throughout chromosome 7, with all major cytogenetic bands represented, except 7p13-p15, 7p11, 7q31.2, and 7q35 . Each EST sequence identified at least one other sequence in publicly available databases (using search tools such as BLASTN, basic local alignment search tool), and many of the ESTs identified cDNAs and several genomic DNA sequences . However, 7 ESTs did not identify highly significant matches (P < 1 x 10(-5)) . Only one (EST01924-D7S2281E) failed to identify any other EST from the dbEST homology searches . BLAST analysis identified at least five genes from EST sequence comparisons: protein tyrosine phosphatase zeta (PTPRZ, also known as RPTPZ) (EST02092), which we had mapped to 7q31.3, in agreement with previous studies; cAMP-dependent protein kinase regulatory subunit bI (EST01644); rat integral membrane glycoprotein (EST00085); human IFNAR gene for interferon alpha/beta receptor (EST00817); and rat 14-3.3 protein gamma subtype (putative protein kinase C regulatory protein) (EST00762) . These ESTs will help to develop the map of chromosome 7, which integrates physical, transcriptional, and cytogenetic data, as well as to provide candidate disease genes for chromosome 7-specific disorders. Dev Biol, 1997 Dec 15, 192(2), 228 - 46 The cut-homeodomain transcriptional activator HNF-6 is coexpressed with its target gene HNF-3 beta in the developing murine liver and pancreas; Rausa F et al.; Murine hepatocyte nuclear factor-3 beta (HNF-3 beta) protein is a member of a large family of developmentally regulated transcription factors that share homology in the winged helix/fork head DNA binding domain and that participate in embryonic pattern formation . HNF-3 beta also mediates cell-specific transcription of genes important for the function of hepatocytes, intestinal and bronchiolar epithelial, and pancreatic acinar cells . We have previously identified a liver-enriched transcription factor, HNF-6, which is required for HNF-3 beta promoter activity and also recognizes the regulatory region of numerous hepatocyte-specific genes . In this study we used the yeast one-hybrid system to isolate the HNF-6 cDNA, which encodes a cut-homeodomain-containing transcription factor that binds with the same specificity as the liver HNF-6 protein . Cotransfection assays demonstrate that HNF-6 activates expression of a reporter gene driven by the HNF-6 binding site from either the HNF-3 beta or transthyretin (TTR) promoter regions . We used interspecific backcross analysis to determine that murine Hnf6 gene is located in the middle of mouse chromosome 9 . In situ hybridization studies of staged specific embryos demonstrate that HNF-6 and its potential target gene, HNF-3 beta, are coexpressed in the pancreatic and hepatic diverticulum . More detailed analysis of HNF-6 and HNF-3 beta's developmental expression patterns provides evidence of colocalization in hepatocytes, intestinal epithelial, and in the pancreatic ductal epithelial and exocrine acinar cells . The expression patterns of these two transcription factors do not overlap in other endoderm-derived tissues or the neurotube . We also found that HNF-6 is also abundantly expressed in the dorsal root ganglia, the marginal layer, and the midbrain . At day 18 of gestation and in the adult pancreas, HNF-6 and HNF-3 beta transcripts colocalize in the exocrine acinar cells, but their expression patterns diverge in other pancreatic epithelium . HNF-6, but not HNF-3 beta, expression continues in the pancreatic ductal epithelium, whereas only HNF-3 beta becomes restricted to the endocrine cells of the islets of Langerhans . We discuss these expression patterns with respect to specification of hepatocytes and differentiation of the endocrine and exocrine pancreas. J Biol Chem, 1998 Jan 16, 273(3), 1749 - 54 HAH1 is a copper-binding protein with distinct amino acid residues mediating copper homeostasis and antioxidant defense; Hung IH et al.; HAH1 is a 68-amino acid protein originally identified as a human homologue of Atx1p, a multi-copy suppressor of oxidative injury in sod1 delta yeast . Molecular modeling of HAH1 predicts a protein structure of two alpha-helices overlaying a four-stranded antiparallel beta-sheet with a potential metal binding site involving two conserved cysteine residues . Consistent with this model, in vitro studies with recombinant HAH1 directly demonstrated binding of Cu(I), and site-directed mutagenesis identified these cysteine residues as copper ligands . Expression of wild type and mutant HAH1 in atx1 delta yeast revealed the essential role of these cysteine residues in copper trafficking to the secretory compartment in vivo, as expression of a Cys-12/Cys-15 double mutant abrogated copper incorporation into the multicopper oxidase Fet3p . In contrast, mutation of the highly conserved lysine residues in the carboxyl terminus of HAH1 had no effect on copper trafficking to the secretory pathway but eliminated the antioxidant function of HAH1 in sod1 delta yeast . Taken together, these data support the concept of a unique copper coordination environment in HAH1 that permits this protein to function as an intracellular copper chaperone mediating distinct biological processes in eucaryotic cells. J Biol Chem, 1998 Jan 16, 273(3), 1591 - 5 Plasma membrane Ca2+ ATPase isoform 4b binds to membrane-associated guanylate kinase (MAGUK) proteins via their PDZ (PSD-95/Dlg/ZO-1) domains; Kim E et al.; Plasma membrane Ca2+ ATPases are P-type pumps important for intracellular Ca2+ homeostasis . The extreme C termini of alternatively spliced "b"-type Ca2+ pump isoforms resemble those of K+ channels and N-methyl-D-aspartate receptor subunits that interact with channel-clustering proteins of the membrane-associated guanylate kinase (MAGUK) family via PDZ domains . Yeast two-hybrid assays demonstrated strong interaction of Ca2+ pump 4b with the PDZ1 + 2 domains of several mammalian MAGUKs . Pump 4b and PSD-95 could be co-immunoprecipitated from COS-7 cells overexpressing these proteins . Surface plasmon resonance revealed that a C-terminal pump 4b peptide interacted with the PDZ1 + 2 domains of hDlg with nanomolar affinity (KD = 1.6 nM), whereas binding to PDZ3 was in the micromolar range (KD = 1.2 microM) . In contrast, the corresponding C-terminal peptide of Ca2+ pump 2b interacted weakly with PDZ1 + 2 and not at all with PDZ3 of hDlg . Ca2+ pump 4b bound strongly to PDZ1 + 2 + 3 of hDlg on filter assays, whereas isoform 2b bound weakly, and the splice variants 2a and 4a failed to bind . Together, these data demonstrate a direct physical binding of Ca2+ pump isoform 4b to MAGUKs via their PDZ domains and reveal a novel role of alternative splicing within the family of plasma membrane Ca2+ pumps . Alternative spl