S. cerevisiae

Proc Natl Acad Sci U S A, 1999 Jan 19, 96(2), 441 - 6
The Cdc6p nucleotide-binding motif is required for loading mcm proteins onto chromatin; Weinreich M et al.; Cdc6p has an essential function in the mechanism and regulation of the initiation of DNA replication . Budding yeast Cdc6p binds to chromatin near autonomously replicating sequence elements in late M to early G1 phase through an interaction with Origin Recognition Complex or another origin-associated factor . It then facilitates the subsequent loading of the Mcm family of proteins near autonomously replicating sequence elements by an unknown mechanism . All Cdc6p homologues contain a bipartite Walker ATP-binding motif that suggests that ATP binding or hydrolysis may regulate Cdc6p activity . To determine whether these motifs are important for Cdc6p activity, mutations were made in conserved residues of the Walker A and B motifs . Substitution of lysine 114 to alanine (K114A) in the Walker A motif results in a temperature-sensitive phenotype in yeast and slower progression into S phase at the permissive temperature . A K114E mutation is lethal . The Cdc6(K114E) protein binds to chromatin but fails to promote loading of the Mcm proteins, suggesting that ATP binding is essential for this activity . The mutant arrests with a G1 DNA content but retains the ability to restrain mitosis in the absence of DNA replication, unlike depletion of Cdc6p . In contrast, Cdc6p containing a double alanine mutation in the Walker B motif, DE(223, 224)AA, is functional, and the mutant exhibits an apparently normal S phase . These results suggest that Cdc6p nucleotide binding is important for establishing the prereplicative complex at origins of DNA replication and that the amino terminus of Cdc6p is required for blocking entry into mitosis.

Proc Natl Acad Sci U S A, 1999 Jan 19, 96(2), 343 - 8
Persistence of an alternate chromatin structure at silenced loci in vitro; Ansari A et al.; In Saccharomyces cerevisiae, transcriptional repression at the HM mating-type loci and telomeres results from the formation of a heterochromatin-like structure . Silencing requires at least three Sir proteins (Sir2p-4p), which are recruited to chromatin by silencers at the HM loci and TG1-3 tracts at telomeres . Sir proteins and telomeres colocalize at the nuclear periphery, suggesting that this subnuclear position may also contribute to transcriptional repression . To evaluate the contribution of nuclear context to silencing, we developed methodology to isolate silent chromatin for analysis in vitro . Site-specific recombination was used in vivo to produce DNA rings from the silent HMR locus, and differential centrifugation was used to isolate the rings from whole-cell lysate . The partially purified rings retained many of the intracellular hallmarks of transcriptionally repressed domains . Specifically, rings from repressed strains were resistant to restriction endonuclease digestion, bore an altered DNA topology, and were associated with Sir3p . The recombination approach also was used to form rings from HMR that lacked silencers . Despite the uncoupling of these cis-acting regulatory elements, similar but nonidentical results were obtained . We conclude that an alternate chromatin structure at silent loci can persist in vitro in the absence of silencers and nuclear compartmentalization.

Nature, 1999 Jan 7, 397(6714), 69 - 72
GABA(A)-receptor-associated protein links GABA(A) receptors and the cytoskeleton; Wang H et al.; Type-A receptors for the neurotransmitter GABA (gamma-aminobutyric acid) are ligand-gated chloride channels that mediate inhibitory neurotransmission . Each subunit of the pentameric receptor protein has ligand-binding sites in the amino-terminal extracellular domain and four membrane-spanning regions, one of which forms a wall of the ion channel . Each subunit also has a large intracellular loop that may be a target for protein kinases and be required for subcellular targeting and membrane clustering of the receptor, perhaps by anchoring the receptor to the cytoskeleton . Neurotransmitter receptors need to be positioned in high density in the cell membrane at sites postsynaptic to nerve terminals releasing that neurotransmitter . Other members of the superfamily of ligand-gated ion-channel receptors associate in postsynaptic-membrane clusters by binding to the proteins rapsyn or gephyrin . Here we identify a new cellular protein, GABA(A)-receptor-associated protein (GABARAP), which can interact with the gamma2 subunit of GABA(A) receptors . GABARAP binds to GABA(A) receptors both in vitro and in vivo, and co-localizes with the punctate staining of GABA(A) receptors on cultured cortical neurons . Sequence analysis shows similarity between GABARAP and light chain-3 of microtubule-associated proteins 1A and 1B . Moreover, the N terminus of GABARAP is highly positively charged and features a putative tubulin-binding motif . The interactions among GABA(A) receptors, GABARAP and tubulin suggest a mechanism for the targeting and clustering of GABA(A) receptors.

Nature, 1999 Jan 7, 397(6714), 66 - 9
The protein MAP-1B links GABA(C) receptors to the cytoskeleton at retinal synapses; Hanley JG et al.; The ionotropic type-A and type-C receptors for the neurotransmitter gamma-aminobutyric acid (GABA(A) and GABA(C) receptors) are the principal sites of fast synaptic inhibition in the central nervous system, but it is not known how these receptors are localized at GABA-dependent synapses . GABA(C) receptors, which are composed of rho-subunits, are expressed almost exclusively in the retina of adult vertebrates, where they are enriched on bipolar cell axon terminals . Here we show that the microtubule-associated protein 1B (MAP-1B) specifically interacts with the GABA(C) rho1 subunit but not with GABA(A) receptor subunits . Furthermore, GABA(C) receptors and MAP-1B co-localize at postsynaptic sites on bipolar cell axon terminals . Co-expression of MAP-1B and the rho1 subunit in COS cells results in a dramatic redistribution of the rho1 subunit . Our observations suggest a novel mechanism for localizing ionotropic GABA receptors to synaptic sites . This mechanism, which is specific for GABA(C) but not GABA(A) receptors, may allow these receptor subtypes, which have distinct physiological and pharmacological properties, to be differentially localized at inhibitory synapses.

Diabetes, 1999 Jan, 48(1), 121 - 7
Regulation of putative fatty acid transporters and Acyl-CoA synthetase in liver and adipose tissue in ob/ob mice; Memon RA et al.; The hyperlipidemia associated with obesity and type 2 diabetes is caused by an increase in hepatic triglyceride synthesis and secretion that is secondary to an increase in de novo lipogenesis, a decrease in fatty acid (FA) oxidation, and an increase in the flux of peripherally derived FA to the liver . The uptake of FA across the plasma membrane may be mediated by three distinct proteins--FA translocase (FAT), plasma membrane FA binding protein (FABP-pm), and FA transport protein (FATP)--that have recently been characterized . Acyl-CoA synthetase (ACS) enhances the uptake of FAs by catalyzing their activation to acyl-CoA esters for subsequent use in anabolic or catabolic pathways . In this study, we examine the mRNA levels of FAT, FABP-pm, FATP, and ACS in the liver and adipose tissue of genetically obese (ob/ob) mice and their control littermates . FAT mRNA levels were 15-fold higher in liver and 60-80% higher in adipose tissue of ob/ob mice . FABP-pm mRNA levels were twofold higher in liver and 50% higher in adipose tissue of ob/ob mice . FATP mRNA levels were not increased in liver or adipose tissue . ACS mRNA levels were higher in adipose tissue but remained unchanged in liver . However, the distribution of ACS activity associated with mitochondria and microsomes in liver was altered in ob/ob mice . In control littermates, 61% of ACS activity was associated with mitochondria and 39% with microsomes, whereas in ob/ob mice 34% of ACS activity was associated with mitochondria and 66% with microsomes; this distribution would make more FA available for esterification, rather than oxidation, in ob/ob mouse liver . Taken together, our results suggest that the upregulation of FAT and FABP-pm mRNAs may increase the uptake of FA in adipose tissue and liver in ob/ob mice, which, coupled with an increase in microsomal ACS activity in liver, will enhance the esterification of FA and support the increased triglyceride synthesis and VLDL production that characterizes obesity and type 2 diabetes.

FEBS Lett, 1998 Dec 28, 441(3), 437 - 40
The C2 cytosolic loop of adenylyl cyclase interacts with the activated form of G alpha s; Torrejon M et al.; Using the yeast two-hybrid system, we studied the physical interaction between the complete C1 and C2 cytosolic domains of Xenopus laevis type 9 (xl9C1, xl9C2) and the C2 domain of rat type 6 (r6C2) adenylyl cyclase (AC) . Heterodimerization between xl9C1 and xl9C2 and homodimerization between C2 (but not C1) domains was observed . Interaction between C2 and human G alpha s (hG alpha s) was also detected and was dependent on G alpha s activation . In contrast X . laevis G alpha s (xlG alpha s), which is 92% identical to hG alpha s, was unable to interact with any of the three AC cytosolic domains tested, corroborating previous findings that showed no effector activation . Through the construction of chimeras, we demonstrated that the amino-terminal half of xlG alpha s was responsible for the lack of interaction with AC . Chimeras between mouse G alpha i2 and G alpha s (N-mG alpha i2/C-G alpha s), that have previously shown to activate AC to a higher extent than wild-type G alpha s, also interacted with the C2 cytosolic domain and with a higher affinity . Interestingly, N-mG alpha i2/C-xlG alpha s chimera was not only able to interact with C2 but also with the C1 cytosolic domain.

Annu Rev Cell Dev Biol, 1998, 14, 459 - 85
Intracellular signaling from the endoplasmic reticulum to the nucleus; Chapman R et al.; Cells respond to an accumulation of unfolded proteins in the endoplasmic reticulum (ER) by increasing transcription of genes encoding ER resident proteins . The information is transmitted from the ER lumen to the nucleus by an intracellular signaling pathway called the unfolded protein response (UPR) . Recent work has shown that this signaling pathway utilizes several novel mechanisms, including translational attenuation and a regulated mRNA splicing step . In this review we aim to integrate these recent advances with current knowledge about maintenance of ER composition and abundance.

Annu Rev Cell Dev Biol, 1998, 14, 305 - 38
Signaling to the actin cytoskeleton; Schmidt A et al.; The actin cytoskeleton is a highly dynamic network composed of actin polymers and a large variety of associated proteins . The main functions of the actin cytoskeleton are to mediate cell motility and cell shape changes during the cell cycle and in response to extracellular stimuli, to organize the cytoplasm, and to generate mechanical forces within the cell . The reshaping and functions of the actin cytoskeleton are regulated by signaling pathways . Here we broadly review the actin cytoskeleton and the signaling pathways that regulate it . We place heavy emphasis on the yeast actin cytoskeleton.

Mol Cell Biol, 1999 Feb, 19(2), 1605 - 15
Human TFIIIC relieves chromatin-mediated repression of RNA polymerase III transcription and contains an intrinsic histone acetyltransferase activity; Kundu TK et al.; Human TFIIIC is a multisubunit factor that is essential for transcription by RNA polymerase III on tRNA and virus-associated RNA genes and initiates preinitiation complex assembly by direct recognition of promoter elements . We show that highly purified TFIIIC, at concentrations above those sufficient for transcription of naked DNA templates, effectively relieves nucleosome-mediated repression on an in vitro-reconstituted chromatin template . Highly purified TFIIIC alone can bind to the A and B boxes of a tRNA gene within a chromatin template and, further, displays a histone acetyltransferase activity that is intrinsic to at least one (and probably three) of its subunits . The possibility of a direct link between TFIIIC-dependent chromatin transcription and acetyltransferase activities is suggested by the partial loss of these activities, but not DNA transcription activity, following pretreatment of TFIIIC with p-hydroxymercuribenzoic acid.

Mol Cell Biol, 1999 Feb, 19(2), 1547 - 57
Interactions between a nuclear transporter and a subset of nuclear pore complex proteins depend on Ran GTPase; Seedorf M et al.; Proteins to be transported into the nucleus are recognized by members of the importin-karyopherin nuclear transport receptor family . After docking at the nuclear pore complex (NPC), the cargo-receptor complex moves through the aqueous pore channel . Once cargo is released, the importin then moves back through the channel for new rounds of transport . Thus, importin and exportin, another member of this family involved in export, are thought to continuously shuttle between the nuclear interior and the cytoplasm . In order to understand how nuclear transporters traverse the NPC, we constructed functional protein fusions between several members of the yeast importin family, including Pse1p, Sxm1p, Xpo1p, and Kap95p, and the green fluorescent protein (GFP) . Complexes containing nuclear transporters were isolated by using highly specific anti-GFP antibodies . Pse1-GFP was studied in the most detail . Pse1-GFP is in a complex with importin-alpha and -beta (Srp1p and Kap95p in yeast cells) that is sensitive to the nucleotide-bound state of the Ran GTPase . In addition, Pse1p associates with the nucleoporins Nsp1p, Nup159p, and Nup116p, while Sxm1p, Xpo1p, and Kap95p show different patterns of interaction with nucleoporins . Association of Pse1p with nucleoporins also depends on the nucleotide-bound state of Ran; when Ran is in the GTP-bound state, the nucleoporin association is lost . A mutant form of Pse1p that does not bind Ran also fails to interact with nucleoporins . These data indicate that transport receptors such as Pse1p interact in a Ran-dependent manner with certain nucleoporins . These nucleoporins may represent major docking sites for Pse1p as it moves in or out of the nucleus via the NPC.

Mol Cell Biol, 1999 Feb, 19(2), 1518 - 25
Nip7p interacts with Nop8p, an essential nucleolar protein required for 60S ribosome biogenesis, and the exosome subunit Rrp43p; Zanchin NI et al.; NIP7 encodes a conserved Saccharomyces cerevisiae nucleolar protein that is required for 60S subunit biogenesis (N . I . T . Zanchin, P . Roberts, A . DeSilva, F . Sherman, and D . S . Goldfarb, Mol . Cell . Biol . 17:5001-5015, 1997) . Rrp43p and a second essential protein, Nop8p, were identified in a two-hybrid screen as Nip7p-interacting proteins . Biochemical evidence for an interaction was provided by the copurification on immunoglobulin G-Sepharose of Nip7p with protein A-tagged Rrp43p and Nop8p . Cells depleted of Nop8p contained reduced levels of free 60S ribosomes and polysomes and accumulated half-mer polysomes . Nop8p-depleted cells also accumulated 35S pre-rRNA and an aberrant 23S pre-rRNA . Nop8p-depleted cells failed to accumulate either 25S or 27S rRNA, although they did synthesize significant levels of 18S rRNA . These results indicate that 27S or 25S rRNA is degraded in Nop8p-depleted cells after the section containing 18S rRNA is removed . Nip7p-depleted cells exhibited the same defects as Nop8p-depleted cells, except that they accumulated 27S precursors . Rrp43p is a component of the exosome, a complex of 3'-to-5' exonucleases whose subunits have been implicated in 5.8S rRNA processing and mRNA turnover . Whereas both green fluorescent protein (GFP)-Nop8p and GFP-Nip7p localized to nucleoli, GFP-Rrp43p localized throughout the nucleus and to a lesser extent in the cytoplasm . Distinct pools of Rrp43p may interact both with the exosome and with Nip7p, possibly both in the nucleus and in the cytoplasm, to catalyze analogous reactions in the multistep process of 60S ribosome biogenesis and mRNA turnover.

Mol Cell Biol, 1999 Feb, 19(2), 1460 - 9
Cyclin E associates with BAF155 and BRG1, components of the mammalian SWI-SNF complex, and alters the ability of BRG1 to induce growth arrest; Shanahan F et al.; SWI-SNF complexes have been implicated in transcriptional regulation by chromatin remodeling . We have identified an interaction between two components of the mammalian SWI-SNF complex and cyclin E, an essential cell cycle regulatory protein required for G1/S transition . BRG1 and BAF155, mammalian homologs of yeast SWI2 and SWI3, respectively, are found in cyclin E complexes and are phosphorylated by cyclin E-associated kinase activity . In this report, we show that overexpression of BRG1 causes growth arrest and induction of senescence-associated beta-galactosidase activity, which can be overcome by cyclin E . Our results suggest that cyclin E may modulate the activity of the SWI-SNF apparatus to maintain the chromatin in a transcriptionally permissive state.

Mol Cell Biol, 1999 Feb, 19(2), 1438 - 49
p73 function is inhibited by tumor-derived p53 mutants in mammalian cells; Di Como CJ et al.; The p53 tumor suppressor protein, found mutated in over 50% of all human tumors, is a sequence-specific transcriptional activator . Recent studies have identified a p53 relative, termed p73 . We were interested in determining the relative abilities of wild-type and mutant forms of p53 and p73alpha and -beta isoforms to transactivate various p53-responsive promoters . We show that both p73alpha and p73beta activate the transcription of reporters containing a number of p53-responsive promoters in the p53-null cell line H1299 . However, a number of significant differences were observed between p53 and p73 and even between p73alpha and p73beta . Additionally, a Saccharomyces cerevisiae-based reporter assay revealed a broad array of transcriptional transactivation abilities by both p73 isoforms at 37 degreesC . Recent data have shown that p73 can associate with p53 by the yeast two-hybrid assay . When we examined complex formation in transfected mammalian cells, we found that p73alpha coprecipitates with mutant but not wild-type p53 . Since many tumor-derived p53 mutants are capable of inhibiting transactivation by wild-type p53, we tested the effects of two representative hot-spot mutants (R175H and R248W) on p73 . By cotransfecting p73alpha along with either p53 mutant and a p53-responsive reporter, we found that both R175H and R248W reduces the transcriptional activity of p73alpha . This decrease in transcriptional activity is correlated with the reduced ability of p73alpha to promote apoptosis in the presence of tumor-derived p53 mutants . Our data suggest the possibility that in some tumor cells, an outcome of the expression of mutant p53 protein may be to interfere with the endogenous p73 protein.

Mol Cell Biol, 1999 Feb, 19(2), 1202 - 9
p53 sites acetylated in vitro by PCAF and p300 are acetylated in vivo in response to DNA damage; Liu L et al.; The p53 tumor suppressor protein is a sequence-specific transcription factor that modulates the response of cells to DNA damage . Recent studies suggest that full transcriptional activity of p53 requires the coactivators CREB binding protein (CBP)/p300 and PCAF . These coactivators interact with each other, and both possess intrinsic histone acetyltransferase activity . Furthermore, p300 acetylates p53 to activate its sequence-specific DNA binding activity in vitro . In this study, we demonstrate that PCAF also acetylates p53 in vitro at a lysine residue distinct from that acetylated by p300 and thereby increases p53's ability to bind to its cognate DNA site . We have generated antibodies to acetylated p53 peptides at either of the two lysine residues that are targeted by PCAF or p300 and have demonstrated that these antibodies are highly specific for both acetylation and the particular site . Using these antibodies, we detect acetylation of these sites in vivo, and interestingly, acetylation at both sites increases in response to DNA-damaging agents . These data indicate that site-specific acetylation of p53 increases under physiological conditions that activate p53 and identify CBP/p300 and PCAF as the probable enzymes that modify p53 in vivo.

Mol Cell Biol, 1999 Feb, 19(2), 1159 - 70
The trithorax group gene moira encodes a brahma-associated putative chromatin-remodeling factor in Drosophila melanogaster; Crosby MA et al.; The genes of the trithorax group (trxG) in Drosophila melanogaster are required to maintain the pattern of homeotic gene expression that is established early in embryogenesis by the transient expression of the segmentation genes . The precise role of each of the diverse trxG members and the functional relationships among them are not well understood . Here, we report on the isolation of the trxG gene moira (mor) and its molecular characterization . mor encodes a fruit fly homolog of the human and yeast chromatin-remodeling factors BAF170, BAF155, and SWI3 . mor is widely expressed throughout development, and its 170-kDa protein product is present in many embryonic tissues . In vitro, MOR can bind to itself and it interacts with Brahma (BRM), an SWI2-SNF2 homolog, with which it is associated in embryonic nuclear extracts . The leucine zipper motif of MOR is likely to participate in self-oligomerization; the equally conserved SANT domain, for which no function is known, may be required for optimal binding to BRM . MOR thus joins BRM and Snf5-related 1 (SNR1), two known Drosophila SWI-SNF subunits that act as positive regulators of the homeotic genes . These observations provide a molecular explanation for the phenotypic and genetic relationships among several of the trxG genes by suggesting that they encode evolutionarily conserved components of a chromatin-remodeling complex.

Mol Cell Biol, 1999 Feb, 19(2), 1056 - 67
A complex containing RNA polymerase II, Paf1p, Cdc73p, Hpr1p, and Ccr4p plays a role in protein kinase C signaling; Chang M et al.; Yeast contains at least two complex forms of RNA polymerase II (Pol II), one including the Srbps and a second biochemically distinct form defined by the presence of Paf1p and Cdc73p (X . Shi et al., Mol . Cell . Biol . 17:1160-1169, 1997) . In this work we demonstrate that Ccr4p and Hpr1p are components of the Paf1p-Cdc73p-Pol II complex . We have found many synthetic genetic interactions between factors within the Paf1p-Cdc73p complex, including the lethality of paf1Delta ccr4Delta, paf1Delta hpr1Delta, ccr4Delta hpr1Delta, and ccr4Delta gal11Delta double mutants . In addition, paf1Delta and ccr4Delta are lethal in combination with srb5Delta, indicating that the factors within and between the two RNA polymerase II complexes have overlapping essential functions . We have used differential display to identify several genes whose expression is affected by mutations in components of the Paf1p-Cdc73p-Pol II complex . Additionally, as previously observed for hpr1Delta, deleting PAF1 or CDC73 leads to elevated recombination between direct repeats . The paf1Delta and ccr4Delta mutations, as well as gal11Delta, demonstrate sensitivity to cell wall-damaging agents, rescue of the temperature-sensitive phenotype by sorbitol, and reduced expression of genes involved in cell wall biosynthesis . This unusual combination of effects on recombination and cell wall integrity has also been observed for mutations in genes in the Pkc1p-Mpk1p kinase cascade . Consistent with a role for this novel form of RNA polymerase II in the Pkc1p-Mpk1p signaling pathway, we find that paf1Delta mpk1Delta and paf1Delta pkc1Delta double mutants do not demonstrate an enhanced phenotype relative to the single mutants . Our observation that the Mpk1p kinase is fully active in a paf1Delta strain indicates that the Paf1p-Cdc73p complex may function downstream of the Pkc1p-Mpk1p cascade to regulate the expression of a subset of yeast genes.

Mol Cell Biol, 1999 Feb, 19(2), 1049 - 55
Selective interaction of vitamin D receptor with transcriptional coactivators by a vitamin D analog; Takeyama K et al.; The nuclear vitamin D receptor (VDR) is a member of a nuclear receptor superfamily and acts as a ligand-dependent transcription factor . A family of cotranscriptional activators (SRC-1, TIF2, and AIB-1) interacts with and activates the transactivation function of nuclear receptors in a ligand-dependent way . We examined interaction of VDR with these coactivators that was induced by several vitamin D analogs, since they exert differential subsets of the biological action of vitamin D through unknown mechanisms . Unlike other vitamin D analogs tested, OCT (22-oxa-1alpha,25-dihydroxyvitamin D3) induced interaction of VDR with TIF2 but not with SRC-1 or AIB-1 . Consistent with these interactions, only TIF2 was able to potentiate the transactivation function of VDR bound to OCT . Thus, the present findings suggest that the structure of VDR is altered in a vitamin D analog-specific way, resulting in selective interactions of VDR with coactivators . Such selective interaction of coactivators with VDR may specify the array of biological actions of a vitamin D analog like OCT, possibly through activating a particular set of target gene promoters.

Mol Cell Biol, 1999 Feb, 19(2), 1025 - 37
Discrimination between NL1- and NL2-mediated nuclear localization of the glucocorticoid receptor; Savory JG et al.; Glucocorticoid receptor (GR) cycles between a free liganded form that is localized to the nucleus and a heat shock protein (hsp)-immunophilin-complexed, unliganded form that is usually localized to the cytoplasm but that can also be nuclear . In addition, rapid nucleocytoplasmic exchange or shuttling of the receptor underlies its localization . Nuclear import of liganded GR is mediated through a well-characterized sequence, NL1, adjacent to the receptor DNA binding domain and a second, uncharacterized motif, NL2, that overlaps with the ligand binding domain . In this study we report that rapid nuclear import (half-life {t1/2} of 4 to 6 min) of agonist- and antagonist-treated GR and the localization of unliganded, hsp-associated GRs to the nucleus in G0 are mediated through NL1 and correlate with the binding of GR to pendulin/importin alpha . By contrast, NL2-mediated nuclear transfer of GR occurred more slowly (t1/2 = 45 min to 1 h), was agonist specific, and appeared to be independent of binding to importin alpha . Together, these results suggest that NL2 mediates the nuclear import of GR through an alternative nuclear import pathway . Nuclear export of GR was inhibited by leptomycin B, suggesting that the transfer of GR to the cytoplasm is mediated through the CRM1-dependent pathway . Inhibition of GR nuclear export by leptomycin B enhanced the nuclear localization of both unliganded, wild-type GR and hormone-treated NL1(-) GR . These results highlight that the subcellular localization of both liganded and unliganded GRs is determined, at least in part, by a flexible equilibrium between the rates of nuclear import and export.

J Biol Chem, 1999 Jan 22, 274(4), 2113 - 7
Activation of transcription in vitro by the BRCA1 carboxyl-terminal domain; Haile DT et al.; The breast and ovarian specific tumor suppressor protein, BRCA1, has been shown to be a transcription factor because its carboxyl terminus, when fused to the GAL4 DNA binding domain, activates gene expression in cells . In this study, purified GAL4-BRCA1 protein functions in transcriptional activation assays using a minimal in vitro system . When compared with a standard activator, GAL4-VP16, the levels of activation produced by the BRCA1 fusion protein were stronger when in the presence of certain coactivators . The transcriptional activation by BRCA1 is maximal when in the presence of the PC4 (positive component 4) coactivator but not HMG2 (high mobility group protein 2) and when the template is negatively supercoiled . By contrast, transcriptional activation by VP16 was highest in the presence of HMG2 as well as PC4 and when DNA templates had linear topology . Activation by VP16 was largely unaffected by the concentration of TFIIH, whereas activation by BRCA1 was strongly affected by TFIIH concentrations . The differing cofactor and template requirements suggest that GAL4-BRCA1 and GAL4-VP16 regulate different steps in the pathways that lead to transcriptional activation.

J Biol Chem, 1999 Jan 22, 274(4), 2060 - 71
Oxygen-regulated and transactivating domains in endothelial PAS protein 1: comparison with hypoxia-inducible factor-1alpha; O'Rourke JF et al.; Endothelial PAS protein 1 (EPAS1) is a basic helix-loop-helix Per-AHR-ARNT-Sim transcription factor related to hypoxia-inducible factor-1alpha (HIF-1alpha) . To analyze EPAS1 domains responsible for transactivation and oxygen-regulated function, we constructed chimeric fusions of EPAS1 with a GAL4 DNA binding domain, plus or minus the VP16 activation domain . Two transactivation domains were defined in EPAS1; a C-terminal domain (amino acids 828-870), and a larger internal domain (amino acids 517-682) . These activation domains were interspersed by functionally repressive sequences, several of which independently conveyed oxygen-regulated activity . Two types of activity were defined . Sequences lying N-terminal to and overlapping the internal transactivation domain conferred regulated repression on the VP16 transactivator . Sequences lying C-terminal to this internal domain conveyed repression and oxygen-regulated activity on the native EPAS1 C-terminal activation domain, but not the Gal/VP16 fusion . Fusions containing internal but not C-terminal regulatory domains manifested regulation of fusion protein level . Comparison of EPAS1 with HIF-1alpha demonstrated a similar organization for both proteins, and for the C terminus defined a conserved RLL motif critical for inducibility . Overall, EPAS1 sequences were less inducible than those of HIF-1alpha, and inducibility was strikingly reduced as their expression level was increased . Despite these quantitative differences, EPAS1 regulation appeared similar to HIF-1alpha, conforming to a model involving the modulation of both protein level and activity, through distinct internal and C-terminal domains.

Brain Res Mol Brain Res, 1999 Jan 22, 64(1), 41 - 51
Molecular cloning and characterization of rat trp homologues from brain; Mizuno N et al.; Identification of trp (transient receptor potential) gene from Drosophila photoreceptor and subsequent molecular cloning of the human cDNA homologues suggest its participation in capacitative calcium entry (CCE) or so called store-operated Ca2+ channel (SOC) . We identified five different trp-related amplifications of reverse-transcription-polymerase chain reaction (RT-PCR) from rat brain; these corresponded to mouse trp homologues, mtrp1,3,4,5,6 and were distributed in various tissues with multiple expression levels . Two cDNAs, homologous to Drosophila trp from rat brain, designated rtrp3 and rtrp6, were isolated and characterized . By RT-PCR analysis, mRNAs of rtrp3 and rtrp6 were found to be expressed differently in brain and other tissues . In situ hybridization analysis revealed that rtrp6 mRNA was preferentially expressed in hippocampal dentate gyrus and cortical layers II and III . Expression of rat TRP3 and TRP6 in COS cells revealed an increase in CCE, as compared to that in the mock-transfected COS cells of the control . Isolation of cDNAs of rat trp gene family provides a useful model for studying mechanism of CCE .

Nucleic Acids Res, 1999 Feb 1, 27(3), 915 - 6
Inclusion of polyvinylpyrrolidone in the polymerase chain reaction reverses the inhibitory effects of polyphenolic contamination of RNA; Koonjul PK et al.; Polysaccharides, secondary metabolites and poly-phenolics are known to co-isolate with nucleic acids from plant tissues resulting in inhibition of molecular manipulations . RNA isolated from the polyphenolic-rich resurrection plant, Myrothamnus flabellifolius, was demonstrated to inhibit a standard polymerase chain reaction used as an assay despite the inclusion of the polyphenolic-binding compound poly(1-vinylpyrrolidone-2) (PVP) into the RNA isolation medium . This inhibition was, however, reversed by the addition of PVP into the PCR mixture itself . Confirmation of the inhibitory effect of polyphenolics on PCR was obtained by addition of green tea polyphenolics to the standard PCR assay . This inhibition was also reversed by the simultaneous inclusion of PVP.

Nucleic Acids Res, 1999 Feb 1, 27(3), 866 - 74
PolyADP-ribose polymerase is a coactivator for AP-2-mediated transcriptional activation; Kannan P et al.; Overexpression of transcription factor AP-2 has been implicated in the tumorigenicity of the human teratocarcinoma cell lines PA-1 that contain an activated ras oncogene . Here we show evidence that overexpression of AP-2 sequesters transcriptional coactivators which results in self-inhibition . We identified AP-2-interacting proteins and determined whether these proteins were coactivators for AP-2-mediated transcription . One such interacting protein is polyADP-ribose polymerase (PARP) . PARP suppresses AP-2 self-inhibition and enhances AP-2 activity in PA-1 cells indicating that it is a coactivator for AP-2-transcription . PARP significantly restores AP-2 transcriptional activity in ras oncogene-transformed cells suggesting that it might suppress transformation in these cells . Another AP-2-interacting protein, RAP74, a subunit of transcription factor TFIIF, does not affect AP-2-mediated transcriptional activation alone or in the presence of RAP30, the other subunit of TFIIF . RAP74 also fails to relieve AP-2-mediated transcriptional self-interference and cross-interference . These studies suggest that the interaction between AP-2 and RAP74 may have functions other than activation of AP-2-mediated transcription.

EMBO J, 1999 Jan 15, 18(2), 457 - 69
Elements essential for accumulation and function of small nucleolar RNAs directing site-specific pseudouridylation of ribosomal RNAs; Bortolin ML et al.; During site-specific pseudouridylation of eukaryotic rRNAs, selection of correct substrate uridines for isomerization into pseudouridine is directed by small nucleolar RNAs (snoRNAs) . The pseudouridylation guide snoRNAs share a common 'hairpin-hinge- hairpin-tail' secondary structure and two conserved sequence motifs, the H and ACA boxes, located in the single-stranded hinge and tail regions, respectively . In the 5'- and/or 3'-terminal hairpin, an internal loop structure, the pseudouridylation pocket, selects the target uridine through formation of base-pairing interactions with rRNAs . Here, essential elements for accumulation and function of rRNA pseudouridylation guide snoRNAs have been analysed by expressing various mutant yeast snR5, snR36 and human U65 snoRNAs in yeast cells . We demonstrate that the H and ACA boxes that are required for formation of the correct 5' and 3' ends of the snoRNA, respectively, are also essential for the pseudouridylation reaction directed by both the 5'- and 3'-terminal pseudouridylation pockets . Similarly, RNA helices flanking the two pseudouridylation pockets are equally essential for pseudouridylation reactions mediated by either the 5' or 3' hairpin structure, indicating that the two hairpin domains function in a highly co-operative manner . Finally, we demonstrate that by manipulating the rRNA recognition motifs of pseudouridylation guide snoRNAs, novel pseudouridylation sites can be generated in yeast rRNAs.

EMBO J, 1999 Jan 15, 18(2), 433 - 43
TATA-binding protein promotes the selective formation of UV-induced (6-4)-photoproducts and modulates DNA repair in the TATA box; Aboussekhra A et al.; DNA-damage formation and repair are coupled to the structure and accessibility of DNA in chromatin . DNA damage may compromise protein binding, thereby affecting function . We have studied the effect of TATA-binding protein (TBP) on damage formation by ultraviolet light and on DNA repair by photolyase and nucleotide excision repair in yeast and in vitro . In vivo, selective and enhanced formation of (6-4)-photoproducts (6-4PPs) was found within the TATA boxes of the active SNR6 and GAL10 genes, engaged in transcription initiation by RNA polymerase III and RNA polymerase II, respectively . Cyclobutane pyrimidine dimers (CPDs) were generated at the edge and outside of the TATA boxes, and in the inactive promoters . The same selective and enhanced 6-4PP formation was observed in a TBP-TATA complex in vitro at sites where crystal structures revealed bent DNA . We conclude that similar DNA distortions occur in vivo when TBP is part of the initiation complexes . Repair analysis by photolyase revealed inhibition of CPD repair at the edge of the TATA box in the active SNR6 promoter in vitro, but not in the GAL10 TATA box or in the inactive SNR6 promoter . Nucleotide excision repair was not inhibited, but preferentially repaired the 6-4PPs . We conclude that TBP can remain bound to damaged promoters and that nucleotide excision repair is the predominant pathway to remove UV damage in active TATA boxes.

EMBO J, 1999 Jan 15, 18(2), 330 - 8
Inhibition of apoptosis and clonogenic survival of cells expressing crmA variants: optimal caspase substrates are not necessarily optimal inhibitors; Ekert PG et al.; To study the role of various caspases during apoptosis, we have designed a series of caspase inhibitors based on the cowpox virus cytokine response modifier A (crmA) protein . Wild-type crmA inhibits caspases 1 and 8 and thereby protects cells from apoptosis triggered by ligation of CD95 or tumour necrosis factor (TNF) receptors, but it does not protect against death mediated by other caspases . By replacing the tetrapeptide pseudosubstrate region of crmA (LVAD) with tetrapeptides that are optimal substrates for the different families of caspases, or with the four residues from the cleavage site of the baculovirus protein p35 (DQMD), we have generated a family of caspase inhibitors that show altered ability to protect against cell death . Although DEVD is the optimal substrate for caspase 3, crmA DEVD was degraded rapidly and was a weaker inhibitor than crmA DQMD, which was not degraded . Unlike wild-type crmA and crmA DEVD, crmA DQMD was able to inhibit apoptosis caused by direct activation of caspase 3 and protected lymphoid cells from death induced by radiation and dexamethasone . Significantly, the protected cells were capable of sustained growth.

EMBO J, 1999 Jan 15, 18(2), 313 - 9
Tim9, a new component of the TIM22.54 translocase in mitochondria; Adam A et al.; We have identified Tim9, a new component of the TIM22.54 import machinery, which mediates transport of proteins into the inner membrane of mitochondria . Tim9, an essential protein of Saccharomyces cerevisiae, shares sequence similarity with Tim10 and Tim12 . Tim9 is located in the mitochondrial intermembrane space and is organized into two distinct hetero-oligomeric assemblies with Tim10 and Tim12 . One complex contains Tim9 and Tim10 . The other complex contains Tim9, Tim10 and Tim12 and is tightly associated with Tim22 in the inner membrane . The TIM9.10 complex is more abundant than the TIM9.10.12 complex and mediates partial translocation of mitochondrial carriers proteins across the outer membrane . The TIM9.10.12 complex assists further translocation into the inner membrane in association with TIM22.54.

Curr Biol, 1999 Jan 14, 9(1), 51 - 4
Dependence on RAD52 and RAD1 for anticancer drug resistance mediated by inactivation of mismatch repair genes; Durant ST et al.; Mismatch repair (MMR) proteins repair mispaired DNA bases and have an important role in maintaining the integrity of the genome {1} . Loss of MMR has been correlated with resistance to a variety of DNA-damaging agents, including many anticancer drugs {2} . How loss of MMR leads to resistance is not understood, but is proposed to be due to loss of futile MMR activity and/or replication stalling {3} {4} . We report that inactivation of MMR genes (MLH1, MLH2, MSH2, MSH3, MSH6, but not PMS1) in isogenic strains of Saccharomyces cerevisiae led to increased resistance to the anticancer drugs cisplatin, carboplatin and doxorubicin, but had no effect on sensitivity to ultraviolet C (UVC) radiation . Sensitivity to cisplatin and doxorubicin was increased in mlh1 mutant strains when the MLH1 gene was reintroduced, demonstrating a direct involvement of MMR proteins in sensitivity to these DNA-damaging agents . Inactivation of MLH1, MLH2 or MSH2 had no significant effect, however, on drug sensitivities in the rad52 or rad1 mutant strains that are defective in mitotic recombination and removing unpaired DNA single strands . We propose a model whereby MMR proteins - in addition to their role in DNA-damage recognition - decrease adduct tolerance during DNA replication by modulating the levels of recombination-dependent bypass . This hypothesis is supported by the finding that, in human ovarian tumour cells, loss of hMLH1 correlated with acquisition of cisplatin resistance and increased cisplatin-induced sister chromatid exchange, both of which were reversed by restoration of hMLH1 expression.

Curr Biol, 1998 Dec 17-31, 8(25), 1395 - 8
Targeted disruption of the gene encoding DNA ligase IV leads to lethality in embryonic mice; Barnes DE et al.; DNA ligase IV is the most recently identified member of a family of enzymes joining DNA strand breaks in mammalian cell nuclei {1} {2} . The enzyme occurs in a complex with the XRCC4 gene product {3}, an interaction mediated via its unique carboxyl terminus {4} {5} . Cells lacking XRCC4 are hypersensitive to ionising radiation and defective in V(D)J recombination {3} {6}, implicating DNA ligase IV in the pathway of nonhomologous end-joining (NHEJ) of DNA double-strand breaks mediated by XRCC4, the Ku70/80 heterodimer and the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) in mammalian cells (reviewed in {7}) . The phenotype of a null mutant of the Saccharomyces cerevisiae DNA ligase IV homologue indicates that the enzyme is non-essential and functions in yeast NHEJ {8} {9} {10} . Unlike other mammalian DNA ligases for which cDNAs have been characterised, DNA ligase IV is encoded by an intronless gene (LIG4) . Here, we show that targeted disruption of LIG4 in the mouse leads to lethality associated with extensive apoptotic cell death in the embryonic central nervous system . Thus, unlike Ku70/80 and DNA-PKcs {11} {12} {13} {14}, DNA ligase IV has an essential function in early mammalian development.

Glia, 1999 Jan, 25(1), 44 - 55
Identification and functional characterization of the mannose receptor in astrocytes; Burudi EM et al.; The immune competence of astrocytes is still ill defined, especially their endocytic capacity, a prerequisite for efficient antigen presentation . We show that mannose receptor, a very important conduit for internalization of infectious agents and self antigens, is functionally expressed in the murine CNS . By in vitro assays, astrocytes and microglia were shown to be the prime cells expressing this receptor . Studies on astrocytes demonstrate that its expression and function are inversely regulated by anti-and pro-inflammatory compounds . Downregulation of the mannose receptor by IFN-gamma is concomitant with the induction of the invariant chain, which is also induced by GM-CSF + IL-4 . Mannose receptor-expressing astrocytes may thus act as scavenger not only in CNS development but also in defense, against soluble and particulate mannosylated pathogens, presenting fragments thereof at strategic locations in the CNS . These findings unravel a new and putatively very important role of astrocytes in innate immunity and possibly development.

Arterioscler Thromb Vasc Biol, 1999 Jan, 19(1), 28 - 38
Growth and cell cycle abnormalities of fibroblasts from Tangier disease patients; Drobnik W et al.; We have investigated the abnormal proliferation and morphology of fibroblasts from patients with Tangier disease (TD), a high density lipoprotein (HDL) deficiency syndrome that is characterized by impairment of HDL3-mediated lipid efflux and Gi-protein-mediated signaling via phosphatidylinositol-specific phospholipase C (PI-PLC) and phospholipase D (PLD) . TD fibroblasts displayed a 30% to 50% reduced in vitro growth rate and a 1.6-fold increased cell surface area . The response to different mitogens was diminished, and asynchronously growing TD fibroblasts showed 4.4+/-0.3% S-phase and 19.1+/-0.5% G2/M-phase cells compared with 9.7+/-0.6% and 7.8+/-0.5%, respectively, in controls . Monensin, but not brefeldin A, induced an S- and G2/M-phase distribution in control cells similar to that found in TD fibroblasts . This effect of monensin was accompanied by an increase of ceramide levels in controls, whereas TD fibroblasts already had a 2.5-fold increased basal ceramide concentration . Incubation of control cells with C2 ceramide and threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) mimicked the effect of monensin on the cell cycle . The inhibition of neither Gi protein function by pertussis toxin nor PLD by butanol resulted in a G2/M-phase arrest . Propranolol, known to increase phosphatidic acid levels, was ineffective in reversing the G2/M-phase arrest in TD fibroblasts . In addition, cDNA sequences and mRNA expression of the participants of PI-PLC or PLD signaling, ie, G-protein subunits alphai1, alphai2, and alphai3; phosphatidylinositol transfer proteins-alpha and -beta; and ADP ribosylation factors 1 and 3 were found to be normal . Thus, growth and cell cycle abnormalities in TD fibroblasts are likely to be related to impaired Golgi function and sphingolipid signaling rather than inoperative G-protein signal transduction . Because PDMP was also found to decrease HDL3-mediated lipid efflux in control but not TD fibroblasts, similar pathways seem to be involved in the disturbances of lipid transport and growth retardation.

Biochemistry, 1999 Jan 12, 38(2), 682 - 95
Assembly of G protein-coupled receptors from fragments: identification of functional receptors with discontinuities in each of the loops connecting transmembrane segments; Martin NP et al.; The alpha-factor receptor of the yeast Saccharomyces cerevisiae is a member of the superfamily of G protein-coupled receptors that mediate signal transduction in response to sensory and chemical stimuli . All members of this superfamily contain seven predicted transmembrane segments . We have created a series of genes encoding alpha-factor receptors with amino- or carboxyl-terminal truncations at each of the loop regions connecting transmembrane segments . Split receptors containing a discontinuity in the peptide backbone were synthesized by coexpressing pairs of truncated receptor fragments in yeast . Complementary pairs of fragments split at sites within each of the cytoplasmic and extracellular loops were capable of assembling and transducing a signal in response to alpha-factor binding . One pair of noncomplementary fragments containing a deletion in the second intracellular loop of the receptor also yielded a functional receptor . Coexpression of certain combinations of overlapping fragments containing supernumerary transmembrane segments also led to formation of functional receptors, apparently because of proteolytic trimming of overlapping regions . Coexpression of truncated receptor fragments with full-length receptors had no effect on signaling by the full-length receptors . These results demonstrate the following: (1) Correct folding of the alpha-factor receptor does not require a covalent connection between any pair of transmembrane segments that are adjacent in the sequence . (2) Most of the second intracellular loop of the receptor is not required for function . (3) The structure of the receptor cannot, in most cases, tolerate the presence of extra transmembrane segments . (4) None of the truncated fragments of the alpha-factor receptor can efficiently oligomerize with normal receptors in such a way as to inhibit receptor function.

Nat Struct Biol, 1999 Jan, 6(1), 64 - 71
Structure of a HAP1-DNA complex reveals dramatically asymmetric DNA binding by a homodimeric protein; King DA et al.; HAP1 is a member of a family of fungal transcription factors that contain a Zn2Cys6 binuclear cluster domain and bind as homodimers to sequences containing two DNA half sites . We have determined the 2.5 A crystal structure of HAP1 bound to a cognate upstream activation sequence from the CYC7 gene . The structure reveals that HAP1 is bound in a dramatically asymmetric manner to the DNA target . This asymmetry aligns the Zn2Cys6 domains in a tandem head-to-tail fashion to contact two DNA half sites, positions an N-terminal arm of one of the protein subunits to interact with the inter-half site base pairs in the DNA minor groove, and suggests a mechanism by which DNA-binding facilitates asymmetric dimerization by HAP1 . Comparisons with the DNA complexes of the related GAL4, PPR1 and PUT3 proteins illustrate how a conserved protein domain can be reoriented to recognize DNA half sites of different polarities and how homodimeric proteins adopt dramatically asymmetric structures to recognize cognate DNA targets.

Nat Struct Biol, 1999 Jan, 6(1), 22 - 7
Structure of HAP1-18-DNA implicates direct allosteric effect of protein-DNA interactions on transcriptional activation; King DA et al.; HAP1 is a yeast transcriptional activator that binds with equal affinity to the dissimilar upstream activation sequences UAS1 and UAS(CYC7), but activates transcription differentially when bound to each site . HAP1-18 harbors an amino acid change in the DNA binding domain . While binding UAS1 poorly, HAP1-18 binds UAS(CYC7) with wild-type properties and activates transcription at elevated levels relative to HAP1 . We have determined the structure of HAP1-18-UAS(CYC7) and have compared it to HAP1-UAS(CYC7) . Unexpectedly, the single amino acid substitution in HAP1-18 nucleates a significantly altered hydrogen bond interface between the protein and DNA resulting in DNA conformational changes and an ordering of one N-terminal arm of the protein dimer along the DNA minor groove . These observations, together with a large subset of transcriptionally defective mutations in the HAP1 DNA-binding domain that map to the HAP1-DNA interface, suggest that protein-DNA interactions may have direct allosteric effects on transcriptional activation.

Mol Cell, 1998 Dec, 2(6), 863 - 7
The ATM-related cofactor Tra1 is a component of the purified SAGA complex; Grant PA et al.; The SAGA histone acetyltransferase/transcriptional adaptor complex is composed of multiple transcriptional regulators including Ada, Spt, and TAFII proteins . Here we identify an additional novel subunit of the complex, Tra1, an ATM/PI-3-kinase-related homolog of the human TRRAP cofactor, which is essential for c-Myc and E2F-mediated oncogenic transformation . Mass spectrometry, immunoblotting, and immunoprecipitation experiments confirm the stable association of this protein within SAGA . In addition, the Tra1 protein is a component of at least two other histone acetyltransferase protein complexes . These results indicate a role for Tra1 in the regulation of transcriptional activation through the recruitment of HAT activity to an activator-bound promoter.

Genes Dev, 1999 Jan 1, 13(1), 125 - 37
PRDI-BF1/Blimp-1 repression is mediated by corepressors of the Groucho family of proteins; Ren B et al.; The PRDI-BF1/Blimp-1 protein is a transcriptional repressor required for normal B-cell differentiation, and it has been implicated in the repression of beta-interferon (IFN-beta) and c-myc gene expression . Here, we show that PRDI-BF1 represses transcription of the IFN-beta promoter and of an artificial promoter through an active repression mechanism . We also identified a minimal repression domain in PRDI-BF1 that is sufficient for transcriptional repression when tethered to DNA as a Gal4 fusion protein . Remarkably, this repression domain interacts specifically with hGrg, TLE1, and TLE2 proteins, all of which are members of the Groucho family of transcriptional corepressors . In addition, the hGrg protein itself can function as a potent repressor when tethered to DNA through the Gal4 DNA-binding domain . We also find that the amino-terminal glutamine-rich domains of hGrg and TLE1 are sufficient to mediate dimerization of the two Groucho family proteins . Proteins containing only this domain can function as a dominant-negative inhibitor of PRDI-BF1 repression, and can significantly increase the IFN-beta promoter activity after virus induction . We conclude that PRDI-BF1/Blimp-1 represses transcription by recruiting a complex of Groucho family proteins to DNA, and suggest that such corepressor complexes are required for the postinduction repression of the IFN-beta promoter.

Genes Dev, 1999 Jan 1, 13(1), 87 - 97
The type 2C Ser/Thr phosphatase PP2Cgamma is a pre-mRNA splicing factor; Murray MV et al.; To identify activities involved in human pre-mRNA splicing, we have developed a procedure to separate HeLa cell nuclear extract into five complementing fractions . An activity called SCF1 was purified from one of these fractions by assaying for reconstitution of splicing in the presence of the remaining four fractions . A component of SCF1 is shown to be PP2Cgamma, a type 2C Ser/Thr phosphatase of previously unknown function . Previous work suggested that dephosphorylation of splicing factors may be important for catalysis after spliceosome assembly, although the identities of the specific phosphatases involved remain unclear . Here we show that human PP2Cgamma is physically associated with the spliceosome in vitro throughout the splicing reaction, but is first required during the early stages of spliceosome assembly for efficient formation of the A complex . The phosphatase activity is required for the splicing function of PP2Cgamma, as an active site mutant does not support spliceosome assembly . The requirement for PP2Cgamma is highly specific, as the closely related phosphatase PP2Calpha cannot substitute for PP2Cgamma . Consistent with a role in splicing, PP2Cgamma localizes to the nucleus in vivo . We conclude that at least one specific dephosphorylation event catalyzed by PP2Cgamma is required for formation of the spliceosome.

Genes Dev, 1999 Jan 1, 13(1), 76 - 86
Keap1 represses nuclear activation of antioxidant responsive elements by Nrf2 through binding to the amino-terminal Neh2 domain; Itoh K et al.; Transcription factor Nrf2 is essential for the antioxidant responsive element (ARE)-mediated induction of phase II detoxifying and oxidative stress enzyme genes . Detailed analysis of differential Nrf2 activity displayed in transfected cell lines ultimately led to the identification of a new protein, which we named Keap1, that suppresses Nrf2 transcriptional activity by specific binding to its evolutionarily conserved amino-terminal regulatory domain . The closest homolog of Keap1 is a Drosophila actin-binding protein called Kelch, implying that Keap1 might be a Nrf2 cytoplasmic effector . We then showed that electrophilic agents antagonize Keap1 inhibition of Nrf2 activity in vivo, allowing Nrf2 to traverse from the cytoplasm to the nucleus and potentiate the ARE response . We postulate that Keap1 and Nrf2 constitute a crucial cellular sensor for oxidative stress, and together mediate a key step in the signaling pathway that leads to transcriptional activation by this novel Nrf2 nuclear shuttling mechanism . The activation of Nrf2 leads in turn to the induction of phase II enzyme and antioxidative stress genes in response to electrophiles and reactive oxygen species.

Mol Cell, 1998 Dec, 2(6), 869 - 75
The 400 kDa subunit of the PCAF histone acetylase complex belongs to the ATM superfamily; Vassilev A et al.; PCAF histone acetylase is found in a complex with more than 20 associated polypeptides . Here we report cloning and characterization of the 400 kDa PCAF-associated factor referred to as PAF400 . PAF400 is almost identical to TRRAP, which binds to c-Myc and E2F, and has significant sequence similarities to the ATM superfamily including FRAP, ATM, ATR, and the catalytic subunit of DNA-PK . Remarkably, PAF400 and FRAP share sequence similarity in broad regions that cover 80% of the entire PAF400 sequence . However, unlike the other members of the ATM superfamily, PAF400 is not a protein kinase as judged from the lack of kinase motif and autophosphorylation activity . We discuss the possibility that PAF400 may play a role in signaling of DNA damage to p53 by stimulation of p53 acetylation.

Mol Cell, 1998 Dec, 2(6), 851 - 61
NURD, a novel complex with both ATP-dependent chromatin-remodeling and histone deacetylase activities; Xue Y et al.; ATP-dependent chromatin-remodeling complexes are known to facilitate transcriptional activation by opening chromatin structures . We report a novel human complex, named NURD, which contains not only ATP-dependent nucleosome disruption activity, but also histone deacetylase activity, which usually associates with transcriptional repression . The deacetylation is stimulated by ATP on nucleosomal templates, suggesting that nucleosome disruption aids the deacetylase to access its substrates . One subunit of NURD was identified as MTA1, a metastasis-associated protein with a region similar to the nuclear receptor core-pressor, N-CoR; and antibodies against NURD partially relieve transcriptional repression by thyroid hormone receptor . These results suggest that ATP-dependent chromatin remodeling can participate in transcriptional repression by assisting repressors in gaining access to chromatin.

Mol Cell, 1998 Dec, 2(6), 709 - 18
The phosphatase Cdc14 triggers mitotic exit by reversal of Cdk-dependent phosphorylation; Visintin R et al.; Exit from mitosis requires the inactivation of mitotic cyclin-dependent kinases (CDKs) by an unknown mechanism . We show that the Cdc14 phosphatase triggers mitotic exit by three parallel mechanisms, each of which inhibits Cdk activity . Cdc14 dephosphorylates Sic1, a Cdk inhibitor, and Swi5, a transcription factor for SIC1, and induces degradation of mitotic cyclins, likely by dephosphorylating the activator of mitotic cyclin degradation, Cdh1/Hct1 . Feedback between these pathways may lead to precipitous collapse of mitotic CDK activity and help coordinate exit from mitosis.

J Cell Sci, 1999 Feb, 112 ( Pt 3), 405 - 14
Involvement of the Ca2+-ATPase PAT1 and the contractile vacuole in calcium regulation in Dictyostelium discoideum; Moniakis J et al.; In Dictyostelium discoideum, the Ca2+-ATPase, PAT1, is localized to membranes of the contractile vacuole and its expression is upregulated substantially when the cells are grown in Ca2+-rich medium . In this study, we have analyzed the cellular/molecular mechanisms regulating PAT1 expression and examined the role of PAT1 and the contractile vacuole in Ca2+ regulation . During both growth and development, Dictyostelium cells respond to low millimolar concentrations of extracellular Ca2+ and upregulate PAT1 in a few hours . This process is dependent on protein synthesis and the serine/threonine phosphatase, calcineurin . Immunofluorescence analysis indicates that the upregulated PAT1 is associated mainly with the contractile vacuole, but it is also on the plasma membrane . This latter finding suggests that the contractile vacuole fuses with the plasma membrane to eliminate excess intracellular Ca2+ . In support of this idea, it was observed that conditions which impair contractile vacuolar function reduce the rate of Ca2+ secretion . It was also found that cells deficient in PAT1, due to the expression of antisense patA RNA or to the presence of calcineurin antagonists, grow normally in low Ca2+ medium but poorly or not at all in high Ca2+ medium . Together, these results suggest that PAT1 and the contractile vacuole are components of a Ca2+ sequestration and excretion pathway, which functions to help maintain Ca2+ homeostasis, especially under conditions of Ca2+ stress.

Glycobiology, 1999 Jan, 9(1), 65 - 72
Nonglucosylated oligosaccharides are transferred to protein in MI8-5 Chinese hamster ovary cells; Quellhorst GJ Jr et al.; A CHO mutant MI8-5 was found to synthesize Man9-GlcNAc2-P-P-dolichol rather than Glc3Man9GlcNAc2-P-P-dolichol as the oligosaccharide-lipid intermediate in N-glycosylation of proteins . MI8-5 cells were incubated with labeled mevalonate, and the prenol was found to be dolichol . The mannose-labeled oligosaccharide released from oligosaccharide-lipid of MI8-5 cells was analyzed by HPLC and alpha-mannosidase treatment, and the data were consistent with a structure of Man9GlcNAc2 . In addition, MI8-5 cells did not incorporate radioactivity into oligosaccharide-lipid during an incubation with tritiated galactose, again consistent with MI8-5 cells synthesizing an unglucosylated oligosaccharide-lipid . MI8-5 cells had parental levels of glucosylphosphoryldolichol synthase activity . However, in two different assays, MI8-5 cells lacked dolichol-P-Glc:Man9GlcNAc2-P-P-dolichol glucosyltransferase activity . MI8-5 cells were found to synthesize glucosylated oligosaccharide after they were transfected with Saccharomyces cerevisiae ALG 6, the gene for dolichol-P-Glc:Man9GlcNAc2-P-P-dolichol glucosyltransferase . MI8-5 cells were found to incorporate mannose into protein 2-fold slower than parental cells and to approximately a 2-fold lesser extent.

Neuron, 1998 Dec, 21(6), 1453 - 63
PDZ proteins bind, cluster, and synaptically colocalize with Eph receptors and their ephrin ligands; Torres R et al.; Localizing cell surface receptors to specific subcellular positions can be critical for their proper functioning, as most notably demonstrated at neuronal synapses . PDZ proteins apparently play critical roles in such protein localizations . Receptor tyrosine kinases have not been previously shown to interact with PDZ proteins in vertebrates . We report that Eph receptors and their membrane-linked ligands all contain PDZ recognition motifs and can bind and be clustered by PDZ proteins . In addition, we find that Eph receptors and ligands colocalize with PDZ proteins at synapses . Thus, PDZ proteins may play critical roles in localizing vertebrate receptor tyrosine kinases and/or their ligands and may be particularly important for Eph function in guidance or patterning or at the synapse.

Phytochemistry, 1998 Dec, 49(7), 1905 - 11
Cloning and characterization of the Arabidopsis thaliana lupeol synthase gene; Herrera JB et al.; A 2274 bp Arabidopsis thaliana cDNA was isolated that encodes a protein 57% identical to cycloartenol synthase from the same organism . The expressed recombinant protein encodes lupeol synthase, which converts oxidosqualene to the triterpene lupeol as the major product . Lupeol synthase is a multifunctional enzyme that forms other triterpene alcohols, including beta-amyrin, as minor products . Sequence analysis suggests that lupeol synthase diverged from cycloartenol synthase after plants diverged from fungi and animals . This evolutionary order is the reason that fungi and animals do not make lupeol.

Biochem J, 1999 Jan 15, 337 ( Pt 2), 179 - 84
Association of protein-tyrosine phosphatase PTP-BAS with the transcription-factor-inhibitory protein IkappaBalpha through interaction between the PDZ1 domain and ankyrin repeats; Maekawa K et al.; PTP-BAS is a membrane-associated protein tyrosine phosphatase containing a band-4.1 homology region and five PDZ (PSD-95 Dlg ZO-1) {discs-large homology region ('DHR')/Gly-Leu-Gly-Phe ('GLGF')} domains . The second and fourth PDZ domains were reported to associate with Fas/CD95 . By using the first PDZ domain as a bait in yeast two-hybrid screening, we have identified IkappaBalpha as a binding protein . IkappaBalpha associated with PDZ1 through the stretch of the N-terminal three ankyrin repeats . The association was also confirmed in HeLa cells by co-immunoprecipitation experiments . Inhibition of PTP-BAS by expression of dominant-negative PTP-BAS mutant resulted in tyrosine-phosphorylation of IkappaBalpha . Tyrosine-phosphorylation of IkappaBalpha is a key event in activation of nuclear factor (NF)-kappaB during reoxygenation . PTP-BAS may thus play a regulatory role in activation of NF-kappaB under high oxidative stress.

Toxicol Appl Pharmacol, 1999 Jan 1, 154(1), 76 - 83
New screening methods for chemicals with hormonal activities using interaction of nuclear hormone receptor with coactivator; Nishikawa J et al.; The endocrine system exerts important functions in a multitude of physiological processes including embryogenesis, differentiation, and homeostasis . Xenobiotics may modify natural endocrine function and so affect human health and wildlife . It is necessary, therefore, to understand the degree to which xenobiotics can disrupt endocrine systems . The key targets of endocrine disruptors are nuclear hormone receptors, which bind to steroid hormones and regulate their gene transcription . We have developed relevant assay systems based on the ligand-dependent interaction between nuclear hormone receptor and coactivator . The coactivators used in this study contained CBP, p300, RIP140, SRC1, TIF1, and TIF2 . By two hybrid assay in yeast, the interactions of estrogen receptor with RIP140, SRC1, TIF1, and TIF2 were detected and they were completely dependent on the presence of estrogen . Specificity of this assay was assessed by determining the effect of steroids, known estrogen receptor agonists, and phytoestrogens . The pattern of response to chemicals were consistent with estrogenic activity measured by other assay systems, indicating that this assay system is reliable for measuring estrogenic activity . In addition, we carried out in vitro binding studies: GST pull-down assay and surface plasmon resonance analysis . The estrogen receptor also bound to coactivator in response to chemicals depending on their estrogenic activity in vitro . These data demonstrate that the measurement of interaction between steroid hormone receptor and coactivator serves as a useful tool for identifying chemicals that interact with steroid receptors .

Protein Expr Purif, 1998 Dec, 14(3), 367 - 70
Protein purification with C-terminal fusion of maltose binding protein; Hennig L et al.; For affinity-chromatography-based purification of proteins that are prone to abnormal termination of translation or that may not be modified at their N-termini, affinity tags are needed which can be fused to the C-terminus . In this publication we describe that maltose binding protein (MBP) fused to the C-terminus of the plant photoreceptor phytochrome B allows purification of the fusion protein via amylose affinity chromatography . After overexpression in yeast a 125-fold enrichment could be achieved . The spectral properties of phytochrome B were not impaired by the fusion and purification . These results demonstrate that not only the widely used N-terminal fusions of MBP but also C-terminal fusions can be employed for protein purification .

Plant J, 1998 Nov, 16(4), 433 - 42
Low temperature regulation of the Arabidopsis CBF family of AP2 transcriptional activators as an early step in cold-induced COR gene expression; Gilmour SJ et al.; Cold-induced expression of the Arabidopsis COR (cold-regulated) genes is mediated by a DNA regulatory element termed the CRT (C-repeat)/DRE (dehydration-responsive element) . Recently, we identified a transcriptional activator, CBF1, that binds to the CRT/DRE and demonstrated that its overexpression in transgenic Arabidopsis plants at non-acclimating temperatures induces COR gene expression and increases plant freezing tolerance . Here we report that CBF1 belongs to a small family of closely related proteins which includes CBF2 and CBF3 . DNA sequencing of an 8.7 kb region of the Arabidopsis genome along with genetic mapping experiments indicated that the three CBF genes are organized in direct repeat on chromosome 4 at 72.8 cM, closely linked to molecular markers PG11 and m600 . Like CBF1, both CBF2 and CBF3 activated expression of reporter genes in yeast that contained the CRT/DRE as an upstream activator sequence . The transcript levels for all three CBF genes increased within 15 min of transferring plants to low temperature, followed by accumulation of COR gene transcripts at about 2 h . CBF transcripts also accumulated rapidly in response to mechanical agitation . The promoter regions of the CBF genes do not contain the CRT sequence, CCGAC, and overexpression of CBF1 did not have a detectable effect on CBF3 transcript levels, suggesting that the CBF gene family is not subject to autoregulation . We propose that cold-induced expression of CRT/DRE-containing COR genes involves a low temperature-stimulated signalling cascade in which CBF gene induction is an early event.

Plant J, 1998 Nov, 16(3), 345 - 53
Reciprocal regulation of distinct asparagine synthetase genes by light and metabolites in Arabidopsis thaliana; Lam HM et al.; In plants, the amino acid asparagine serves as an important nitrogen transport compound whose levels are dramatically regulated by light in many plant species, including Arabidopsis thaliana . To elucidate the mechanisms regulating the flux of assimilated nitrogen into asparagine, we examined the regulation of the gene family for asparagine synthetase in Arabidopsis . In addition to the previously identified ASN1 gene, we identified a novel class of asparagine synthetase genes in Arabidopsis (ASN2 and ASN3) by functional complementation of a yeast asparagine auxotroph . The proteins encoded by the ASN2/3 cDNAs contain a Pur-F type glutamine-binding triad suggesting that they, like ASN1, encode glutamine-dependent asparagine synthetase isoenzymes . However, the ASN2/3 isoenzymes form a novel dendritic group with monocot AS genes which is distinct from all other dicot AS genes including Arabidopsis ASN1 . In addition to these distinctions in sequence, the ASN1 and ASN2 genes are reciprocally regulated by light and metabolites . Time-course experiments reveal that light induces levels of ASN2 mRNA while it represses levels of ASN1 mRNA in a kinetically reciprocal fashion . Moreover, the levels of ASN2 and ASN1 mRNA are also reciprocally regulated by carbon and nitrogen metabolites . The distinct regulation of ASN1 and ASN2 genes combined with their distinct encoded isoenzymes suggest that they may play different roles in nitrogen metabolism, as discussed in this paper.

J Biochem (Tokyo), 1999 Jan, 125(1), 130 - 7
Identification of SEC12, SED4, truncated SEC16, and EKS1/HRD3 as multicopy suppressors of ts mutants of Sar1 GTPase; Saito Y et al.; The yeast SAR1 gene encodes a low-molecular-weight GTPase which is essential for the formation of transport vesicles from the endoplasmic reticulum (ER) . To understand how the Sar1p function is regulated in its GTPase cycle, we searched for multicopy suppressors of sar1 temperature-sensitive mutants and identified SEC12, SED4, truncated SEC16, and EKS1 . EKS1 turns out to be identical to HRD3, which was independently isolated as a gene implicated in the degradation of an HMG-CoA reductase isozyme, Hmg2p . In this paper, we show that the product of EKS1/HRD3 is a type-I transmembrane glycoprotein and resides in the ER . The eks1/hrd3 disrupted cells are normal in growth and transport of cargo proteins, but missecrete BiP (Kar2p) . The overexpression of EKS1/HRD3, which stabilizes Hmg2p, did not affect the stability of wild-type or mutant Sar1p or any early Sec proteins we examined . These results suggest that the role of Eks1p/Hrd3p is not involved in the ER protein degradation in general but rather required for the maintenance of the ER membrane functions . The novel genetic interactions unveiled between SAR1, SEC12, SEC16, and SED4 will provide useful information as to how the complex machinery of vesicle budding operates.

J Biol Chem, 1999 Jan 15, 274(3), 1628 - 34
High mobility group protein 1 interacts specifically with the core domain of human TATA box-binding protein and interferes with transcription factor IIB within the pre-initiation complex; Sutrias-Grau M et al.; The high mobility group (HMG) box domain has defined a family of proteins, mostly transcription factors, that specifically interacts with DNA on the minor groove and sharply bends it . The founding member of the family, HMG1, does not specifically recognize regular B-DNA but is recruited to DNA by interaction with other transcription factors and TATA box-binding protein (TBP) . However, conflicting effects of HMG1 on transcription have been reported . We show that the interaction between HMG1 and TBP is species-specific . This interaction in turn affects the interaction of TBP with transcription factor (TF) IIB and is competed by TFIIA . A primary binding site was mapped to the H2' alpha-helix in the highly conserved core domain of human TBP . On HMG1, the primary binding site was only in the HMG box A, and HMG box A was also sufficient to interact with native TFIID . Both HMG boxes efficiently repressed transcription in vitro as fusions to the Gal4-DNA binding domain . Additionally, HMG box B showed a weak level of activation at very low amounts . These results suggest a general involvement of HMG1 at the early stages of polymerase II transcription that may result in subtle activation or repression of individual genes.

J Biol Chem, 1999 Jan 15, 274(3), 1533 - 40
Cloning of AIP1, a novel protein that associates with the apoptosis-linked gene ALG-2 in a Ca2+-dependent reaction; Vito P et al.; ALG-2 is a 22-kDa calcium-binding protein necessary for cell death induced by different stimuli in 3DO T-cell hybridoma . 3DO cell clones depleted of ALG-2 protein exhibit normal caspases activation, suggesting that ALG-2 function is required downstream or is independent of caspase proteases activity for apoptosis to occur . Using the yeast two-hybrid screening system, we have isolated and characterized the mouse cDNA encoding for ALG-2 interacting protein 1 (AIP1), a novel protein that interacts with ALG-2 . ALG-2 and AIP1 colocalize in the cytosol and the presence of calcium is an indispensable requisite for their association . Sequence alignment shows that AIP1 is highly similar to BRO1, a yeast protein related to components of the Pkc1p-MAP kinase cascade . Overexpression of a truncated form of AIP1 protects two different cell types from death induced by trophic factors withdrawal; thus, our data indicate that AIP1 cooperates with ALG-2 in executing the calcium-dependent requirements along the cell death pathway.

J Biol Chem, 1999 Jan 15, 274(3), 1189 - 92
Overlapping but distinct patterns of histone acetylation by the human coactivators p300 and PCAF within nucleosomal substrates; Schiltz RL et al.; A number of transcriptional coactivators possess intrinsic histone acetylase activity, providing a direct link between hyperacetylated chromatin and transcriptional activation . We have determined the core histone residues acetylated in vitro by recombinant p300 and PCAF within mononucleosomes . p300 specifically acetylates all sites of histones H2A and H2B known to be acetylated in bulk chromatin in vivo but preferentially acetylates lysines 14 and 18 of histone H3 and lysines 5 and 8 of histone H4 . PCAF primarily acetylates lysine 14 of H3 but also less efficiently acetylates lysine 8 of H4 . PCAF in its native form, which is present in a stable multimeric protein complex lacking p300/CBP, primarily acetylates H3 to a monoacetylated form, suggesting that PCAF-associated polypeptides do not alter the substrate specificity . These distinct patterns of acetylation by the p300 and PCAF may contribute to their differential roles in transcriptional regulation.

Biochem Biophys Res Commun, 1998 Dec 18, 253(2), 358 - 63
The interaction of the vitamin D receptor with nuclear receptor corepressors and coactivators; Tagami T et al.; The vitamin D receptor (VDR), thyroid hormone receptor (TR), and retinoic acid receptor (RAR) are ligand-dependent transcription factors that function via the formation of heterodimeric complexes with retinoid X receptor (RXR) . Although TR and RAR are known to act as transcriptional repressors in the absence of cognate ligands, it is not clear whether VDR exhibits this property . Recently, transcriptional repression (basal silencing) by TR and RAR was shown to be mediated by nuclear receptor corepressors (CoRs), such as NCoR and SMRT . In this report, we examined the silencing ability of VDR and its interaction with NCoR and SMRT using mammalian two-hybrid assays . The Gal4-VDR fusion protein silenced the basal expression of a reporter that contains Gal4 binding sites, but the degree of silencing activity was weaker than that of Gal4-TR . In mammalian two-hybrid assays, the interaction of VP16-SMRT or VP16-NCoR was also stronger with Gal4-TR than with Gal4-VDR . Similar results were obtained when the assay was performed using the opposite configuration . Gal4-SMRT or Gal4-NCoR interacted better with VP16-TR than with VP16-VDR . These interactions were disrupted by the addition of cognate ligands . In contrast, VP16-VDR interacted better than VP16-TR when studied with a coactivator, Gal4-SRC1, or with the heterodimeric partner, Gal4-RXR . Consistent with these findings, relatively weak transcriptional silencing by the native VDR was observed using the osteopontin VDRE . Thus, in comparison to TR, VDR exhibits relatively weak ligand-independent transcriptional silencing, but it possesses strong dimerization with RXR and ligand-induced binding to transcriptional coactivators .

Plant Physiol, 1999 Jan, 119(1), 353 - 62
Microsomal electron transfer in higher plants: cloning and heterologous expression of NADH-cytochrome b5 reductase from Arabidopsis; Fukuchi-Mizutani M et al.; AtCBR, a cDNA encoding NADH-cytochrome (Cyt) b5 reductase, and AtB5-A and AtB5-B, two cDNAs encoding Cyt b5, were isolated from Arabidopsis . The primary structure deduced from the AtCBR cDNA was 40% identical to those of the NADH-Cyt b5 reductases of yeast and mammals . A recombinant AtCBR protein prepared using a baculovirus system exhibited typical spectral properties of NADH-Cyt b5 reductase and was used to study its electron-transfer activity . The recombinant NADH-Cyt b5 reductase was functionally active and displayed strict specificity to NADH for the reduction of a recombinant Cyt b5 (AtB5-A), whereas no Cyt b5 reduction was observed when NADPH was used as the electron donor . Conversely, a recombinant NADPH-Cyt P450 reductase of Arabidopsis was able to reduce Cyt b5 with NADPH but not with NADH . To our knowledge, this is the first evidence in higher plants that both NADH-Cyt b5 reductase and NADPH-Cyt P450 reductase can reduce Cyt b5 and have clear specificities in terms of the electron donor, NADH or NADPH, respectively . This substrate specificity of the two reductases is discussed in relation to the NADH- and NADPH-dependent activities of microsomal fatty acid desaturases.

Plant Physiol, 1999 Jan, 119(1), 49 - 56
Regulation and functional expression of cinnamate 4-hydroxylase from parsley; Koopmann E et al.; A previously isolated parsley (Petroselinum crispum) cDNA with high sequence similarity to cinnamate 4-hydroxylase (C4H) cDNAs from several plant sources was expressed in yeast (Saccharomyces cerevisiae) containing a plant NADPH:cytochrome P450 oxidoreductase and verified as encoding a functional C4H (CYP73A10) . Low genomic complexity and the occurrence of a single type of cDNA suggest the existence of only one C4H gene in parsley . The encoded mRNA and protein, in contrast to those of a functionally related NADPH:cytochrome P450 oxidoreductase, were strictly coregulated with phenylalanine ammonia-lyase mRNA and protein, respectively, as demonstrated by coinduction under various conditions and colocalization in situ in cross-sections from several different parsley tissues . These results support the hypothesis that the genes encoding the core reactions of phenylpropanoid metabolism form a tight regulatory unit.

Mol Biochem Parasitol, 1998 Nov 30, 97(1-2), 55 - 68
A tissue specific approach for analysis of membrane and secreted protein antigens from Haemonchus contortus gut and its application to diverse nematode species; Rehman A et al.; General methods to conduct tissue specific analysis are largely lacking for nematodes . An approach is described that focused on isolation of membrane and secreted protein genes from the gut of the parasitic nematode Haemonchus contortus . The approach capitalized on a monoclonal antibody that recognizes multiple membrane and secreted worm proteins . Polyclonal antisera made against these proteins were used to screen expression cDNA libraries made either from adult worm gut or whole worm . The genes identified encode predicted or known membrane and secreted proteins from gut, including a cysteine protease, a zinc metallopeptidase and a previously described GA1 protein . Another gene, Hc40, was isolated from the whole worm cDNA library and is nearly identical to a vaccine patent sequence pBTA879 . Tissue analysis demonstrated the intended focus on membrane and secreted proteins from parasite gut was achieved . Proteins related to each of those described were identified from other nematode species through data base analysis . Additionally, this analysis led to (1) identification of homologues of each gene in C . elegans; (2) deduction of a dimorphic structure in the Hc40 protein; (3) recognition of both monomorphic and dimorphic families of Hc40-related proteins; and (4) detection of two apparent classes of transcripts (mep1a and mep1b) that would each encode a divergent version of the putative zinc metallopeptidase MEP1 . The tissue specific approach and information base described should generally contribute to investigations on nutrient digestion and related secretory processes in nematode gut.

J Am Soc Mass Spectrom, 1998 Mar, 9(3), 208 - 15
Epitope mapping of monoclonal antibodies by mass spectrometry: identification of protein antigens in complex biological systems; Yu L et al.; We describe the application of immunoaffinity extraction and mass spectrometry to the analysis of Ty1 Gag protein in lysates of Saccharomyces cerevisiae . A magnetic bead-conjugated monoclonal antibody was used to achieve selective extraction, the specificity of which was established by matrix-assisted laser desorption/ionization mass spectrometric (MS) analysis of an extract of the lysate of cells overexpressing the Ty1 Gag protein . MS analysis of similar extracts of lysates following tryptic hydrolysis confirmed selective extraction of the epitope-containing peptide fragment . Sufficient sensitivity was achieved to allow the application of this approach to the analysis of lysates of wild-type cells . Furthermore, the sequence of the epitope-containing peptide was confirmed by electrospray-tandem MS . To our knowledge, this constitutes the first report of the application of immunoaffinity extraction and tandem MS analysis to the characterization of an antigen recovered from a complex cellular system.

Biochim Biophys Acta, 1999 Jan 6, 1426(2), 359 - 72
ALG gene expression and cell cycle progression; Kukuruzinska MA et al.; The evolutionarily conserved ALG genes function in the dolichol pathway in the synthesis of the lipid-linked oligosaccharide precursor for protein N-glycosylation . Increasing evidence suggests a role for these genes in the cell cycle . In Saccharomyces cerevisiae, coordinate regulation of the ALG genes makes up the primary genomic response to growth stimulation; several features of the ALG genes' expression resemble mammalian early growth response genes . However, only the first gene in the pathway, ALG7, is downregulated in response to an antimitogenic signal that leads to cell cycle arrest and differentiation, suggesting that selective inhibition of the first gene may be sufficient to regulate the dolichol pathway for the withdrawal from the cell cycle . The availability of mutants in the early essential ALG genes has established functional relationships between these genes' expression and G1/S transition, budding, progression through G2 and withdrawal from the cell cycle . Analysis of the regulation of ALG7 has provided insights into how this gene's expression is controlled at the molecular level . Recent studies have also begun to reveal how ALG7 expression is linked to cell cycle arrest in response to antimitogenic cues and have identified G1 cyclins as some of its downstream targets . Since the functions of the ALG genes appear to be as conserved among eukaryotes as the cell cycle machinery, it is likely that these genes play a similar role in mammalian cell proliferation and differentiation.

J Mol Biol, 1998 Dec 18, 284(5), 1307 - 22
Genetic analysis of Xenopus transcription factor IIIA; Bumbulis MJ et al.; We describe a method for the genetic analysis of the DNA-binding properties of Xenopus transcription factor IIIA (TFIIIA) . In this approach, a transcriptional activator with the DNA-binding specificity of Xenopus TFIIIA is expressed in yeast cells, where it specifically activates expression of a beta-galactosidase reporter gene containing one or more Xenopus 5 S rRNA genes that function as upstream activator sequences . This transcription-promoting activity was used as the basis for a genetic assay of Xenopus TFIIIA's DNA-binding function in yeast, an assay that we show can be calibrated quantitatively to allow the affinity of the Xenopus TFIIIA-5 S rRNA gene interaction to be deduced from measurements of beta-galactosidase activity . We have combined this genetic assay with a simple and efficient method of mutagenesis that makes use of error-prone PCR and homologous recombination to generate and screen large numbers of TFIIIA mutants for those with altered 5 S rRNA gene-binding affinity . Over 30 such mutants have been identified and partially characterized . The mutants we have obtained provide strong support for the application to intact TFIIIA of recent structural models of the N-terminal zinc fingers of the protein bound to fragments of the 5 S rRNA gene . Other mutants permit identification of important residues in more C-terminal zinc fingers of TFIIIA for which high-resolution structural information is not currently available . Finally, our results have interesting implications with respect to the mechanism of activation of transcription by RNA polymerase II in yeast .

Genomics, 1998 Dec 15, 54(3), 494 - 504
Identification and characterization of human cDNAs specific to BCS1, PET112, SCO1, COX15, and COX11, five genes involved in the formation and function of the mitochondrial respiratory chain; Petruzzella V et al.; We have successfully applied a strategy based on the "cyberscreening" of the expressed sequence tags database using yeast protein sequences as "probes" to identify the human gene orthologs to BCS1, COX15, PET112, COX11, and SCO1, five yeast genes involved in the biogenesis of the mitochondrial respiratory chain complexes . In yeast, BCS1 is involved mainly in the assembly of complex III, while the other genes appear to control the structure/function of cytochrome-c oxidase . Significant amino acid identity and similarity were demonstrated by comparison of the human with the corresponding yeast polypeptides . Sequence alignment revealed numerous colinear identical regions and the conservation of functional domains . Mitochondrial targeting of the human gene products, suggested by computer analysis of the protein sequences, was confirmed by an in vitro import and protease-protection assay . These data strongly suggest that the human gene products share similar or identical functions with their yeast homologues . Genes controlling the structure/function of the respiratory chain complexes are attractive candidates for human mitochondrial disorders such as Leigh disease . However, both sequence analysis and functional complementation assays on an index patient do not support an etiological role for any of these genes .

Genomics, 1998 Dec 15, 54(3), 477 - 83
Characterization of gene expression, genomic structure, and chromosomal localization of Hells (Lsh); Geiman TM et al.; Hells (Lsh) is a lymphoid-specific presumptive helicase with highest expression in lymphoid precursor cells . Other members of the helicase family participate in maintenance of genome stability, DNA repair, and transcriptional control . Here we report the structure and chromosomal location of the Hells gene . The open reading frame of the murine Hells gene spans at least 26.6 kb of chromosomal DNA and is composed of 18 exons . The genomic structure of the seven helicase domains closely resembles that of mammalian Rad54, a gene whose product appears to be involved in recombination and double-strand break repair . The human homologue, the HELLS gene, has a mRNA expression pattern that is similar to murine Hells expression . Low-stringency hybridization in a Southern analysis reveals homologous Hells genes in a variety of species including Saccharomyces cerevisiae . FISH analysis maps the murine Hells gene to region C3-D1 on chromosome 19 . The human homologue maps to a region of synteny on chromosome 10q23-q24, a breakpoint region frequently involved in human leukemia .

Genomics, 1998 Dec 15, 54(3), 460 - 8
The quiescin Q6 gene (QSCN6) is a fusion of two ancient gene families: thioredoxin and ERV1; Coppock DL et al.; Cell and tissue growth is a dynamic process determined by the fraction of cells in the proliferative cycle, the fraction of cells in quiescence, and the rate of cell death . Genes whose expression is induced at the beginning of the transition from the proliferative cell cycle to quiescence may play an important role in this process . We have identified a gene, Quiescin Q6 (QSCN6), whose expression is induced just as fibroblasts begin to leave the proliferative cycle and enter quiescence . QSCN6 is located on human chromosome 1q24, near the putative hereditary prostate cancer locus (HPC1) . A triplet repeat (CTG)n encodes a putative signal sequence . The gene encodes a 582-amino-acid open reading frame that has domains that are members of two ancient gene families . These domains apparently underwent a gene fusion event during metazoan evolution to create QSCN6 . QSCN6 is most closely related to three genes of unknown function from Caenorhabditis elegans as well as a gene from guinea pig . Analysis of this relationship showed nine Quiescin homology zones (QHZ) . QHZ 0 is the putative signal sequence, QHZ 1 is homologous to a thioredoxin domain, and QHZ 2, 3, 4, and 8 are homologous only to themselves, while QHZ 5, 6, and 7 are homologous to the ERV1 gene of Saccharomyces cerevisiae . In both thioredoxin and ERV1 gene superfamilies, QSCN6 sequences appear to be on distinct branches of their respective phylogenetic trees, consistent with an ancient origin of the QSCN6 gene . We present a model of the origin of QSCN6 and discuss its potential role in growth regulation .

EMBO J, 1999 Jan 4, 18(1), 258 - 69
The STAR protein, GLD-1, is a translational regulator of sexual identity in Caenorhabditis elegans; Jan E et al.; The Caenorhabditis elegans sex determination gene, tra-2, is translationally regulated by elements in the 3'-untranslated region called TGEs . TGEs govern the translation of mRNAs in both invertebrates and vertebrates, indicating that this is a highly conserved mechanism for controlling gene activity . A factor called DRF, found in worm extracts binds the TGEs and may be a repressor of translation . Using the yeast three-hybrid screen and RNA gel shift analysis, we have found that the protein GLD-1, a germline-specific protein and a member of the STAR family of RNA-binding proteins, specifically binds to the TGEs . GLD-1 is essential for oogenesis, and is also necessary for spermatogenesis and inhibition of germ cell proliferation . Several lines of evidence demonstrate that GLD-1 is a translational repressor acting through the TGEs to repress tra-2 translation . GLD-1 can repress the translation of reporter RNAs via the TGEs both in vitro and in vivo, and is required to maintain low TRA-2A protein levels in the germline . Genetic analysis indicates that GLD-1 acts upstream of the TGE control . Finally, we show that endogenous GLD-1 is a component of DRF . The conservation of the TGE control and the STAR family suggests that at least a subset of STAR proteins may work through the TGEs to control translation.

EMBO J, 1999 Jan 4, 18(1), 212 - 28
Integration of a growth-suppressing BTB/POZ domain protein with the DP component of the E2F transcription factor; de la Luna S et al.; Transcription factor E2F plays an important role in orchestrating early cell cycle progression through its ability to co-ordinate and integrate the cell cycle with the transcription apparatus . Physiological E2F arises when members of two distinct families of proteins interact as E2F-DP heterodimers, in which the E2F component mediates transcriptional activation and the physical interaction with pocket proteins, such as the tumour suppressor protein pRb . In contrast, a discrete role for the DP subunit has not been defined . We report the identification and characterization of DIP, a novel mammalian protein that can interact with the DP component of E2F . DIP was found to contain a BTB/POZ domain and shows significant identity with the Drosophila melanogaster germ cell-less gene product . In mammalian cells, DIP is distributed in a speckled pattern at the nuclear envelope region, and can direct certain DP subunits and the associated heterodimeric E2F partner into a similar pattern . DIP-dependent growth arrest is modulated by the expression of DP proteins, and mutant derivatives of DIP that are compromised in cell cycle arrest exhibit reduced binding to the DP subunit . Our study defines a new pathway of growth control that is integrated with the E2F pathway through the DP subunit of the heterodimer.

EMBO J, 1999 Jan 4, 18(1), 179 - 87
XIAP, a cellular member of the inhibitor of apoptosis protein family, links the receptors to TAB1-TAK1 in the BMP signaling pathway; Yamaguchi K et al.; Signals elicited by transforming growth factor-beta (TGF-beta) superfamily ligands are generated following the formation of heteromeric receptor complexes consisting of type I and type II receptors . TAK1, a member of the MAP kinase kinase kinase family, and its activator, TAB1, participate in the bone morphogenetic protein (BMP) signaling pathway involved in mesoderm induction and patterning in early Xenopus embryos . However, the events leading from receptor activation to TAK1 activation remain to be identified . A yeast interaction screen was used to search for proteins that function in the pathway linking the receptors and TAB1-TAK1 . The human X-chromosome-linked inhibitor of apoptosis protein (XIAP) was isolated as a TAB1-binding protein . XIAP associated not only with TAB1 but also with the BMP receptors in mammalian cells . Injection of XIAP mRNA into dorsal blastomeres enhanced the ventralization of Xenopus embryos in a TAB1-TAK1-dependent manner . Furthermore, a truncated form of XIAP lacking the TAB1-binding domain partially blocked the expression of ventral mesodermal marker genes induced by a constitutively active BMP type I receptor . These results suggest that XIAP participates in the BMP signaling pathway as a positive regulator linking the BMP receptors and TAB1-TAK1.

EMBO J, 1999 Jan 4, 18(1), 75 - 84
Compartmentation of protein folding in vivo: sequestration of non-native polypeptide by the chaperonin-GimC system; Siegers K et al.; The functional coupling of protein synthesis and chaperone-assisted folding in vivo has remained largely unexplored . Here we have analysed the chaperonin-dependent folding pathway of actin in yeast . Remarkably, overexpression of a heterologous chaperonin which traps non-native polypeptides does not interfere with protein folding in the cytosol, indicating a high-level organization of folding reactions . Newly synthesized actin avoids the chaperonin trap and is effectively channelled from the ribosome to the endogenous chaperonin TRiC . Efficient actin folding on TRiC is critically dependent on the hetero-oligomeric co-chaperone GimC . By interacting with folding intermediates and with TRiC, GimC accelerates actin folding at least 5-fold and prevents the premature release of non-native protein from TRiC . We propose that TRiC and GimC form an integrated 'folding compartment' which functions in cooperation with the translation machinery . This compartment sequesters newly synthesized actin and other aggregation-sensitive polypeptides from the crowded macromolecular environment of the cytosol, thereby allowing their efficient folding.

FEBS Lett, 1998 Dec 11, 441(1), 127 - 31
A novel human RasGAP-like gene that maps within the prostate cancer susceptibility locus at chromosome 1q25; Noto S et al.; We report the molecular cloning of a human cDNA that encodes a molecule having striking homology with Ras-specific GTPase-activating proteins (RasGAPs) . Among previously described RasGAPs, the cDNA product is most closely related to Caenorhabditis elegans GAP-2, including a predicted coiled-coil structure near the carboxyl terminus . Expression of the cDNA in Saccharomyces cerevisiae defective in one of two RasGAPs, Ira2, complemented loss of the Ira2 function, indicating that the cDNA product functions as a RasGAP . The RasGAP-like gene is located on the human chromosome 1q25, the locus that appears to contain a hereditary prostate cancer susceptible gene, HPC1.

Cell Mol Neurobiol, 1998 Dec, 18(6), 721 - 9
Abnormalities in stress proteins in prion diseases; Tatzelt J et al.; 1 . Prion diseases include kuru, Creutzfeldt-Jakob disease (CJD), Gerstmann-Straussler-Scheinker disease (GSS), and fatal familia insomnia (FFI) of humans, as well as scrapie and bovine spongiform encephalopathy (BSE) of animals . 2 . All these disorders involve conversion of the normal, cellular prion protein (PrPC) into the corresponding scrapie isoform (PrPSc) . PrPC adopts a structure rich in alpha-helices and devoid of beta-sheet, in contrast to PrPSc, which has a high beta-sheet content and is resistant to limited digestion by proteases . That a conformational transition features in the conversion of PrPC into PrPSc implies that prion diseases are disorders of protein conformation . 3 . This concept has been extended by our studies with heat shock proteins (Hsp), many of which are thought to function as molecular chaperones . We found that the induction of some Hsps but not others was profoundly altered in scrapie-infected cells and that the distribution of Hsp73 is unusual in these cells . 4 . Whether the conversion of PrPC into PrPSc is assisted by molecular chaperones, or if the accumulation of the abnormally folded PrPSc is complexed with Hsps remains to be established.

J Mass Spectrom, 1998 Dec, 33(12), 1246 - 55
Investigation of the covalent modification of the catalytic triad of human cytomegalovirus protease by pseudo-reversible beta-lactam inhibitors and a peptide chloromethylketone; Haley TM et al.; An investigation into the interaction between human cytomegalovirus (HCMV) protease and several beta-lactams, with characterization of the resulting acylenzymes using mass spectrometry, is reported . The time dependence of the inhibitors is highlighted by making comparisons of values obtained for inhibition and acylation . Analysis of inactivated HCMV protease revealed a beta-lactam: protease stoichiometry of 1 . Subsequent enzymatic digestion with trypsin, peptide mapping using liquid chromatography coupled with electrospray ionization mass spectrometry and sequencing by nanoelectrospray tandem mass spectrometry (NanoES-MS/MS) allowed the identification of the site of covalent modification and confirmed Ser 132 as the active site hydroxyl nucleophile . Further, treatment of the protease with a peptide chloromethylketone and sequence analysis using NanoES-MS/MS of the alkylated enzyme confirmed His 63 as the active site imidazole nucleophile.

Crit Care Med, 1998 Dec, 26(12), 1972 - 6
Effects of hyperbaric oxygen exposure on a zymosan-induced shock model; Luongo C et al.; OBJECTIVE: To evaluate the effects of hyperbaric oxygen (HBO) therapy on zymosan-induced shock in rats . Zymosan, a cell wall component of the yeast Saccharomyces cerevisiae, induces inflammation by causing the production of various cytokines and pro-inflammatory mediators . The administration of zymosan to rats represents a new experimental shock model by inducing acute peritonitis, severe hypotension, and signs of systemic illness . However, it has been recently proposed that the zymosan-induced shock, like septic shock, may be mediated by overproduction of nitric oxide . DESIGN: Experimental study . SETTING: Institute of Pharmacology and Toxicology, 2nd University of Naples, Naples, Italy . SUBJECTS: Male rats were treated with zymosan (500 mg/kg) by intraperitoneal route, with HBO (2 Absolute Atmosphere) or with zymosan and HBO (2 Absolute Atmosphere) . MEASUREMENTS AND MAIN RESULTS: Peritoneal exudate, plasma, and peritoneal nitric oxide metabolites (NOx) and zymosan determined a time-dependent increase in peritoneal and plasma NOx concentrations, and peritoneal leukocytes were determined . Moreover, symptomatology was observed . The administration of zymosan caused the appearance of a severe illness in the rats characterized by ruffled fur, lethargy, conjunctivitis, diarrhea, and a significant loss of body weight . All zymosan-treated rats developed an acute peritonitis, producing turbid exudate . Zymosan determined a time-dependent increase in peritoneal, plasma NOx, and tumor necrosis factor (TNF)-alpha concentrations . Morbidity of zymosan shocked rats has been attenuated and no mortality was observed by treatment with HBO . These findings were associated with a significant reduction either of peritoneal leukocytes and exudate, or plasma and peritoneal NOx concentrations . Moreover, TNF-alpha levels were significantly reduced in animals shocked by zymosan and treated with HBO.

Cell, 1998 Dec 23, 95(7), 891 - 902
A critical role for DNA end-joining proteins in both lymphogenesis and neurogenesis; Gao Y et al.; XRCC4 was identified via a complementation cloning method that employed an ionizing radiation (IR)-sensitive hamster cell line . By gene-targeted mutation, we show that XRCC4 deficiency in primary murine cells causes growth defects, premature senescence, IR sensitivity, and inability to support V(D)J recombination . In mice, XRCC4 deficiency causes late embryonic lethality accompanied by defective lymphogenesis and defective neurogenesis manifested by extensive apoptotic death of newly generated postmitotic neuronal cells . We find similar neuronal developmental defects in embryos that lack DNA ligase IV, an XRCC4-associated protein . Our findings demonstrate that differentiating lymphocytes and neurons strictly require the XRCC4 and DNA ligase IV end-joining proteins and point to the general stage of neuronal development in which these proteins are necessary.

Proc Natl Acad Sci U S A, 1999 Jan 5, 96(1), 203 - 7
The BCR-ABL oncoprotein potentially interacts with the xeroderma pigmentosum group B protein; Takeda N et al.; The previously uncharacterized CDC24 homology domain of BCR, which is missing in the P185 BCR-ABL oncogene of Philadelphia chromosome (Ph1)-positive acute lymphocytic leukemia but is retained in P210 BCR-ABL of chronic myelogeneous leukemia, was found to bind to the xeroderma pigmentosum group B protein (XPB) . The binding appeared to be required for XPB to be tyrosine-phosphorylated by BCR-ABL . The interaction not only reduced both the ATPase and the helicase activities of XPB purified in the baculovirus system but also impaired XPB-mediated cross-complementation of the repair deficiency in rodent UV-sensitive mutants of group 3 . The persistent dysfunction of XPB may in part underlie genomic instability in blastic crisis.

Proc Natl Acad Sci U S A, 1999 Jan 5, 96(1), 109 - 14
Maturation of the tyrosine kinase c-src as a kinase and as a substrate depends on the molecular chaperone Hsp90; Xu Y et al.; Although Hsp90 displays general chaperone activity in vitro, few substrates of the chaperone have been identified in vivo, and the characteristics that render these substrates dependent on Hsp90 remain elusive . To investigate this issue, we exploited a paradoxical observation: several unrelated oncogenic viral tyrosine kinases, including v-src, attain their native conformation after association with Hsp90, yet their nearly identical cellular homologs interact only weakly with the chaperone . It has been controversial whether Hsp90 is vital for normal maturation of the cellular kinases or is simply binding a misfolded subfraction of the proteins . By modulating Hsp90 levels in Saccharomyces cerevisiae, we determined that Hsp90 is indeed necessary for the maturation of c-src (the normal homolog of v-src) . c-src maturation is, however, less sensitive to Hsp90 perturbations than is v-src maturation . Dependence of the two proteins on Hsp90 does not correspond to their relative efficiency in reaching their final destination (the plasma membrane); we observed that in yeast, unlike in vertebrate cells, neither c-src nor v-src concentrate in the membrane . Expression of different v/c-src chimeras in cells carrying wild-type or temperature-sensitive Hsp90 alleles revealed that the difference between the proteins instead arises from multiple, naturally occurring mutations in the C-terminal region of v-src.

Proc Natl Acad Sci U S A, 1999 Jan 5, 96(1), 67 - 72
Mediator protein mutations that selectively abolish activated transcription; Myers LC et al.; Deletion of any one of three subunits of the yeast Mediator of transcriptional regulation, Med2, Pgd1 (Hrs1), and Sin4, abolished activation by Gal4-VP16 in vitro . By contrast, other Mediator functions, stimulation of basal transcription and of TFIIH kinase activity, were unaffected . A different but overlapping Mediator subunit dependence was found for activation by Gcn4 . The genetic requirements for activation in vivo were closely coincident with those in vitro . A whole genome expression profile of a Deltamed2 strain showed diminished transcription of a subset of inducible genes but only minor effects on "basal" transcription . These findings make an important connection between transcriptional activation in vitro and in vivo, and identify Mediator as a "global" transcriptional coactivator.

Proc Natl Acad Sci U S A, 1999 Jan 5, 96(1), 55 - 60
Homotypic interaction and multimerization of nucleocapsid protein of tomato spotted wilt tospovirus: identification and characterization of two interacting domains; Uhrig JF et al.; The nucleocapsid protein (N) of tomato spotted wilt tospovirus (TSWV) plays a central role in the viral life cycle . With the aid of the yeast two-hybrid system and surface plasmon resonance analysis, homotypic interaction and multimerization of the N protein was detected . Analysis of deletion mutants identified two binding regions in the protein, located at the N terminus (amino acids 1-39) and the C terminus (amino acids 233-248), respectively, implying a "head-to-tail" interaction of the N terminus with the C terminus to form a multimeric chain . Further characterization of the binding domains was performed by site-directed mutagenesis . Two phenylalanines (F242 and F246) highly conserved in the N proteins within the Tospovirus genus were shown to play a crucial role in the interaction.

Eur J Biochem, 1998 Dec 1, 258(2), 846 - 53
Enhancement of phosphoinositide 3-kinase (PI 3-kinase) activity by membrane curvature and inositol-phospholipid-binding peptides; Hubner S et al.; The phosphorylation of phosphatidylinositol (PtdIns) on the 3' position of the inositol ring by phosphoinositide 3-kinase (PI 3-kinase) is shown to depend strongly on the curvature of liposomes containing a mixture of phosphatidylcholine (PtdCho) and PtdIns . Vesicles with an average diameter of 50 nm are phosphorylated 100 times faster than chemically identical vesicles with an average diameter greater than 300 nm . The low reactivity of large vesicles is not due to the difference in vesicle number for large and small vesicles at constant total lipid, nor to occlusion of lipid surfaces in multilammelar structures, and can be reversed by addition of low (< 1:100) molar ratios of either the PtdIns transfer protein sec14p or a ten-residue peptide derived from the inositol-phospholipid-binding site of gelsolin . Similar measurements using PI 4-kinase showed a weak dependence on vesicle size . The strong dependence of PI 3-kinase function on membrane curvature suggests possible localization of PI 3-kinase activity at sites where clustering of receptors, for example, may locally deform the membrane, and suggests that once PI 3-kinase is localized and activated at surface sites, the reaction may become self-accelerating.

Eur J Biochem, 1998 Dec 1, 258(2), 387 - 95
Rejoining of DNA double-strand breaks in vitro by single-strand annealing; Gottlich B et al.; Nonhomologous DNA end joining (NHEJ) is considered the major pathway of double-strand break (DSB) repair in vertebrate cells . Various studies indicated the existence of at least two different NHEJ pathways; one that joins DNA ends accurately and depends on Ku, a protein heterodimer that binds to DNA ends, and one that generates deletions and is independent of Ku . While the former pathway has been characterised in some detail, only little is known about the latter error-prone . We have partially purified such an NHEJ activity from extracts of Xenopus laevis eggs . End-joined junctions formed in the most extensively purified protein fraction displayed deletions containing short patches of sequence homology at their break points, a feature characteristic of single-strand annealing (SSA) . Detailed biochemical characterisation revealed the presence of DNA ligase III, DNA polymerase epsilon, FEN-1 endonuclease, and exonuclease activities of 5'-3' and 3'-5' directionality . We show that these activities are able to correctly process proposed intermediates of SSA . Interestingly, neither Ku nor the associated DNA-dependent protein kinase were detected, indicating that the mechanism can dispense with Ku . Our findings provide evidence for the existence of an error-prone NHEJ pathway that creates deletions by microhomology-driven SSA.

Hybridoma, 1998 Oct, 17(5), 413 - 20
Characteristic immunolocalization of Ku protein as nuclear matrix; Yu E et al.; Two hybridoma clones, NMB1 and NML90, were established using nuclear matrix proteins from normal human thymi or malignant lymphoma as immunogens . They reacted with human Ku70 and Ku80, respectively, by immunoblotting . When HeLa cell nuclear proteins were fractionated and applied to immunoblotting, both Ku70 and Ku80 were detected in the nuclear matrix as well as the soluble nuclear protein fractions . By confocal scanning microscopy, the immunoreactivity of Ku70 and Ku80 was localized to distinct nucleoplasmic fibrillar network and fine granules in the interphase cell nuclei . When HeLa cells were fractionated in situ using DNase I and buffers containing 0.25 M (NH4)2SO4 and 2 M NaCl, the nucleoplasmic reticular structure was largely preserved, but granules disappeared . The nucleoplasmic distribution of Ku in the tissue and in cultured cells was distinct from each other . In the adult tissue, it consisted mostly of either distinct curvilinear lines along the nuclear periphery or of tangled, beaded lines throughout the nuclei . When xenotransplants of HeLa cell in Scid mice were examined, the "tissue type" immunolocalization pattern was reproduced consistently . In most fetal tissues, "tissue type" and "cell type" patterns were admixed . Monoclonal antibodies described here are useful tools for studying the structure and function of the nuclear matrix.

Bioorg Med Chem Lett, 1998 Sep 22, 8(18), 2577 - 82
New antifungals selected by molecular topology; Pastor L et al.; Molecular topology has been applied to find the new lead antimycotic compounds . Among the selected compounds stands out 3,3'-(4,4'-Biphenylene)bis(2,5-diphenyl-2H-tetrazolium chloride), Benztropine mesylate and Dicyclopentamethylenethiuram disulphide, with minimum inhibitory concentrations between 1.6 and 2 micrograms/mL.

Bioorg Med Chem Lett, 1998 Sep 8, 8(17), 2303 - 8
Exploring structure-activity relationships around the phosphomannose isomerase inhibitor AF14049 via combinatorial synthesis; Bhandari A et al.; Phosphomannose Isomerase (PMI) has been shown by genetic methods to be an essential enzyme in fungal cell wall biosynthesis . The PMI inhibitor AF14049 was discovered as an unanticipated side product from high-throughput library screening against the enzyme from C, albicans . Solid-phase synthetic methods were developed and a series of libraries and discrete analogs synthesized to explore SAR around AF14049.

J Biol Chem, 1999 Jan 8, 274(2), 595 - 606
Mxi1 is a repressor of the c-Myc promoter and reverses activation by USF; Lee TC et al.; The basic region/helix-loop-helix/leucine zipper (B-HLH-LZ) oncoprotein c-Myc is abundant in proliferating cells and forms heterodimers with Max protein that bind to E-box sites in DNA and stimulate genes required for proliferation . A second B-HLH-LZ protein, Mxi1, is induced during terminal differentiation, and forms heterodimers with Max that also bind E-boxes but tether the mSin3 transcriptional repressor protein along with histone deacetylase thereby antagonizing Myc-dependent activation . We show that Mxi1 also antagonizes Myc by a second pathway, repression of transcription from the major c-myc promoter, P2 . Repression was independent of Mxi1 binding to mSin3 but dependent on the Mxi1 LZ and COOH-terminal sequences, including putative casein kinase II phosphorylation sites . Repression targeted elements of the myc P2 promoter core (-35/+10), where it reversed transactivation by the constitutive transcription factor, USF . We show that Zn2+ induction of a stably transfected, metallothionein promoter-regulated mxi1 gene blocked the ability of serum to induce transcription of the endogenous c-myc gene and cell entry into S phase . Thus, induction of Mxi1 in terminally differentiating cells may block Myc function by repressing the c-myc gene P2 promoter, as well as by antagonizing Myc-dependent transactivation through E-boxes.

Am J Respir Crit Care Med, 1999 Jan, 159(1), 100 - 6
Iron uptake promotes hyperoxic injury to alveolar macrophages; Wesselius LJ et al.; Iron uptake by cells may increase the intracellular pool of prooxidant iron prior to storage of iron within ferritin . Because hyperoxia is toxic to alveolar macrophages (AM) via mechanisms involving oxidant stress, we hypothesized that iron uptake by AM might promote hyperoxia-induced injury . To assess this hypothesis, we cultured AM recovered from healthy volunteers under conditions of normoxia or hyperoxia (60% or 95% oxygen) in media of varying iron content, including control media (3 microM iron) and media supplemented with iron (FeCl3; total iron 10, 20, or 40 microM) . AM injury was assessed by measuring release of lactate dehydrogenase (LDH), phagocytic activity for yeast, and cytosolic concentrations of calcium ({Ca2+}i) as determined by ratio image analysis of AM loaded with the fluorescent calcium probe indo-1 . There was dose-dependent accumulation of iron and ferritin synthesis in AM exposed to iron-supplemented media . Exposure of AM to hyperoxia (60% and 95% oxygen, 18 h) in control media increased LDH release and impaired phagocytic activity for yeast; however, similar hyperoxic exposures in iron-supplemented media significantly increased the cells' LDH release and decreased phagocytosis . Exposure to 95% oxygen increased the {Ca2+}i of AM over 18 h, but similar exposure in iron-supplemented media induced greater increases in {Ca2+}i . As compared with exposure to normoxia, exposure to hyperoxia (60% and 95% oxygen) also decreased iron uptake and, to a greater extent, ferritin synthesis by AM in iron-supplemented media . These data suggest that: (1) iron uptake promotes hyperoxic injury to AM; and (2) hyperoxia impairs the capacity of AM to sequester iron in ferritin.

FEBS Lett, 1998 Dec 4, 440(3), 291 - 6
Mutations at position 1122 in the catalytic domain of the mouse ras-specific guanine nucleotide exchange factor CDC25Mm originate both loss-of-function and gain-of-function proteins; Carrera V et al.; The role of two residues within the catalytic domain of CDC25Mm, a mouse ras-specific guanine nucleotide exchange factor (GEF), was investigated by site-directed mutagenesis . The function of the mutant proteins was tested in vivo in both a Saccharomyces cerevisiae cdc25 complementation assay and in a mammalian fos-luciferase assay, and in in vitro assays on human and yeast Ras proteins . Mutants CDC25Mm(E1048K) and CDC25Mm(S1122V) were shown to be (partly) inactive proteins, similar to their yeast homologs . Mutant CDC25Mm(S1122A) showed higher nucleotide exchange activity than the wild type protein on the basis of both in vitro and in vivo assays . Thus, alanine and valine substitutions at position 1122 within the GEF catalytic domain originate mutations with opposite biological properties, indicating an important role for position 1122 in GEF function.

Oncogene, 1998 Dec 17, 17(24), 3187 - 97
Isolation of growth suppressors from a cDNA expression library; Pestov DG et al.; We describe an experimental procedure for the isolation of growth inhibitory sequences from a complex cDNA library . This approach first takes advantage of the SETGAP technique (selectable expression of transient growth arrest phenotype) to enrich for growth inhibitory sequences, followed by a screening procedure to identify individual cDNAs that inhibit cell proliferation . Here we provide a detailed description of the experimental protocol and report the characterization of two cDNA sequences isolated in our initial screen of a mouse cDNA library . One of these cDNAs encodes the mouse ubiquitin-conjugation enzyme UbcM2 . The other encodes a truncated form of a novel WD40 repeat protein, named Bopl, which is conserved from yeast to human . Together, these results demonstrate a new approach for the isolation of growth suppressors from cDNA libraries, and identify a previously unknown gene likely to be involved in growth control.

Bioorg Med Chem Lett, 1998 May 5, 8(9), 1113 - 6
An enantioselective fluorimetric assay for alcohol dehydrogenases using albumin-catalyzed beta-elimination of umbelliferone; Klein G et al.; 3-hydroxybutyl umbelliferyl ethers (R)-1 and (S)-1 are fluorogenic substrates for alcohol dehydrogenases . Their oxidation forms ketone 2, which undergoes beta-elimination of umbelliferone under catalysis by bovine serum albumin, leading to a > 20-fold fluorescence increase at lambda em = 460 +/- 20 nm (lambda ex = 360 +/- 20 nm) . Enantioselectivity is determined in two separate tests with each enantiomeric substrate.

Bioorg Med Chem Lett, 1998 Apr 7, 8(7), 705 - 10
Syntheses and kinetic evaluation of hydroxamate-based peptide inhibitors of glyoxalase I; Ly HD et al.; Hydroxamate-containing tripeptide analogs resembling a reactive intermediate in glyoxalase I catalysis were prepared by solution methods and were found to be competitive inhibitors of the enzyme from Saccharomyces cerevisiae . Electronic properties of the hydroxamate functionality as well as those of the expected intermediates in the enzyme-catalyzed reaction were compared.

J Chromatogr A, 1998 Nov 27, 826(2), 167 - 81
Examination of micro-tip reversed-phase liquid chromatographic extraction of peptide pools for mass spectrometric analysis; Erdjument-Bromage H et al.; Mass spectrometry occupies a central position in most current protein identification schemes . So-called 'mass fingerprinting' techniques rely on composite mass patterns of proteolytic fragments, or dissociation products thereof, to query databases . Keys to successful analysis of ever smaller amounts are sensitivity and complete spectral information, both of which depend for a large part on proper sample preparation . Clean-up and concentration of peptide mixtures over eppendorf gel loading tips filled with chromatographic media (i.e . 'micro-tips') are believed to be quite useful in this regard . We have studied quantitative and qualitative aspects of polypeptide extraction using these small manual devices . Optimization of sample volume and additives, micro-tip bed volume, and eluent composition and volume, all contribute to effective recovery (approximately 65-70%, on average) . Improper digest conditions can, in fact, lead to far bigger losses, suggesting the need for at least trace amounts of Zwittergent 3-16 . Of particular interest is our finding that partial fractionation, obtained by two-step micro-tip elution, generally results in more and better signals during subsequent mass analysis . Thus, by using optimized micro-tips, in combination with adequate sample handling and instrumentation, direct mass spectrometric identification can be routinely and successfully done in any resource facility type setting.

Genes Dev, 1998 Dec 15, 12(24), 3843 - 56
Maintenance of sister-chromatid cohesion at the centromere by the Drosophila MEI-S332 protein; Tang TT et al.; Sister-chromatid cohesion is essential for the faithful segregation of chromosomes during cell division . Recently biochemical analysis with Xenopus extracts suggests that cohesion is established during S phase by a cohesion complex but that other proteins must maintain it in mitosis . The Drosophila melanogaster MEI-S332 protein is present on centromeres in mitosis and meiosis and is essential for cohesion at the centromeres in meiosis II . Here, we analyze the timing of MEI-S332 assembly onto centromeres and the functional domains of the MEI-S332 protein . We find that MEI-S332 is first detectable on chromosomes during prometaphase, and this localization is independent of microtubules . MEI-S332 contains two separable functional domains, as mutations within these domains show intragenic complementation . The carboxy-terminal basic region is required for chromosomal localization . The amino-terminal coiled-coil domain may facilitate protein-protein interactions between MEI-S332 and male meiotic proteins . MEI-S332 interacts with itself in the yeast two-hybrid assay and in immunoprecipitates from Drosophila oocyte and embryo extracts . Thus it appears that MEI-S332 assembles into a multimeric protein complex that localizes to centromeric regions during prometaphase and is required for the maintenance of sister-chromatid cohesion until anaphase, rather than its establishment in S phase.

Genes Dev, 1998 Dec 15, 12(24), 3803 - 8
Transactivation-defective c-MycS retains the ability to regulate proliferation and apoptosis; Xiao Q et al.; Transcriptional activation by c-Myc through specific E box elements is thought to be essential for its biological role . However, c-MycS is unable to activate transcription through these elements and yet retains the ability to stimulate proliferation, induce anchorage-independent growth, and induce apoptosis . In addition, c-MycS retains the ability to repress transcription of several specific promoters . Furthermore, c-MycS can rescue the c-myc null phenotype in fibroblasts with homozygous deletion of c-myc . Taken together, our data argue against the paradigm that all of the biological functions of c-Myc are mediated by transcriptional activation of specific target genes through E box elements.

Plant Mol Biol, 1998 Dec, 38(6), 919 - 27
Protein phosphatase 2C (PP2C) function in higher plants; Rodriguez PL; In the past few years, molecular cloning studies have revealed the primary structure of plant protein serine/threonine phosphatases . Two structurally distinct families, the PP1/PP2A family and the PP2C family, are present in plants as well as in animals . This review will focus on the plant PP2C family of protein phosphatases . Biochemical and molecular genetic studies in Arabidopsis have identified PP2C enzymes as key players in plant signal transduction processes . For instance, the ABI1/ABI2 PP2Cs are central components in abscisic acid (ABA) signal transduction . Arabidopsis mutants containing a single amino acid exchange in ABI1 or ABI2 show a reduced response to ABA . Another member of the PP2C family, kinase-associated protein phosphatase (KAPP), appears to be an important element in some receptor-like kinase (RLK) signalling pathways . Finally, an alfalfa PP2C acts as a negative regulator of a plant mitogen-activated protein kinase (MAPK) pathway . Thus, the plant PP2Cs function as regulators of various signal transduction pathways.

Jpn J Pharmacol, 1998 Nov, 78(3), 355 - 64
Cholesterol-lowering effects of NTE-122, a novel acyl-CoA:cholesterol acyltransferase (ACAT) inhibitor, on cholesterol diet-fed rats and rabbits; Azuma Y et al.; Pharmacological characterization of NTE-122 (trans-1,4-bis{{1-cyclohexyl-3-(4-dimethylamino phenyl)ureido}methyl}cyclohexane), a novel acyl-CoA:cholesterol acyltransferase (ACAT) inhibitor, was performed with both in vitro and in vivo assay systems . NTE-122 inhibited microsomal ACAT activities of various tissues (liver of rabbit and rat, small intestine of rabbit and rat, and aorta of rabbit) and cultured cells (HepG2 and CaCo-2), with IC50 values from 1.2 to 9.6 nM . The inhibition mode of NTE-122 was competitive for HepG2 ACAT . NTE-122 had no effect on other lipid metabolizing enzymes, such as 3-hydroxy-3-methylglutaryl-CoA reductase, acyl-CoA synthetase, cholesterol esterase, lecithin:cholesterol acyltransferase, acyl-CoA:sn-glycerol-3-phosphate acyltransferase and cholesterol 7alpha-hydroxylase up to 10 microM . When NTE-122 was administered to the cholesterol diet-fed rats, serum and liver cholesterol levels were markedly reduced with an ED50 of 0.12 and 0.44 mg/kg/day, respectively . In the cholesterol diet-fed rabbits, NTE-122 significantly lowered plasma and liver cholesterol levels at more than 2 mg/kg/day . These results indicate that NTE-122 is a potent, selective and competitive inhibitor of ACAT, making it a worth while therapeutic agent for hypercholesterolemia and atherosclerosis.

J Biol Chem, 1999 Jan 1, 274(1), 218 - 26
Evidence for (Mac1p)2.DNA ternary complex formation in Mac1p-dependent transactivation at the CTR1 promoter; Joshi A et al.; The Mac1 protein in Saccharomyces cerevisiae is required for the expression CTR1 and FRE1, which, respectively, encode the copper permease and metal reductase that participate in copper uptake . Mac1p binds to a core GCTC sequence present as a repeated unit in the promoters of both genes . We show here that Mac1p DNA binding required an intact N-terminal protein domain that includes a likely zinc finger motif . This binding was enhanced by the presence of a TATTT sequence immediately 5' to the core GCTC, in contrast to a TTTTT one . This increased binding was demonstrated clearly in vitro in electrophoretic mobility shift assays that showed Mac1p.DNA complex formation to a single TATTTGCTC element but not to a TTTTTGCTC one . Furthermore, the fraction of Mac1p in a ternary (Mac1p)2.DNA complex in comparison to a binary Mac1p.DNA complex increased when the DNA included two TATTTGCTC elements . A similar increase in ternary complex formation was demonstrated upon homologous mutation of the FRE1 Mac1p-dependent promoter element . The in vivo importance of this ternary complex formation at the CTR1 promoter was indicated by the stronger trans-activity of this promoter mutated to contain two TATTT elements and the attenuated activity of a mutant promoter containing two TTTTT elements that in vitro supported only a weak ternary complex signal in the shift assay . The stronger binding to TATTT appeared due to a more favorable protein contact with adenine in comparison to thymine at this position . An in vivo two-hybrid analysis demonstrated a Mac1p-Mac1p protein-protein interaction . This Mac1p-Mac1p interaction may promote (Mac1p)2.DNA ternary complex formation at Mac1p-responsive upstream activating sequences.

J Biol Chem, 1999 Jan 1, 274(1), 205 - 10
The Tup1-Cyc8 protein complex can shift from a transcriptional co-repressor to a transcriptional co-activator; Conlan RS et al.; Cyc8(Ssn6)-Tup1, a general co-repressor complex, is recruited to promoter DNA via interactions with DNA-binding regulatory proteins and inhibits the transcription of many different yeast genes . Previous studies have established that repression function of the complex is performed by one subunit of the complex, the Tup1 protein, and requires specific components of the RNA polymerase II holoenzyme such as Sin4 and Rgr1 . In this study we test the transcriptional activity of the Cyc8 subunit using a lexA operator-containing reporter . We show that a LexA-Cyc8 hybrid stimulates transcription when expressed in a tup1Delta, a sin4Delta, or a rgr1Delta strain, suggesting that transcriptional activation is an intrinsic property of the Cyc8-Tup1 co-repressor . In support of this notion we demonstrate that Cyc8-Tup1 has a dual function on CIT2, a gene encoding a citrate synthase that is expressed upon mitochondrial dysfunction . First, we show that Cyc8-Tup1 is tethered to CIT2 promoter by interacting with the activation domain of Rtg3, a bHLH/L-Zip DNA-binding transactivator of CIT2 . Next we demonstrate that Cyc8-Tup1 activates CIT2 transcription in response to mitochondrial dysfunction, and this stimulatory effect is mediated by Cyc8 . In contrast, basal (noninduced) expression of this gene is inhibited by Tup1 . These findings establish a positive role for the Cyc8-Tup1 complex in transcription and support a model by which specific metabolic signals may convert the Cyc8-Tup1 transcriptional co-repressor to a co-activator of certain promoters.

J Biol Chem, 1999 Jan 1, 274(1), 196 - 204
The association of initiation factor 4F with poly(A)-binding protein is enhanced in serum-stimulated Xenopus kidney cells; Fraser CS et al.; Serum stimulation of cultured Xenopus kidney cells results in enhanced phosphorylation of the translational initiation factor (eIF) 4E and promotes a 2.8-fold increase in the binding of the adapter protein eIF4G to eIF4E, to form the functional initiation factor complex eIF4F . Here we demonstrate the serum-stimulated co-isolation of the poly(A)-binding protein (PABP) with the eIF4F complex . This apparent interaction of PABP with eIF4F suggests that a mechanism shown to be important in the control of translation in the yeast Saccharomyces cerevisiae also operates in vertebrate cells . We also present evidence that the signaling pathways modulating eIF4E phosphorylation and function in Xenopus kidney cells differ from those in several mammalian cell types studied previously . Experiments with the immunosuppressant rapamycin suggest that the mTOR signaling pathway is involved in serum-promoted eIF4E phosphorylation and association with eIF4G . Moreover, we could find little evidence for regulation of eIF4E function via interaction with the specific binding proteins 4E-BP1 or 4E-BP2 in these cells . Although rapamycin abrogated serum-enhanced rates of protein synthesis and the interaction of eIF4G with eIF4E, it did not prevent the increase in association of eIF4G with PABP . This suggests that serum stimulates the interaction between eIF4G and PABP by a distinct mechanism that is independent of both the mTOR pathway and the enhanced association of eIF4G with eIF4E.

J Biol Chem, 1999 Jan 1, 274(1), 183 - 8
Analysis of the MRP8-MRP14 protein-protein interaction by the two-hybrid system suggests a prominent role of the C-terminal domain of S100 proteins in dimer formation; Propper C et al.; Calcium-binding S100 proteins are thought to play a central role in calcium-mediated signal transduction pathways . They consist of two helix-loop-helix, calcium-binding EF-hand domains . A characteristic feature is their tendency to form homo- and/or heterodimeric complexes . This report presents for the first time a functional "in vivo" approach to the analysis of S100 protein dimerization . Using the two-hybrid system we analyzed the dimerization of MRP8 (S100A8) and MRP14 (S100A9), two S100 proteins expressed in myeloid cells . It is reported that the MRP8-MRP14 heteromer is the clearly preferred complex in both man and mouse . The ability to homodimerize, however, appears to be restricted to the murine MRPs . Interaction analysis of chimeric murine/human MRP14 proteins indicates, that the C-terminal EF-hand domain plays a prominent role in MRP8-MRP14 interaction and determines the specificity of dimerization . Site-directed mutagenesis of four evolutionary conserved hydrophobic amino acids, which have been recently supposed to be essential for S100 protein dimerization, suggests that at least one of these, namely the most N-terminal located residue, is not critical for dimerization.

J Biol Chem, 1999 Jan 1, 274(1), 11 - 9
Interaction of the integrin beta1 cytoplasmic domain with ICAP-1 protein; Zhang XA et al.; In a yeast two-hybrid screen, a protein named ICAP-1 (beta1 integrin cytoplasmic domain associated protein) associated with the integrin beta1 cytoplasmic tail but not with tails from three other integrin beta subunits (beta2, beta3, and beta5) or from seven different alpha subunits . Likewise in human cells, ICAP-1 associated specifically with the beta1 but not beta2, beta3, or beta5 tails . The carboxyl-terminal 14 amino acids of beta1 were critical for ICAP-1 interaction . ICAP-1 is a ubiquitously expressed protein of 27 and 31 kDa, with the smaller form being preferentially solubilized by Triton X-100 . Phosphorylation of both 27- and 31-kDa forms was constitutive but was increased by 1.5-2-fold upon cell spreading on fibronectin, compared with poly-L-lysine . Also, ICAP-1 contributes to beta1 integrin-dependent migration because (i) ICAP-1 transfection markedly increased chemotactic migration of COS7 cells through fibronectin-coated but not vitronectin-coated porous filters, and (ii) support of beta1-dependent cell migration (in Chinese hamster ovary cells transfected with various wild type and mutant beta1 forms) correlated with ICAP-1 association . In summary, ICAP-1 (i) associates specifically with beta1 integrins, (ii) is phosphorylated upon beta1 integrin-mediated adhesion, and (iii) may regulate beta1-dependent cell migration.

Photochem Photobiol, 1998 Dec, 68(6), 857 - 63
Recombinant phytochrome of the moss Ceratodon purpureus: heterologous expression and kinetic analysis of Pr-->Pfr conversion; Zeidler M et al.; The phytochrome-encoding gene Cerpu;PHY;2 (CP2) of the moss Ceratodon purpureus was heterologously expressed in Saccharomyces cerevisiae as a polyhistidine-tagged apoprotein and assembled with phytochromobilin (P phi B) and phycocyanobilin (PCB) . Nickel-affinity chromatography yielded a protein fraction containing approximately 80% phytochrome . The holoproteins showed photoreversibility with both chromophores . Difference spectra gave maxima at 644/716 nm (red-absorbing phytochrome {Pr}/far-red-absorbing phytochrome {Pfr}) for the PCB adduct, and 659/724 nm for the P phi B-adduct, the latter in close agreement with values for phytochrome extracted from Ceratodon itself, implying that P phi B is the native chromophore in this moss species . Immunoblots stained with the antiphytochrome antibody APC1 showed that the recombinant phytochrome had the same molecular size as phytochrome from Ceratodon extracts . Further, the mobility of recombinant CP2 holophytochrome on native size-exclusion chromatography was similar to that of native oat phytochrome, implying that CP2 forms a dimer . Kinetics of absorbance changes during the Pr-->Pfr photoconversion of the PCB adduct, monitored between 620 and 740 nm in the microsecond range, revealed the rapid formation of a red-shifted intermediate (I700), decaying with a time constant of approximately 110 microseconds . This is similar to the behavior of phytochromes from higher plants when assembled with the same chromophore . When following the formation of the Pfr state, two major processes were identified (with time constants of 3 and 18 ms) that are followed by slow reactions in the range of 166 ms and 8 s, respectively, albeit with very small amplitudes.

Anal Biochem, 1998 Dec 1, 265(1), 167 - 75
Detection of metallothionein isoforms from three different species using on-line capillary electrophoresis-mass spectrometry; Knudsen CB et al.; An on-line capillary electrophoresis-mass spectrometry method (CE-MS) for the detection of metallothionein (MT) isoforms is described . The detected masses were usually within 1-1.5 mass units of the expected molecular weights . MT-containing samples from rabbit, sheep, and yeast (Saccharomyces cerevisiae) were subjected to CE-MS analysis . The analysis of rabbit liver MT revealed the masses of 10 proteins/peptides . Five of the detected masses corresponded well with the expected masses calculated from the amino acid sequence of previously described MT isoforms, one was suspected to be a deacetylated form of MT-2A, one was presumed to be a yet unknown isoform, and three masses were classified as non-MT compounds . From the analysis of a fetal sheep liver extract six proteins were detected of which three masses corresponded to previously described MT isoforms . Two purified MT subforms from S . cerivisiae (encoded by the CUP1 locus) were analyzed for their copper content and both forms were found to contain eight copper atoms per molecule.

Anal Biochem, 1998 Nov 15, 264(2), 158 - 64
Mapping a protein-binding site on straightened DNA by atomic force microscopy; Yokota H et al.; We have developed an Atomic Force Microscopy (AFM)-based method for mapping protein-binding sites on individual, long DNA molecules (> 5 kb) at nanometer resolution . The protein is clearly detected at the apex of the bent DNA molecules . Randomly coiled DNA molecules or protein:DNA complexes were extended by a motor-controlled moving meniscus on an atomically flat surface . The immobilized molecules were detected by AFM . The straightened DNA displayed a sharp bend at the site of bound protein with the two DNA segments linearly extending from the protein-binding site . Using GAL4, a yeast transcription factor, we demonstrate good agreement of the position of the observed binding site on straightened DNA templates to the predicted binding site . The technique is expected to have significant implications in elucidating DNA and protein interactions in general, and specifically, for the measurement of promoter occupancy with unlabeled regulatory proteins at the single-molecule level.

Mol Biol Evol, 1998 Dec, 15(12), 1674 - 84
Cytosolic isocitrate dehydrogenase in humans, mice, and voles and phylogenetic analysis of the enzyme family; Nekrutenko A et al.; In this study, we report cDNA sequences of the cytosolic NADP-dependent isocitrate dehydrogenase for humans, mice, and two species of voles (Microtus mexicanus and Microtus ochrogaster) . Inferred amino acid sequences from these taxa display a high level of amino acid sequence conservation, comparable to that of myosin beta heavy chain, and share known structural features . A Caenorhabditis elegans enzyme that was previously identified as a protein similar to isocitrate dehydrogenase is most likely the NADP-dependent cytosolic isocitrate dehydrogenase enzyme equivalent, based on amino acid similarity to mammalian enzymes and phylogenetic analysis . We also suggest that NADP-dependent isocitrate dehydrogenases characterized from alfalfa, soybean, and eucalyptus are most likely cytosolic enzymes . The phylogenetic tree of various isocitrate dehydrogenases from eukaryotic sources revealed that independent gene duplications may have given rise to the cytosolic and mitochondrial forms of NADP-dependent isocitrate dehydrogenase in animals and fungi . There appears to be no statistical support for a hypothesis that the mitochondrial and cytosolic forms of the enzyme are orthologous in these groups . A possible scenario of the evolution of NADP-dependent isocitrate dehydrogenases is proposed.

Microcirculation, 1998, 5(4), 239 - 57
The mechanics of vascular cell motility; Shuster CB et al.; Alterations in vascular cell shape and motility occur during developmental processes and in response to injury . Similarly, during tumor vascularization and atherogenesis, endothelial and smooth muscle cells undergo motile and proliferative responses to extracellular cues . Recent inroads into our understanding of signal transduction have identified several candidate pathways by which the extracellular matrix- and growth factor-mediated stimulation of vascular cell motility may be mediated . The multiple and divergent extracellular stimuli that stimulate vascular motile responses may converge on the cytoskeleton via a family of ras-related GTPases . Biochemical analyses as well as examination of cytoskeletal dynamics in vivo indicate that actin polymerization at the forward aspects of spreading cytoplasm is capable of driving forward protrusion formation in the absence of a conventional actin motor . Actin polymerization at the plasma membrane of leading lamellae may be mediated both by de novo nucleation of actin filaments and the generation of free filament ends by uncapping the barbed ends of existing actin filaments . This review summarizes the most recent findings in extracellular-cytoskeletal-signal transduction, therein, providing a framework to explain the remarkable remodeling seen in the vasculature during developmental and disease-related processes.

Cell, 1998 Dec 11, 95(6), 847 - 58
Fab1p PtdIns(3)P 5-kinase function essential for protein sorting in the multivesicular body; Odorizzi G et al.; Sorting of signal-transducing cell surface receptors within multivesicular bodies (MVBs) is required for their rapid down-regulation and degradation within lysosomes . Yeast mutants defective in late stages of transport to the vacuole/lysosome accumulate MVBs . We demonstrate that the membrane glycoprotein carboxypeptidase S and the G protein-coupled receptor Ste2p are targeted into the vacuole lumen, and this process requires a subset of VPS gene products essential for normal endosome function . The PtdIns(3)P 5-kinase activity of Fab1p, which converts the product of the Vps34p PtdIns 3-kinase PtdIns(3)P into PtdIns(3,5)P2, also is required for cargo-selective sorting into the vacuole lumen . These findings demonstrate a role for phosphoinositide signaling at distinct stages of vacuolar/lysosomal protein transport and couple PtdIns(3,5)P2 synthesis to regulation of MVB sorting.

Biol Chem, 1998 Nov, 379(11), 1319 - 22
Cryoinactivation and conformational drift of glyceraldehyde-3-phosphate dehydrogenase from rabbit muscle; Tian S et al.; The cryoinactivation of glyceraldehyde-3-phosphate dehydrogenase from rabbit muscle (GAPDH-rabbit) was studied . It was found that the inactivation of GAPDH-rabbit at 0 degrees C was much faster than that of GAPDH from yeasts, and showed obvious time and concentration dependence . The spectral properties, enzyme activity and behavior under pressure, of GAPDH-rabbit treated either by cryoinactivation, or pressure-induced dissociation and reassociation, were very similar . These results provided evidence to support the idea that cryoinactivation of oligomeric proteins, might take place through a cycle of dissociation-reassociation accompanied with the so-called conformational drift postulated by King and Weber (1986).

J Cell Biol, 1998 Dec 28, 143(7), 1947 - 60
Dual function of Cyk2, a cdc15/PSTPIP family protein, in regulating actomyosin ring dynamics and septin distribution; Lippincott J et al.; We previously showed that the budding yeast Saccharomyces cerevisiae assembles an actomyosin-based ring that undergoes a contraction-like size change during cytokinesis . To learn more about the biochemical composition and activity of this ring, we have characterized the in vivo distribution and function of Cyk2p, a budding yeast protein that exhibits significant sequence similarity to the cdc15/PSTPIP family of cleavage furrow proteins . Video microscopy of cells expressing green fluorescent protein (GFP)-tagged Cyk2p revealed that Cyk2p forms a double ring that coincides with the septins through most of the cell cycle . During cytokinesis, however, the Cyk2 double ring merges with the actomyosin ring and exhibits a contraction-like size change that is dependent on Myo1p . The septin double ring, in contrast, does not undergo the contraction-like size change but the separation between the two rings increases during cytokinesis . These observations suggest that the septin-containing ring is dynamically distinct from the actomyosin ring and that Cyk2p transits between the two types of structures . Gene disruption of CYK2 does not affect the assembly of the actomyosin ring but results in rapid disassembly of the ring during the contraction phase, leading to incomplete cytokinesis, suggesting that Cyk2p has an important function in modulating the stability of the actomyosin ring during contraction . Overexpression of Cyk2p also blocks cytokinesis, most likely due to a loss of the septins from the bud neck, indicating that Cyk2p may also play a role in regulating the localization of the septins.

J Cell Biol, 1998 Dec 28, 143(7), 1919 - 30
Visualization and molecular analysis of actin assembly in living cells; Schafer DA et al.; Actin filament assembly is critical for eukaryotic cell motility . Arp2/3 complex and capping protein (CP) regulate actin assembly in vitro . To understand how these proteins regulate the dynamics of actin filament assembly in a motile cell, we visualized their distribution in living fibroblasts using green flourescent protein (GFP) tagging . Both proteins were concentrated in motile regions at the cell periphery and at dynamic spots within the lamella . Actin assembly was required for the motility and dynamics of spots and for motility at the cell periphery . In permeabilized cells, rhodamine-actin assembled at the cell periphery and at spots, indicating that actin filament barbed ends were present at these locations . Inhibition of the Rho family GTPase rac1, and to a lesser extent cdc42 and RhoA, blocked motility at the cell periphery and the formation of spots . Increased expression of phosphatidylinositol 5-kinase promoted the movement of spots . Increased expression of LIM-kinase-1, which likely inactivates cofilin, decreased the frequency of moving spots and led to the formation of aggregates of GFP-CP . We conclude that spots, which appear as small projections on the surface by whole mount electron microscopy, represent sites of actin assembly where local and transient changes in the cortical actin cytoskeleton take place.

J Cell Biol, 1998 Dec 28, 143(7), 1813 - 30
Specific binding of the karyopherin Kap121p to a subunit of the nuclear pore complex containing Nup53p, Nup59p, and Nup170p; Marelli M et al.; We have identified a specific karyopherin docking complex within the yeast nuclear pore complex (NPC) that contains two novel, structurally related nucleoporins, Nup53p and Nup59p, and the NPC core protein Nup170p . This complex was affinity purified from cells expressing a functional Nup53p-protein A chimera . The localization of Nup53p, Nup59p, and Nup170p within the NPC by immunoelectron microscopy suggests that the Nup53p-containing complex is positioned on both the cytoplasmic and nucleoplasmic faces of the NPC core . In association with the isolated complex, we have also identified the nuclear transport factor Kap121p (Pse1p) . Using in vitro binding assays, we showed that each of the nucleoporins interacts with one another . However, the association of Kap121p with the complex is mediated by its interaction with Nup53p . Moreover, Kap121p is the only beta-type karyopherin that binds Nup53p suggesting that Nup53p acts as a specific Kap121p docking site . Kap121p can be released from Nup53p by the GTP bound form of the small GTPase Ran . The physiological relevance of the interaction between Nup53p and Kap121p was further underscored by the observation that NUP53 mutations alter the subcellular distribution of Kap121p and the Kap121p- mediated import of a ribosomal L25 reporter protein . Interestingly, Nup53p is specifically phosphorylated during mitosis . This phenomenon is correlated with a transient decrease in perinuclear-associated Kap121p.

J Cell Biol, 1998 Dec 28, 143(7), 1763 - 74
INCENP centromere and spindle targeting: identification of essential conserved motifs and involvement of heterochromatin protein HP1; Ainsztein AM et al.; The inner centromere protein (INCENP) has a modular organization, with domains required for chromosomal and cytoskeletal functions concentrated near the amino and carboxyl termini, respectively . In this study we have identified an autonomous centromere- and midbody-targeting module in the amino-terminal 68 amino acids of INCENP . Within this module, we have identified two evolutionarily conserved amino acid sequence motifs: a 13-amino acid motif that is required for targeting to centromeres and transfer to the spindle, and an 11-amino acid motif that is required for transfer to the spindle by molecules that have targeted previously to the centromere . To begin to understand the mechanisms of INCENP function in mitosis, we have performed a yeast two-hybrid screen for interacting proteins . These and subsequent in vitro binding experiments identify a physical interaction between INCENP and heterochromatin protein HP1(Hsalpha) . Surprisingly, this interaction does not appear to be involved in targeting INCENP to the centromeric heterochromatin, but may instead have a role in its transfer from the chromosomes to the anaphase spindle.

Nucleic Acids Res, 1999 Jan 15, 27(2), 656 - 64
A novel assay for examining the molecular reactions at the eukaryotic replication fork: activities of replication protein A required during elongation; Walther AP et al.; Studies to elucidate the reactions that occur at the eukaryotic replication fork have been limited by the model systems available . We have established a method for isolating and characterizing Simian Virus 40 (SV40) replication complexes . SV40 rolling circle complexes are isolated using paramagnetic beads and then incubated under replication conditions to obtain continued elongation . In rolling circle replication, the normal mechanism for termination of SV40 replication does not occur and the elongation phase of replication is prolonged . Thus, using this assay system, elongation phase reactions can be examined in the absence of initiation or termination . We show that the protein requirements for elongation of SV40 rolling circles are equivalent to complete SV40 replication reactions . The DNA produced by SV40 rolling circles is double-stranded, unmethylated and with a much longer length than the template DNA . These properties are similar to those of physiological replication forks . We show that proteins associated with the isolated rolling circles, including SV40 T antigen, DNA polymerase alpha, replication protein A (RPA) and RF-C, are necessary for continued DNA synthesis . PCNA is also required but is not associated with the isolated complexes . We present evidence suggesting that synthesis of the leading and lagging strands are co-ordinated in SV40 rolling circle replication . We have used this system to show that both RPA-protein and RPA-DNA interactions are important for RPA's function in elongation.

Nucleic Acids Res, 1999 Jan 15, 27(2), 573 - 80
Tandem repeats finder: a program to analyze DNA sequences; Benson G; A tandem repeat in DNA is two or more contiguous, approximate copies of a pattern of nucleotides . Tandem repeats have been shown to cause human disease, may play a variety of regulatory and evolutionary roles and are important laboratory and analytic tools . Extensive knowledge about pattern size, copy number, mutational history, etc . for tandem repeats has been limited by the inability to easily detect them in genomic sequence data . In this paper, we present a new algorithm for finding tandem repeats which works without the need to specify either the pattern or pattern size . We model tandem repeats by percent identity and frequency of indels between adjacent pattern copies and use statistically based recognition criteria . We demonstrate the algorithm's speed and its ability to detect tandem repeats that have undergone extensive mutational change by analyzing four sequences: the human frataxin gene, the human beta T cellreceptor locus sequence and two yeast chromosomes . These sequences range in size from 3 kb up to 700 kb . A World Wide Web server interface atc3.biomath.mssm.edu/trf.html has been established for automated use of the program.

Nucleic Acids Res, 1999 Jan 15, 27(2), 562 - 72
Structure and function of a small RNA that selectively inhibits internal ribosome entry site-mediated translation; Venkatesan A et al.; A 60 nt long RNA termed IRNA, isolated from the yeast Saccharomyces cerevesiae, was previously shown to selectively block internal ribosome entry site (IRES)-mediated translation without interfering with cap-dependent translation of cellular mRNAs both in vivo and in vitro . IRNA specifically bound cellular proteins believed to be important for IRES-mediated translation . We demonstrate here that a complementary copy of IRNA (cIRNA) is also active in blocking IRES-mediated translation and that it binds many of the same cellular proteins that IRNA does . We have probed the secondary structure of both IRNA and cIRNA using single-strand- and double-strand-specific nucleases as well as using oligonucleotide hybridization followed by RNase H digestion . Both IRNA and cIRNA share secondary structural homology, although distinct differences do exist between the two structures . Mutational analysis of IRNA shows that sequences that form both the main stem and one loop are critical for its translation inhibitory activity . Maintenance of the established secondary structure appears to be required for both IRNA's ability to bind cellular trans -acting proteins believed to be required for IRES-mediated translation and its ability to block IRES-mediated translation.

Plant Mol Biol, 1998 Nov, 38(5), 879 - 83
Molecular cloning in Arabidopsis thaliana of a new protein phosphatase 2C (PP2C) with homology to ABI1 and ABI2; Rodriguez PL et al.; We report the cloning of both the cDNA and the corresponding genomic sequence of a new PP2C from Arabidopsis thaliana, named AtP2C-HA (for homology to ABI1/ABI2) . The AtP2C-HA cDNA contains an open reading frame of 1536 bp and encodes a putative protein of 511 amino acids with a predicted molecular mass of 55.7 kDa . The AtP2C-HA protein is composed of two domains, a C-terminal PP2C catalytic domain and a N-terminal extension of ca . 180 amino acid residues . The deduced amino acid sequence is 55% and 54% identical to ABI1 and ABI2, respectively . Comparison of the genomic structure of the ABI1, ABI2 and AtP2C-HA genes suggests that they belong to a multigene family . The expression of the AtP2C-HA gene is up-regulated by abscisic acid (ABA) treatment.

FEBS Lett, 1998 Nov 27, 440(1-2), 141 - 6
Identification of the copper chaperone, CUC-1, in Caenorhabditis elegans: tissue specific co-expression with the copper transporting ATPase, CUA-1; Wakabayashi T et al.; A cDNA encoding a putative copper chaperone protein, CUC-1, was cloned from Caenorhabditis elegans . CUC-1 had the characteristic motifs of MTCXXC and KKTGK, and showed 49.3 and 39.1% sequence identity with yeast Atx1p and human HAH1, respectively . Expression of CUC-1 cDNA complemented a null atx1 mutant, the yeast copper chaperone gene, thus demonstrating that CUC-1 is a functional copper chaperone . Studies with transgenic worms indicated that cuc-1 and cua-1, which encodes the copper transporting ATPase, are expressed together in intestinal cells of adult and hypodermal cells in the larvae . cua-1 was also expressed in pharyngeal muscle but cuc-1 was not . These results suggest that CUC-1 and CUA-1 constitute a copper trafficking pathway similar to the yeast counterparts in intestinal and hypodermal cells, and CUA-1 may have a different function in pharyngeal muscle.

FEBS Lett, 1998 Nov 27, 440(1-2), 135 - 40
A phage-based system to select multiple protein-protein interactions simultaneously from combinatorial libraries; Rudert F et al.; Selectively infective phage (SIP) can be used to identify protein-protein interactions . SIP was modified to facilitate the simultaneous selection of interacting protein pairs from large combinatorial libraries . An interference-resistant phage was constructed which non-covalently, but stably links the genetic information of an interacting pair, encoded separately on phage and phagemid vectors, by co-packaging into heteropolyphages . In a model system, the interaction between a SIP-selected peptide and the intracellular domain of the p75 neurotrophin receptor was detected in the presence of a 10(4)-fold excess of a non-interacting control pair (jun leucine zipper and p75 intracellular domain) via SIP hetero-polyphage transductants . To minimize the redundancy of transductants and to minimize possible ligand exchange generated in a solution-based SIP screening, a filter-based in situ infectivity screening was developed . The combination of the above techniques may provide a powerful system for rapid screening of very large sequence spaces.

Proc Natl Acad Sci U S A, 1998 Dec 22, 95(26), 15559 - 64
Analysis of variable (diversity) joining recombination in DNAdependent protein kinase (DNA-PK)-deficient mice reveals DNA-PK-independent pathways for both signal and coding joint formation; Bogue MA et al.; Previous studies have suggested that ionizing radiation causes irreparable DNA double-strand breaks in mice and cell lines harboring mutations in any of the three subunits of DNA-dependent protein kinase (DNA-PK) (the catalytic subunit, DNA-PKcs, or one of the DNA-binding subunits, Ku70 or Ku86) . In actuality, these mutants vary in their ability to resolve double-strand breaks generated during variable (diversity) joining {V(D)J} recombination . Mutant cell lines and mice with targeted deletions in Ku70 or Ku86 are severely compromised in their ability to form coding and signal joints, the products of V(D)J recombination . It is noteworthy, however, that severe combined immunodeficient (SCID) mice, which bear a nonnull mutation in DNA-PKcs, are substantially less impaired in forming signal joints than coding joints . The current view holds that the defective protein encoded by the murine SCID allele retains enough residual function to support signal joint formation . An alternative hypothesis proposes that DNA-PKcs and Ku perform different roles in V(D)J recombination, with DNA-PKcs required only for coding joint formation . To resolve this issue, we examined V(D)J recombination in DNA-PKcs-deficient (SLIP) mice . We found that the effects of this mutation on coding and signal joint formation are identical to the effects of the SCID mutation . Signal joints are formed at levels 10-fold lower than in wild type, and one-half of these joints are aberrant . These data are incompatible with the notion that signal joint formation in SCID mice results from residual DNA-PKcs function, and suggest a third possibility: that DNA-PKcs normally plays an important but nonessential role in signal joint formation.

Proc Natl Acad Sci U S A, 1998 Dec 22, 95(26), 15400 - 5
Differential regulation of transcription: repression by unactivated mitogen-activated protein kinase Kss1 requires the Dig1 and Dig2 proteins; Bardwell L et al.; Kss1, a yeast mitogen-activated protein kinase (MAPK), in its unphosphorylated (unactivated) state binds directly to and represses Ste12, a transcription factor necessary for expression of genes whose promoters contain filamentous response elements (FREs) and genes whose promoters contain pheromone response elements (PREs) . Herein we show that two nuclear proteins, Dig1 and Dig2, are required cofactors in Kss1-imposed repression . Dig1 and Dig2 cooperate with Kss1 to repress Ste12 action at FREs and regulate invasive growth in a naturally invasive strain . Kss1-imposed Dig-dependent repression of Ste12 also occurs at PREs . However, maintenance of repression at PREs is more dependent on Dig1 and/or Dig2 and less dependent on Kss1 than repression at FREs . In addition, derepression at PREs is more dependent on MAPK-mediated phosphorylation than is derepression at FREs . Differential utilization of two types of MAPK-mediated regulation (binding-imposed repression and phosphorylation-dependent activation), in combination with distinct Ste12-containing complexes, contributes to the mechanisms by which separate extracellular stimuli that use the same MAPK cascade can elicit two different transcriptional responses.

Proc Natl Acad Sci U S A, 1998 Dec 22, 95(26), 15287 - 92
Cloning and functional expression of a cDNA encoding a pheromone gland-specific acyl-CoA Delta11-desaturase of the cabbage looper moth, Trichoplusia ni; Knipple DC et al.; Desaturation of coenzyme-A esters of saturated fatty acids is a common feature of sex pheromone biosynthetic pathways in the Lepidoptera . The enzymes that catalyze this step share several biochemical properties with the ubiquitous acyl-CoA Delta9-desaturases of animals and fungi, suggesting a common ancestral origin . Unlike metabolic acyl-CoA Delta9-desaturases, pheromone desaturases have evolved unusual regio- and stereoselective activities that contribute to the remarkable diversity of chemical structures used as pheromones in this large taxonomic group . In this report, we describe the isolation of a cDNA encoding a pheromone gland desaturase from the cabbage looper moth, Trichoplusia ni, a species in which all unsaturated pheromone products are produced via a Delta11Z-desaturation mechanism . The largest ORF of the approximately 1,250-bp cDNA encodes a 349-aa apoprotein (PDesat-Tn Delta11Z) with a predicted molecular mass of 40,240 Da . Its hydrophobicity profile is similar overall to those of rat and yeast Delta9-desaturases, suggesting conserved transmembrane topology . A 182-aa core domain delimited by conserved histidine-rich motifs implicated in iron-binding and catalysis has 72 and 58% similarity (including conservative substitutions) to acyl-CoA Delta9Z-desaturases of rat and yeast, respectively . Northern blot analysis revealed an approximately 1,250-nt PDesat-Tn Delta11Z mRNA that is consistent with the spatial and temporal distribution of Delta11-desaturase enzyme activity . Genetic transformation of a desaturase-deficient strain of the yeast Saccharomyces cerevisiae with an expression plasmid encoding PDesat-Tn Delta11Z resulted in complementation of the strain's fatty acid auxotrophy and the production of Delta11Z-unsaturated fatty acids.

Proc Natl Acad Sci U S A, 1998 Dec 22, 95(26), 15281 - 6
RNA polymerase subunit RPB5 plays a role in transcriptional activation; Miyao T et al.; A mutation in RPB5 (rpb5-9), an essential RNA polymerase subunit assembled into RNA polymerases I, II, and III, revealed a role for this subunit in transcriptional activation . Activation by GAL4-VP16 was impaired upon in vitro transcription with mutant whole-cell extracts . In vivo experiments using inducible reporter plasmids and Northern analysis support the in vitro data and demonstrate that RPB5 influences activation at some, but not all, promoters . Remarkably, this mutation maps to a conserved region of human RPB5 implicated by others to play a role in activation . Chimeric human-yeast RPB5 containing this conserved region now can function in place of its yeast counterpart . The defects noted with rpb5-9 are similar to those seen in truncation mutants of the RPB1-carboxyl terminal domain (CTD) . We demonstrate that RPB5 and the RPB1-CTD have overlapping roles in activation because the double mutant is synthetically lethal and has exacerbated activation defects at the GAL1/10 promoter . These studies demonstrate that there are multiple activation targets in RNA polymerase II and that RPB5 and the CTD have similar roles in activation.

Proc Natl Acad Sci U S A, 1998 Dec 22, 95(26), 15253 - 8
The biochemical properties of the ATPase activity of a 70-kDa heat shock protein (Hsp70) are governed by the C-terminal domains; Lopez-Buesa P et al.; The cytosolic 70-kDa heat shock proteins (Hsp70s), Ssa and Ssb, of Saccharomyces cerevisiae are functionally distinct . Here we report that the ATPase activities of these two classes of Hsp70s exhibit different kinetic properties . The Ssa ATPase has properties similar to those of other Hsp70s studied, such as DnaK and Hsc70 . Ssb, however, has an unusually low steady-state affinity for ATP but a higher maximal velocity . In addition, the ATPase activity of Hsp70s, like that of Ssa1, depends on the addition of K+ whereas Ssb activity does not . Suprisingly, the isolated 44-kDa ATPase domain of Ssb has a Km and Vmax for ATP hydrolysis similar to those of Ssa, rather than those of full length Ssb . Analysis of Ssa/Ssb fusion proteins demonstrates that the Ssb peptide-binding domain fused to the Ssa ATPase domain generates an ATPase of relatively high activity and low steady-state affinity for ATP similar to that of native Ssb . Therefore, at least some of the biochemical differences between the ATPases of these two classes of Hsp70s are not intrinsic to the ATPase domain itself . The differential influence of the peptide-binding domain on the ATPase domain may, in part, explain the functional uniqueness of these two classes of Hsp70s.

Nature, 1998 Dec 10, 396(6711), 587 - 90
Decoupling of nucleotide- and microtubule-binding sites in a kinesin mutant; Song H et al.; Molecular motors require ATP to move along microtubules or actin filaments . To understand how molecular motors function, it is crucial to know how binding of the motor to its filamentous track stimulates the hydrolysis of ATP by the motor, enabling it to move along the filament . A mechanism for the enhanced ATP hydrolysis has not been elucidated, but it is generally accepted that conformational changes in the motor proteins occur when they bind to microtubules or actin filaments, facilitating the release of ADP . Here we report that a mutation in the motor domain of the microtubule motor proteins Kar3 and Ncd uncouples nucleotide- and microtubule-binding by the proteins, preventing activation of the motor ATPase by microtubules . Unlike the wild-type motors, the mutants bind tightly to both ADP and microtubules, indicating that interactions between the nucleotide- and microtubule-binding sites are blocked . The region of the motor that includes the mutated amino acid could transmit or undergo a conformational change required to convert the motor ATPase into a microtubule-stimulated state.

Nature, 1998 Dec 10, 396(6711), 575 - 80
Ca2+/calmodulin signals the completion of docking and triggers a late step of vacuole fusion; Peters C et al.; The basic reaction mechanisms for membrane fusion in the trafficking of intracellular membranes and in exocytosis are probably identical . But in contrast to regulated exocytosis, intracellular fusion reactions are referred to as 'constitutive' as no final Ca2+-dependent triggering step has been observed . Although transport from the endoplasmic reticulum to the Golgi apparatus in the cell depends on Ca2+, as does endosome fusion and assembly of the nuclear envelope, it is unclear whether Ca2+ triggers these events . Membrane fusion involves several subreactions: priming, tethering and docking . Proteins that are needed for fusion include p115, SNAPs, NSF, SNAREs and small GTPases, which operate in these early reactions, but the machinery that catalyses the final mixing of biological membranes is still unknown . Here we show that Ca2+ is released from the vacuolar lumen following completion of the docking step . We have identified calmodulin as the putative Ca2+ sensor and as the first component required in the post-docking phase of vacuole fusion . Calmodulin binds tightly to vacuoles upon Ca2+ release . Unlike synaptotagmin or syncollin in exocytosis, calmodulin does not act as a fusion clamp but actively promotes bilayer mixing . Hence, activation of SNAREs is not sufficient to drive bilayer mixing between physiological membranes . We propose that Ca2+ control of the latest phase of membrane fusion may be a conserved feature, relevant not only for exocytosis, but also for intracellular, 'constitutive' fusion reactions . However, the origin of the Ca2+ signal, its receptor and its mode of processing differ.

Nature, 1998 Dec 10, 396(6711), 543 - 8
Defining the functions of trans-SNARE pairs; Ungermann C et al.; The homotypic fusion of yeast vacuoles includes a 'docking' step, which we show here to consist of two sequential reactions: a reversible 'tethering' mediated by the GTPase Ypt7, and 'SNARE pairing', in which SNARE proteins from opposite membranes form a complex in trans . The function of this trans-SNARE complex must be transient, as the complex can be disassembled by excess Sec18 in the presence of Sec17 and ATP without influencing the fusion rate . These data indicate that SNARE pairing may transiently signal to downstream factors, leading to fusion.

Mech Dev, 1998 Nov, 78(1-2), 113 - 8
Gal4 in the Drosophila female germline; Rorth P; The modular Gal4 system has proven to be an extremely useful tool for conditional gene expression in Drosophila . One limitation has been the inability of the system to work in the female germline . A modified Gal4 system that works throughout oogenesis is presented here . To achieve germline expression, it was critical to change the basal promoter and 3'-UTR in the Gal4-responsive expression vector (generating UASp) . Basal promoters and heterologous 3'-UTRs are often considered neutral, but as shown here, can endow qualitative tissue-specificity to a chimeric transcript . The modified Gal4 system was used to investigate the role of the Drosophila FGF homologue branchless, ligand for the FGF receptor breathless, in border cell migration . FGF signaling guides tracheal cell migration in the embryo . However, misexpression of branchless in the ovary had no effect on border cell migration . Thus border cells and tracheal cells appear to be guided differently .

Mech Dev, 1998 Nov, 78(1-2), 97 - 111
Direct binding between two PDZ domain proteins Canoe and ZO-1 and their roles in regulation of the jun N-terminal kinase pathway in Drosophila morphogenesis; Takahashi K et al.; During Drosophila embryogenesis, the ventral epidermis dorsally expands and the left and right epithelial sheets meet and fuse along the dorsal midline . For this dorsal closure to occur, two PDZ domain proteins, Cno and ZO-1, are required . The dorsal epidermis remains open when the expression of ZO-1 and Cno are reduced simultaneously by hypomorphic mutations in the relevant loci . ZO-1 and Cno colocalize at adherens junctions in embryonic epithelia, and form a protein complex upon binding to each other . Genetic analysis showed that Cno is involved in the Jun N-terminal kinase (JNK) pathway for dorsal closure, as a modulator acting upstream of, or in parallel with, the small GTPase Drac1 . The ZO-1-Cno complex may be involved in dynamic changes in cytoskeletal organization and cell adhesion during morphogenetic events associated with dorsal closure in the Drosophila embryo .

Mol Cell Biol, 1999 Jan, 19(1), 882 - 8
Activation of transcription by metabolic intermediates of the pyrimidine biosynthetic pathway; Flynn PJ et al.; Saccharomyces cerevisiae responds to pyrimidine starvation by increasing the expression of four URA genes, encoding the enzymes of de novo pyrimidine biosynthesis, three- to eightfold . The increase in gene expression is dependent on a transcriptional activator protein, Ppr1p . Here, we investigate the mechanism by which the transcriptional activity of Ppr1p responds to the level of pyrimidine biosynthetic intermediates . We find that purified Ppr1p is unable to promote activation of transcription in an in vitro system . Transcriptional activation by Ppr1p can be observed, however, if either dihydroorotic acid (DHO) or orotic acid (OA) is included in the transcription reactions . The transcriptional activation function and the DHO/OA-responsive element of Ppr1p localize to the carboxyl-terminal 134 amino acids of the protein . Thus, Ppr1p directly senses the level of early pyrimidine biosynthetic intermediates within the cell and activates the expression of genes encoding proteins required later in the pathway . These results are discussed in terms of (i) regulation of the pyrimidine biosynthetic pathway and (ii) a novel mechanism of regulating gene expression.

Mol Cell Biol, 1999 Jan, 19(1), 855 - 63
Activation domain-specific and general transcription stimulation by native histone acetyltransferase complexes; Ikeda K et al.; Recent progress in identifying the catalytic subunits of histone acetyltransferase (HAT) complexes has implicated histone acetylation in the regulation of transcription . Here, we have analyzed the function of two native yeast HAT complexes, SAGA (Spt-Ada-Gcn5 Acetyltransferase) and NuA4 (nucleosome acetyltransferase of H4), in activating transcription from preassembled nucleosomal array templates in vitro . Each complex was tested for the ability to enhance transcription driven by GAL4 derivatives containing either acidic, glutamine-rich, or proline-rich activation domains . On nucleosomal array templates, the SAGA complex selectively stimulates transcription driven by the VP16 acidic activation domain in an acetyl coenzyme A-dependent manner . In contrast, the NuA4 complex facilitates transcription mediated by any of the activation domains tested if allowed to preacetylate the nucleosomal template, indicating a general stimulatory effect of histone H4 acetylation . However, when the extent of acetylation by NuA4 is limited, the complex also preferentially stimulates VP16-driven transcription . SAGA and NuA4 interact directly with the VP16 activation domain but not with a glutamine-rich or proline-rich activation domain . These data suggest that recruitment of the SAGA and NuA4 HAT complexes by the VP16 activation domain contributes to HAT-dependent activation . In addition, extensive H4/H2B acetylation by NuA4 leads to a general activation of transcription, which is independent of activator-NuA4 interactions.

Mol Cell Biol, 1999 Jan, 19(1), 817 - 25
The two forms of karyogamy transcription factor Kar4p are regulated by differential initiation of transcription, translation, and protein turnover; Gammie AE et al.; Kar4p is a transcription factor in Saccharomyces cerevisiae that is required for the expression of karyogamy-specific genes during mating, for the efficient transit from G1 during mitosis, and for essential functions during meiosis . Kar4p exists in two forms: a constitutive slower-migrating form, which predominates during vegetative growth, and a faster-migrating form, which is highly induced by mating pheromone . Transcript mapping of KAR4 revealed that the constitutive mRNA was initiated upstream of two in-frame ATG initiation codons, while the major inducible mRNA originated between them . Thus, the two forms of Kar4p are derived from the translation of alternative transcripts, which possess different AUG initiation codons . Site-directed mutations were constructed to inactivate one or the other of the initiation codons, allowing the expression of the two Kar4p forms separately . At normal levels of expression, the constitutive form of Kar4p did not support wild-type levels of mating . However, the two forms of Kar4p could also be expressed separately from the regulatable GAL1 promoter, and no functional difference was detected when they were expressed at equivalent levels . Pulse-chase experiments showed that the induced form of Kar4p was highly expressed and stable during mating but rapidly turned over in vegetative cells . In contrast, the constitutively expressed longer form showed the same rate of turnover regardless of the growth condition . Furthermore, overexpression of either form of Kar4p in vegetative cells was toxic . Thus, the elaborate regulation of the two forms of Kar4p at the levels of transcription, translation, and protein turnover reflects the requirement for high levels of the protein during mating and for low levels during the subsequent phases of the cell cycle.

Mol Cell Biol, 1999 Jan, 19(1), 796 - 806
Histone acetyltransferase and protein kinase activities copurify with a putative Xenopus RNA polymerase I holoenzyme self-sufficient for promoter-dependent transcription; Albert AC et al.; Mounting evidence suggests that eukaryotic RNA polymerases preassociate with multiple transcription factors in the absence of DNA, forming RNA polymerase holoenzyme complexes . We have purified an apparent RNA polymerase I (Pol I) holoenzyme from Xenopus laevis cells by sequential chromatography on five columns: DEAE-Sepharose, Biorex 70, Sephacryl S300, Mono Q, and DNA-cellulose . Single fractions from every column programmed accurate promoter-dependent transcription . Upon gel filtration chromatography, the Pol I holoenzyme elutes at a position overlapping the peak of Blue Dextran, suggesting a molecular mass in the range of approximately 2 MDa . Consistent with its large mass, Coomassie blue-stained sodium dodecyl sulfate-polyacrylamide gels reveal approximately 55 proteins in fractions purified to near homogeneity . Western blotting shows that TATA-binding protein precisely copurifies with holoenzyme activity, whereas the abundant Pol I transactivator upstream binding factor does not . Also copurifying with the holoenzyme are casein kinase II and a histone acetyltransferase activity with a substrate preference for histone H3 . These results extend to Pol I the suggestion that signal transduction and chromatin-modifying activities are associated with eukaryotic RNA polymerases.

Mol Cell Biol, 1999 Jan, 19(1), 577 - 84
Elevated levels of a U4/U6.U5 snRNP-associated protein, Spp381p, rescue a mutant defective in spliceosome maturation; Lybarger S et al.; U4 snRNA release from the spliceosome occurs through an essential but ill-defined Prp38p-dependent step . Here we report the results of a dosage suppressor screen to identify genes that contribute to PRP38 function . Elevated expression of a previously uncharacterized gene, SPP381, efficiently suppresses the growth and splicing defects of a temperature-sensitive (Ts) mutant prp38-1 . This suppression is specific in that enhanced SPP381 expression does not alter the abundance of intronless RNA transcripts or suppress the Ts phenotypes of other prp mutants . Since SPP381 does not suppress a prp38::LEU2 null allele, it is clear that Spp381p assists Prp38p in splicing but does not substitute for it . Yeast SPP381 disruptants are severely growth impaired and accumulate unspliced pre-mRNA . Immune precipitation studies show that, like Prp38p, Spp381p is present in the U4/U6.U5 tri-snRNP particle . Two-hybrid analyses support the view that the carboxyl half of Spp381p directly interacts with the Prp38p protein . A putative PEST proteolysis domain within Spp381p is dispensable for the Spp381p-Prp38p interaction and for prp38-1 suppression but contributes to Spp381p function in splicing . Curiously, in vitro, Spp381p may not be needed for the chemistry of pre-mRNA splicing . Based on the in vivo and in vitro results presented here, we propose that two small acidic proteins without obvious RNA binding domains, Spp381p and Prp38p, act in concert to promote U4/U5.U6 tri-snRNP function in the spliceosome cycle.

Mol Cell Biol, 1999 Jan, 19(1), 441 - 9
Receptor inhibition of pheromone signaling is mediated by the Ste4p Gbeta subunit; Kim J et al.; The pheromone response pathway of the yeast Saccharomyces cerevisiae is initiated in MATa cells by binding of alpha-factor to the alpha-factor receptor . MATa cells in which the a-factor receptor is inappropriately expressed exhibit reduced pheromone signaling, a phenomenon termed receptor inhibition . In cells undergoing receptor inhibition, activation of the signaling pathway occurs normally at early time points but decreases after prolonged exposure to pheromone . Mutations that suppress the effects of receptor inhibition were obtained in the STE4 gene, which encodes the beta-subunit of the G protein that transmits the pheromone response signal . These mutations mapped to the N terminus and second WD repeat of Ste4p in regions that are not part of its Galpha binding surface . A STE4 allele containing several of these mutations, called STE4(SD13), reversed the signaling defect seen at late times in cells undergoing receptor inhibition but had no effect on the basal activity of the pathway . Moreover, the signaling properties of STE4(SD13) were indistinguishable from those of STE4 in wild-type MATa and MATalpha cells . These results demonstrate that the effect of the STE4(SD13) allele is specific to the receptor inhibition function of STE4 . STE4(SD13) suppressed the signaling defect conferred by receptor inhibition in a MATa strain containing a deletion of GPA1, the G protein alpha-subunit gene; however, STE4(SD13) had no effect in a MATalpha strain containing a GPA1 deletion . Suppression of receptor inhibition by STE4(SD13) in a MATa strain containing a GPA1 deletion was unaffected by deletion of STE2, the alpha-factor receptor gene . The results presented here are consistent with a model in which an a-specific gene product other than Ste2p detects the presence of the a-factor receptor and blocks signaling by inhibiting the function of Ste4p.

Mol Cell Biol, 1999 Jan, 19(1), 412 - 23
Testing for DNA tracking by MOT1, a SNF2/SWI2 protein family member; Auble DT et al.; Proteins in the SNF2/SWI2 family use ATP hydrolysis to catalyze rearrangements in diverse protein-DNA complexes . How ATP hydrolysis is coupled to these rearrangements is unknown, however . One attractive model is that these ATPases are ATP-dependent DNA-tracking enzymes . This idea was tested for the SNF2/SWI2 protein family member MOT1 . MOT1 is an essential Saccharomyces cerevisiae transcription factor that uses ATP to dissociate TATA binding protein (TBP) from DNA . By using a series of DNA templates with one or two TATA boxes in combination with binding sites for heterologous DNA binding "roadblock" proteins, the ability of MOT1 to track along DNA was assayed . The results demonstrate that, following ATP-dependent TBP-DNA dissociation, MOT1 dissociates rapidly from the DNA by a mechanism that does not require a DNA end . Template commitment footprinting experiments support the conclusion that ATP-dependent DNA tracking by MOT1 does not occur . These results support a model in which MOT1 drives TBP-DNA dissociation by a mechanism that involves a transient, ATP-dependent interaction with TBP-DNA which does not involve ATP-dependent DNA tracking.

Mol Cell Biol, 1999 Jan, 19(1), 342 - 52
Functional domains of the Rsp5 ubiquitin-protein ligase; Wang G et al.; RSP5, an essential gene of Saccharomyces cerevisiae, encodes a hect domain E3 ubiquitin-protein ligase . Hect E3 proteins have been proposed to consist of two broad functional domains: a conserved catalytic carboxyl-terminal domain of approximately 350 amino acids (the hect domain) and a large, nonconserved amino-terminal domain containing determinants of substrate specificity . We report here the mapping of the minimal region of Rsp5 necessary for its essential in vivo function, the minimal region necessary to stably interact with a substrate of Rsp5 (Rpb1, the large subunit of RNA polymerase II), and the finding that the hect domain, by itself, is sufficient for formation of the ubiquitin-thioester intermediate . Mutations within the hect domain that affect either the ability to form a ubiquitin-thioester or to catalyze substrate ubiquitination abrogate in vivo function, strongly suggesting that the ubiquitin-protein ligase activity of Rsp5 is intrinsically linked to its essential function . The amino-terminal region of Rsp5 contains three WW domains and a C2 calcium-binding domain . Two of the three WW domains are required for the essential in vivo function, while the C2 domain is not, and requirements for Rpb1 binding and ubiquitination lie within the region required for in vivo function . Together, these results support the two-domain model for hect E3 function and indicate that the WW domains play a role in the recognition of at least some of the substrates of Rsp5, including those related to its essential function . In addition, we show that haploid yeast strains bearing complete disruptions of either of two other hect E3 genes of yeast, designated HUL4 (YJR036C) and HUL5 (YGL141W), are viable.

Mol Cell Biol, 1999 Jan, 19(1), 182 - 93
Alternative splicing results in differential expression, activity, and localization of the two forms of arginyl-tRNA-protein transferase, a component of the N-end rule pathway; Kwon YT et al.; The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue . The underlying ubiquitin-dependent proteolytic system, called the N-end rule pathway, is organized hierarchically: N-terminal aspartate and glutamate (and also cysteine in metazoans) are secondary destabilizing residues, in that they function through their conjugation, by arginyl-tRNA-protein transferase (R-transferase), to arginine, a primary destabilizing residue . We isolated cDNA encoding the 516-residue mouse R-transferase, ATE1p, and found two species, termed Ate1-1 and Ate1-2 . The Ate1 mRNAs are produced through a most unusual alternative splicing that retains one or the other of the two homologous 129-bp exons, which are adjacent in the mouse Ate1 gene . Human ATE1 also contains the alternative 129-bp exons, whereas the plant (Arabidopsis thaliana) and fly (Drosophila melanogaster) Ate1 genes encode a single form of ATE1p . A fusion of ATE1-1p with green fluorescent protein (GFP) is present in both the nucleus and the cytosol, whereas ATE1-2p-GFP is exclusively cytosolic . Mouse ATE1-1p and ATE1-2p were examined by expressing them in ate1Delta Saccharomyces cerevisiae in the presence of test substrates that included Asp-betagal (beta-galactosidase) and Cys-betagal . Both forms of the mouse R-transferase conferred instability on Asp-betagal (but not on Cys-betagal) through the arginylation of its N-terminal Asp, the ATE1-1p enzyme being more active than ATE1-2p . The ratio of Ate1-1 to Ate1-2 mRNA varies greatly among the mouse tissues; it is approximately 0.1 in the skeletal muscle, approximately 0.25 in the spleen, approximately 3.3 in the liver and brain, and approximately 10 in the testis, suggesting that the two R-transferases are functionally distinct.

EMBO J, 1998 Dec 15, 17(24), 7430 - 41
The role of exportin-t in selective nuclear export of mature tRNAs; Arts GJ et al.; Exportin-t (Xpo-t) is a vertebrate nuclear export receptor for tRNAs that binds tRNA cooperatively with GTP-loaded Ran . Xpo-t antibodies are shown to efficiently block tRNA export from Xenopus oocyte nuclei suggesting that it is responsible for at least the majority of tRNA export in these cells . We examine the mechanism by which Xpo-t-RanGTP specifically exports mature tRNAs rather than other forms of nuclear RNA, including tRNA precursors . Chemical and enzymatic footprinting together with phosphate modification interference reveals an extensive interaction between the backbone of the TPsiC and acceptor arms of tRNAPhe and Xpo-t-RanGTP . Analysis of mutant or precursor tRNA forms demonstrates that, aside from these recognition elements, accurate 5' and 3' end-processing of tRNA affects Xpo-t-RanGTP interaction and nuclear export, while aminoacylation is not essential . Intron-containing, end-processed, pre-tRNAs can be bound by Xpo-t-RanGTP and are rapidly exported from the nucleus if Xpo-t is present in excess . These results suggest that at least two mechanisms are involved in discrimination of pre-tRNAs and mature tRNAs prior to nuclear export.

EMBO J, 1998 Dec 15, 17(24), 7219 - 29
Cdc6 protein causes premature entry into S phase in a mammalian cell-free system; Stoeber K et al.; We exploit an improved mammalian cell-free DNA replication system to analyse quiescence and Cdc6 function . Quiescent 3T3 nuclei cannot initiate replication in S phase cytosol from HeLa or 3T3 cells . Following release from quiescence, nuclei become competent to initiate semiconservative DNA replication in S phase cytosol, but not in G0 phase cytosol . Immunoblots show that quiescent cells lack Cdc6 and that minichromosome maintenance (MCM) proteins are not associated with chromatin . Competence of G1 phase nuclei to replicate in vitro coincides with maximum Cdc6 accumulation and MCM protein binding to chromatin in vivo . Addition of recombinant Cdc6 to permeabilized, but not intact, G1 nuclei causes up to 82% of the nuclei to initiate and accelerates G1 progression, making nuclei competent to replicate prematurely.

J Biol Chem, 1998 Dec 25, 273(52), 35347 - 54
Functional domains of the alpha1 catalytic subunit of the AMP-activated protein kinase; Crute BE et al.; The AMP-activated protein kinase is a heterotrimeric enzyme, important in cellular adaptation to the stress of nutrient starvation, hypoxia, increased ATP utilization, or heat shock . This mammalian enzyme is composed of a catalytic alpha subunit and noncatalytic beta and gamma subunits and is a member of a larger protein kinase family that includes the SNF1 kinase of Saccharomyces cerevisiae . In the present study, we have identified by truncation and site-directed mutagenesis several functional domains of the alpha1 catalytic subunit, which modulate its activity, subunit association, and protein turnover . C-terminal truncation of the 548-amino acid (aa) wild-type alpha1 protein to aa 312 or 392 abolishes the binding of the beta/gamma subunits and dramatically increases protein expression . The full-length wild-type alpha1 subunit is only minimally active in the absence of co-expressed beta/gamma, and alpha1(1-392) likewise has little activity . Further truncation to aa 312, however, is associated with a large increase in enzyme specific activity, thus revealing an autoinhibitory sequence between aa 313 and 392 . alpha-1(1-312) still requires the phosphorylation of the activation loop Thr-172 for enzyme activity, yet is now independent of the allosteric activator, AMP . The increased levels of protein expression on transient transfection of either truncated alpha subunit cDNA are because of a decrease in enzyme turnover by pulse-chase analysis . Taken together, these data indicate that the alpha1 subunit of AMP-activated protein kinase contains several features that determine enzyme activity and stability . A constitutively active form of the kinase that does not require participation by the noncatalytic subunits provides a unique reagent for exploring the functions of AMP-activated protein kinase.

J Biol Chem, 1998 Dec 25, 273(52), 34837 - 42
Single point mutations distributed in 10 soluble and membrane regions of the Nicotiana plumbaginifolia plasma membrane PMA2 H+-ATPase activate the enzyme and modify the structure of the C-terminal region; Morsomme P et al.; The Nicotiana plumbaginifolia pma2 (plasma membrane H+-ATPase) gene is capable of functionally replacing the H+-ATPase genes of the yeast Saccharomyces cerevisiae, provided that the external pH is kept above 5.0 . Single point mutations within the pma2 gene were previously identified that improved H+-ATPase activity and allowed yeast growth at pH 4.0 . The aim of the present study was to identify most of the PMA2 positions, the mutation of which would lead to improved growth and to determine whether all these mutations result in similar enzymatic and structural modifications . We selected additional mutants in total 42 distinct point mutations localized in 30 codons . They were distributed in 10 soluble and membrane regions of the enzyme . Most mutant PMA2 H+-ATPases were characterized by a higher specific activity, lower inhibition by ADP, and lower stimulation by lysophosphatidylcholine than wild-type PMA2 . The mutants thus seem to be constitutively activated . Partial tryptic digestion and immunodetection showed that the PMA2 mutants had a conformational change making the C-terminal region more accessible . These data therefore support the hypothesis that point mutations in various H+-ATPase parts displace the inhibitory C-terminal region, resulting in enzyme activation . The high density of mutations within the first half of the C-terminal region suggests that this part is involved in the interaction between the inhibitory C-terminal region and the rest of the enzyme.

Mol Cells, 1998 Oct 31, 8(5), 606 - 13
Isoform-specific interaction of the cytoplasmic domains of Na,K-ATPase; Yoon T et al.; The Na,K-ATPase is a heterodimer consisting of an alpha and a beta subunit, which exchanges intracellular Na+ for extracellular K+ using the energy of ATP hydrolysis . Several studies have demonstrated that the enzyme exists as an (alphabeta)2 heterotetramer, an oligomer of alphabeta dimers within the cell membrane, at least during some portion of the transport cycle although its functional significance is unknown . In our study, we employed the yeast two-hybrid system to identify the cytoplasmic domains of the Na,K-ATPase which might be involved in intersubunit and/or intrasubunit interactions to form higher order oligmers . Our data demonstrate that the N-terminus and the cytoplasmic loop 1 of the alpha2 subunit interact with each other, while those of the alpha1 subunit do not, suggesting that the interaction is isoform-specific . Therefore, the N-terminal and the cytoplasmic loop 1 might be the regions where the alpha2 subunit, which are involved in alpha alpha interactions, stabilize Na,K-ATPase as alphabeta protomer, diprotomer, or higher order oligomer because the interaction can be intrasubunit as well as intersubunit interactions . Our study suggests that there may be an isoform-specific difference in the alpha-alpha interaction and that the isoform-specific interaction may contribute significantly to the differences of the physiological function and regulation among the alpha isoforms.

Mol Cells, 1998 Oct 31, 8(5), 518 - 23
Son of sevenless binds to the SH3 domain of src-type tyrosine kinase; Park C et al.; To identify molecules which bind to the SH3 domains of p56lck, we screened a mouse T-cell lymphoma cDNA library using the yeast two-hybrid system . As a result, we obtained several positive clones including the Son of Sevenless gene which encodes a mammalian homolog of Drosophila Ras GDP/GTP exchange factor . In a subsequent analysis with the yeast two-hybrid system, Sos associated only with the constitutively active form of p56lck (F505) but not with wild type p56lck (Y505), indicating the requirement for an active conformation of p56lck for binding to Sos . Subsequently, we have demonstrated in vitro that the SH3 domain of p56lck as well as the proline-rich sequences of Sos are responsible for this association . In addition, the proline-rich domain of Sos also bound to the SH3 domains of other src-type tyrosine kinases, src and fyn, but not to that of PLC-gamma . More importantly, the p56lck SH3-Sos interaction was enhanced by serum stimulation, suggesting the possibility that the direct interaction between p56lck SH3 and Sos may contribute to the regulation of the Ras pathway.

Carcinogenesis, 1998 Nov, 19(11), 1931 - 7
Resistance to 6-thioguanine in mismatch repair-deficient human cancer cell lines correlates with an increase in induced mutations at the HPRT locus; Glaab WE et al.; Although the resistance to the cytotoxic response of certain DNA damaging agents has been well characterized in cells deficient in mismatch repair, little is known about how such resistance affects mutagenesis . Using human cancer cell lines defective in mismatch repair (MMR) and complementary cell lines in which the MMR defects were corrected by chromosome transfer, we present the cytotoxic effect and the mutagenic response at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus following exposure to the chemotherapeutic agent, 6-thioguanine (6-TG) . Upon exposure to 6-TG, there was a differential cytotoxic response . The MMR-deficient cells were resistant to 6-TG exposure up to 5 microM, whereas the MMR-proficient cell lines were significantly more sensitive at the same levels of exposure . Furthermore, the mutagenic response at HPRT induced by 6-TG was substantially increased in the MMR-deficient lines relative to the MMR-proficient cell lines . These findings support the notion that cytotoxicity to 6-TG is mediated through functional MMR and that resistance to the cytotoxic effects of 6-TG is directly associated with an increase in induced mutations in MMR-defective cells . These data suggest that the use of 6-TG as a chemotherapeutic agent may result in the selection of MMR-defective cells, thereby predisposing the patient to an increased risk for developing secondary tumors.

Comp Biochem Physiol B Biochem Mol Biol, 1998 Aug, 120(4), 639 - 46
Comparative studies of suidatrestin, a specific inhibitor of trehalases; Knuesel I et al.; Suidatrestin, isolated from a Streptomyces strain, was characterized as a new trehalase inhibitor . Its inhibitory potential was 7 to 50-fold higher than that of validamycin when tested against insect, fungal and mammalian trehalases . The kinetic properties of suidatrestin were studied in vitro with trehalases from flight muscle mitochondria of the fly, Protophormia terraenovae, from larval midgut of the moth, Spodoptera littoralis, and from porcine kidney, as well as with maltase from yeast . Suidatrestin was inactive on maltase but inhibited all trehalases with IC50 values of 0.08-0.1 microM; Ki values ranged from 0.02 to 0.05 microM . The very low Ki/K(m) ratios (3.9 x 10(-6) -4.9 x 10(-6)) indicated excellent in vitro inhibitory action of suidatrestin . When injected into larvae of S . littoralis, suidatrestin required high and repetitive doses which lead to reversible inhibition of larval growth only . Consecutive omission of the inhibitor even stimulated weight increase above that of controls . Significant mortality was achieved at a rather high dose only . Injection of a growth-inhibiting dose of suidatrestin did not change hemolymph osmolality as a measure of sugar concentration . The discrepancy between in vitro and in vivo potency of suidatrestin may be understood once its chemical structure is fully known.

Biochem J, 1999 Jan 1, 337 ( Pt 1), 37 - 43
Catalytic triad of microsomal epoxide hydrolase: replacement of Glu404 with Asp leads to a strongly increased turnover rate; Arand M et al.; Microsomal epoxide hydrolase (mEH) belongs to the superfamily of alpha/beta-hydrolase fold enzymes . A catalytic triad in the active centre of the enzyme hydrolyses the substrate molecules in a two-step reaction via the intermediate formation of an enzyme-substrate ester . Here we show that the mEH catalytic triad is composed of Asp226, Glu404 and His431 . Replacing either of these residues with non-functional amino acids results in a complete loss of activity of the enzyme recombinantly expressed in Saccharomyces cerevisiae . For Glu404 and His431 mutants, their structural integrity was demonstrated by their retained ability to form the substrate ester intermediate, indicating that the lack of enzymic activity is due to an indispensable function of either residue in the hydrolytic step of the enzymic reaction . The role of Asp226 as the catalytic nucleophile driving the formation of the ester intermediate was substantiated by the isolation of a peptide fraction carrying the 14C-labelled substrate after cleavage of the ester intermediate with cyanogen bromide . Sequence analysis revealed that one of the two peptides within this sample harboured Asp226 . Surprisingly, the replacement of Glu404 with Asp greatly increased the Vmax of the enzyme with styrene 7,8-oxide (23-fold) and 9, 10-epoxystearic acid (39-fold) . The increase in Vmax was paralleled by an increase in Km with both substrates, in line with a selective enhancement of the second, rate-limiting step of the enzymic reaction . Owing to its enhanced catalytic properties, the Glu404-->Asp mutant might represent a versatile tool for the enantioselective bio-organic synthesis of chiral fine chemicals . The question of why all native mEHs analysed so far have a Glu in place of the acidic charge relay residue is discussed.

Biol Pharm Bull, 1998 Nov, 21(11), 1215 - 7
Evaluation of fluorescence polarization method for binding study in carbohydrate-lectin interaction; Oda Y et al.; The fluorescence polarization (FP) technique was evaluated to determine molecular interaction between plant lectins and polysaccharides, yeast cells and glycopeptide after labeling the lectins with fluorescein isothiocyanate . Use of Lycoris radiata agglutinin allowed determination of the molecular interactions with large biomolecules containing mannose oligomers and polymers . Another example using a fluorescein-labeled glycopeptide also indicated that use of the FP method would allow easy observation of the molecular interactions on the quantitative base . The present technique is highly sensitive and facile because it does not require any washing procedures before measurement.

J Hum Genet, 1998, 43(4), 268 - 71
Cloning, expression analysis, and chromosomal localization of HIP1R, an isolog of huntingtin interacting protein (HIP1); Seki N et al.; Huntington disease (HD) is an inherited neurodegenerative disorder which is associated with CAG expansion in the coding region of the gene for huntingtin protein . Recently, a huntingtin interacting protein, HIP1, was isolated by the yeast two-hybrid system . Here we report the isolation of a cDNA clone for HIP1R (huntingtin interacting protein-1 related), which encodes a predicted protein product sharing a striking homology with HIP1 . RT-PCR analysis showed that the messenger RNA was ubiquitously expressed in various human tissues . Based on PCR-assisted analysis of a radiation hybrid panel and fluorescence in situ hybridization, HIP1R was localized to the q24 region of chromosome 12.

Int J Mol Med, 1998 May, 1(5), 797 - 9
The presenilin 2 loop domain interacts with the mu-calpain C-terminal region; Shinozaki K et al.; Presenilin 2 (PS2) is a gene responsible for the early-onset familial Alzheimer's disease (AD) . PS2 mutations are considered to be closely related to the pathogenesis of AD . We screened for proteins that interact with PS2 to understand its pathological and physiological functions . Using the PS2 loop domain as the bait, the yeast two-hybrid system was used for screening, and mu-calpain was identified as a PS2 binding protein . In COS-1 cells, the interaction of PS2 with mu-calpain was confirmed by immunoprecipitation . These results suggested that PS2 and mu-calpain interact with each other, and might regulate each other's functions.

J Cell Biol, 1998 Dec 14, 143(6), 1505 - 21
Recycling of golgi-resident glycosyltransferases through the ER reveals a novel pathway and provides an explanation for nocodazole-induced Golgi scattering; Storrie B et al.; During microtubule depolymerization, the central, juxtanuclear Golgi apparatus scatters to multiple peripheral sites . We have tested here whether such scattering is due to a fragmentation process and subsequent outward tracking of Golgi units or if peripheral Golgi elements reform through a novel recycling pathway . To mark the Golgi in HeLa cells, we stably expressed the Golgi stack enzyme N-acetylgalactosaminyltransferase-2 (GalNAc-T2) fused to the green fluorescent protein (GFP) or to an 11-amino acid epitope, VSV-G (VSV), and the trans/TGN enzyme beta1,4-galactosyltransferase (GalT) fused to GFP . After nocodazole addition, time-lapse microscopy of GalNAc-T2-GFP and GalT-GFP revealed that scattered Golgi elements appeared abruptly and that no Golgi fragments tracked outward from the compact, juxtanuclear Golgi complex . Once formed, the scattered structures were relatively stable in fluorescence intensity for tens of minutes . During the entire process of dispersal, immunogold labeling for GalNAc-T2-VSV and GalT showed that these were continuously concentrated over stacked Golgi cisternae and tubulovesicular Golgi structures similar to untreated cells, suggesting that polarized Golgi stacks reform rapidly at scattered sites . In fluorescence recovery after photobleaching over a narrow (FRAP) or wide area (FRAP-W) experiments, peripheral Golgi stacks continuously exchanged resident proteins with each other through what appeared to be an ER intermediate . That Golgi enzymes cycle through the ER was confirmed by microinjecting the dominant-negative mutant of Sar1 (Sar1pdn) blocking ER export . Sar1pdn was either microinjected into untreated or nocodazole-treated cells in the presence of protein synthesis inhibitors . In both cases, this caused a gradual accumulation of GalNAc-T2-VSV in the ER . Few to no peripheral Golgi elements were seen in the nocodazole-treated cells microinjected with Sar1pdn . In conclusion, we have shown that Golgi-resident glycosylation enzymes recycle through the ER and that this novel pathway is the likely explanation for the nocodazole-induced Golgi scattering observed in interphase cells.

J Cell Biol, 1998 Dec 14, 143(6), 1447 - 55
A novel nuclear import pathway for the transcription factor TFIIS; Albertini M et al.; We have identified a novel pathway for protein import into the nucleus . We have shown that the previously identified but uncharacterized yeast protein Nmd5p functions as a karyopherin . It was therefore designated Kap119p (karyopherin with Mr of 119 kD) . We localized Kap119p to both the nucleus and the cytoplasm . We identified the transcription elongation factor TFIIS as its major cognate import substrate . The cytoplasmic Kap119p exists as an approximately stoichiometric complex with TFIIS . RanGTP, not RanGDP, dissociated the isolated Kap119p/TFIIS complex and bound to Kap119p . Kap119p also bound directly to a number of peptide repeat containing nucleoporins in overlay assays . In wild-type cells, TFIIS was primarily localized to the nucleus . In a strain where KAP119 has been deleted, TFIIS was mislocalized to the cytoplasm indicating that TFIIS is imported into the nucleus by Kap119p . The transport of various substrates that use other karyopherin-mediated import or export pathways was not affected in a kap119Delta strain . Hence Kap119p is a novel karyopherin that is responsible for the import of the transcription elongation factor TFIIS.

J Biol Chem, 1998 Dec 18, 273(51), 34653 - 60
Promoter structure and transcriptional activation with chromatin templates assembled in vitro . A single Gal4-VP16 dimer binds to chromatin or to DNA with comparable affinity; Pazin MJ et al.; To gain a better understanding of the role of chromatin in the regulation of transcription by RNA polymerase II, we examined the relation between promoter structure and the ability of Gal4-VP16 to function with chromatin templates assembled in vitro . First, to investigate whether there are synergistic interactions among multiple bound factors, we studied promoter constructions containing one or five Gal4 sites and found that a single recognition site is sufficient for Gal4-VP16 to bind to chromatin, to induce nucleosome rearrangement, and to activate transcription . Notably, we observed that Gal4-VP16 binds to a single site in chromatin with affinity comparable with that which it binds to naked DNA, even in the absence of ATP-dependent nucleosome remodeling activity . Second, to explore the relation between translational nucleosome positioning and transcriptional activation, we analyzed a series of promoter constructions in which nucleosomes were positioned by Gal4-VP16 at different locations relative to the RNA start site . These experiments revealed that the positioning of a nucleosome over the RNA start site is not an absolute barrier to transcriptional activation . Third, to determine the contribution of core promoter elements to transcriptional activation with chromatin templates, we tested the ability of Gal4-VP16 to activate transcription with TATA box- versus DPE-driven core promoters and found that the TATA box is not required to achieve transcriptional activation by Gal4-VP16 with chromatin templates . These results suggest that a single protomer of a strong activator is able to bind to chromatin, to induce nucleosome remodeling, and to activate transcription in conjunction with a broad range of chromatin structures and core promoter elements.

J Biol Chem, 1998 Dec 18, 273(51), 34341 - 8
The cellular localization of the murine serine/arginine-rich protein kinase CLK2 is regulated by serine 141 autophosphorylation; Nayler O et al.; Pre-mRNA splicing is catalyzed by a multitude of proteins including serine/arginine-rich (SR) proteins, which are thought to play a crucial role in the formation of spliceosomes and in the regulation of alternative splicing . SR proteins are highly phosphorylated, and their kinases are believed to regulate the recruitment of SR proteins from nuclear storage compartments known as speckles . Recently, a family of autophosphorylating kinases termed CLK (CDC2/CDC28-like kinases) was shown to phosphorylate SR proteins and to influence alternative splicing in overexpression systems . Here we used endogenous CLK2 protein to demonstrate that it displays different biochemical characteristics compared with its overexpressed protein and that it is differentially phosphorylated in vivo . Furthermore, CLK2 changed its nuclear localization upon treatment with the kinase inhibitor 5, 6-dichloro-1-beta-D-ribofuranosylbenzimidazole . We have also identified a CLK2 autophosphorylation site, which is highly conserved among all CLK proteins, and we show by site-directed mutagenesis that its phosphorylation influences the subnuclear localization of CLK2 . Our data suggest that CLK2 localization and possibly activity are influenced by a balance of CLK2 autophosphorylation and the regulation by CLK2 kinases and phosphatases.

J Biol Chem, 1998 Dec 18, 273(51), 34310 - 5
Structural and thermodynamic responses of mutations at a Ca2+ binding site engineered into human lysozyme; Kuroki R et al.; Structural determinants of Ca2+ binding sites within proteins typically comprise several acidic residues in appropriate juxtaposition . Three residues (Ala-83, Gln-86, and Ala-92) in human lysozyme are characteristically mutated to Lys, Asp, and Asp, respectively, in natural Ca2+ binding lysozymes and alpha-lactalbumins . The effects of these mutations on the stability and Ca2+ binding properties of human lysozyme were investigated using calorimetry and were interpreted with crystal structures . The double mutant, in which Glu-86 and Ala-92 were replaced with Asp, clearly showed Ca2+ binding affinity, whereas neither point mutant showed Ca2+ affinity, indicating that both residues are essential . The further mutation of Ala-83 --> Lys did not affect the Ca2+ binding of the double mutant . The point mutations Ala-83 --> Lys and Glu-86 --> Asp did not affect the stability, whereas the mutation Ala-92 --> Asp was about 1.3 kcal/mol less stable . Structural analyses showed that both Asp-86 and Lys-83 were exposed to solvent . Side chains of Asp-86 and Asp-91 were rotated in opposite directions about chi1 angle, as if to reduce the electrostatic repulsion . The charged amino acids at the Ca2+ binding site did not significantly affect stability of the protein, possibly because of the local conformational change of the side chains.

J Biol Chem, 1998 Dec 18, 273(51), 34302 - 9
E3-ubiquitin ligase/E6-AP links multicopy maintenance protein 7 to the ubiquitination pathway by a novel motif, the L2G box; Kuhne C et al.; Ubiquitin ligases are generally assumed to play a major role in substrate recognition and thus provide specificity to a particular ubiquitin modification system . The multicopy maintenance protein (Mcm) 7 subunit of the replication licensing factor-M was identified as a substrate of the E3-ubiquitin ligase/E6-AP by its interaction with human papillomavirus-18E6 . Mcm7 is ubiquitinated in vivo in both an E6-AP-dependent and -independent manner . E6-AP functions in these reactions independently of the viral oncogene E6 . We show that recognition of Mcm7 by E6-AP is mediated by a homotypic interaction motif present in both proteins, called the L2G box . These findings served as the basis for the definition of substrate specificity for E6-AP . A small cluster of proteins whose function is intimately associated with the control of cell growth and/or proliferation contains the L2G box and is thereby implicated in an E6-AP and, by default, HPV-E6-dependent ubiquitination pathway.

Genes Dev, 1998 Dec 1, 12(23), 3650 - 62
The essential Gcd10p-Gcd14p nuclear complex is required for 1-methyladenosine modification and maturation of initiator methionyl-tRNA; Anderson J et al.; Gcd10p and Gcd14p are essential proteins required for the initiation of protein synthesis and translational repression of GCN4 mRNA . The phenotypes of gcd10 mutants were suppressed by high-copy-number IMT genes, encoding initiator methionyl tRNA (tRNAiMet), or LHP1, encoding the yeast homolog of the human La autoantigen . The gcd10-504 mutation led to a reduction in steady-state levels of mature tRNAiMet, attributable to increased turnover rather than decreased synthesis of pre-tRNAiMet . Remarkably, the lethality of a GCD10 deletion was suppressed by high-copy-number IMT4, indicating that its role in expression of mature tRNAiMet is the essential function of Gcd10p . A gcd14-2 mutant also showed reduced amounts of mature tRNAiMet, but in addition, displayed a defect in pre-tRNAiMet processing . Gcd10p and Gcd14p were found to be subunits of a protein complex with prominent nuclear localization, suggesting a direct role in tRNAiMet maturation . The chromatographic behavior of elongator and initiator tRNAMet on a RPC-5 column indicated that both species are altered structurally in gcd10Delta cells, and analysis of base modifications revealed that 1-methyladenosine (m1A) is undetectable in gcd10Delta tRNA . Interestingly, gcd10 and gcd14 mutations had no effect on processing or accumulation of elongator tRNAMet, which also contains m1A at position 58, suggesting a unique requirement for this base modification in initiator maturation.

Exp Cell Res, 1998 Dec 15, 245(2), 282 - 9
Evidence for different MCM subcomplexes with differential binding to chromatin in Xenopus; Coue M et al.; MCM proteins are molecular components of the DNA replication licensing system in Xenopus . These proteins comprise a conserved family made up of six distinct members which have been found to associate in large protein complexes . We have used a combination of biochemical and cytological methods to study the association of soluble and chromatin-bound Xenopus MCM proteins during the cell cycle . In interphase, soluble MCM proteins are found organized in a core salt-resistant subcomplex that includes MCM subunits which are known to have high affinity for histones . The interphasic complex is modified at mitosis and the subunit composition of the resulting mitotic subcomplexes is distinct, indicating that the stability of the MCM complex is under cell cycle control . Moreover, we provide evidence that the binding of MCM proteins to chromatin may occur in sequential steps involving the loading of distinct MCM subunits . Comparative analysis of the chromatin distribution of MCM2, 3, and 4 shows that the binding of MCM4 is distinct from that of MCM2 and 3 . Altogether, these data suggest that licensing of chromatin by MCMs occurs in an ordered fashion involving discrete subcomplexes .

Eur J Biochem, 1998 Nov 15, 258(1), 100 - 6
Self-association of the C-terminal domain of the hepatitis-C virus core protein; Yan BS et al.; The N-terminal region of the hepatitis-C virus (HCV) core protein is rich in basic residues, while the C-terminal end of the protein comprises of a stretch of hydrophobic amino acids . Between these two extremes is an amphipathic region with two predicted alpha-helical segments . This region embodies Leu or hydrophobic residues in positions of heptad repeats and is possibly capable of self-association . To investigate this possibility, the core sequence was divided into two fragments and expressed separately as recombinant proteins . Recombinant proteins with the N-terminal fragment remained as monomers even at high concentrations in SDS/PAGE . Recombinant protein with the C-terminal fragment appeared largely monomeric on denaturing gels but some oligomers were also detected . Furthermore, proline mutations in either one of the predicted alpha helices adversely affected the observed oligomerization . The self-association capacity of the core protein C-terminal region was further supported by results from a yeast two-hybrid system . To affirm our conclusion, a peptide covering the heptad repeats and the predicted alpha helices was synthesized . Data from mass spectrometry and gel-filtration chromatography concluded that this peptide readily self-associated into the homodimer . Therefore, our results suggest that the oligomerization motifs of the HCV core protein may not be limited to the previously suggested N-terminal region.

FEMS Microbiol Lett, 1998 Dec 1, 169(1), 1 - 8
Isolation of a class IV chitin synthase gene from a zygomycete fungus, Rhizopus oligosporus; Motoyama T et al.; We found the presence of DNA sequence which shows sequence similarity to the class IV chitin synthase gene (CHS3) of Saccharomyces cerevisiae in the genome of 14 Rhizopus species which belong to zygomycetes . We cloned a gene (chs3), which might correspond to one of these homologous sequences, from Rhizopus oligosporus by low stringency plaque hybridization probed with CHS3 . The deduced amino acid sequence of this gene showed highest similarity to the class IV chitin synthase of Neurospora crassa (46.7% identity over 1087 amino acids), showing that this gene encodes a class IV chitin synthase . Northern analysis revealed the differential expression pattern of this gene in the asexual life cycle with highest expression in the early stage of asexual spore formation . This is the first report of the isolation and analysis of a class IV chitin synthase gene from zygomycete fungi.

Mol Endocrinol, 1998 Dec, 12(12), 1914 - 30
Agonist and antagonists induce homodimerization and mixed ligand heterodimerization of human progesterone receptors in vivo by a mammalian two-hybrid assay; Leonhardt SA et al.; This study utilizes the mammalian two-hybrid system to examine the role of ligand in the dimerization of human progesterone receptor (hPR) . The GAL4 DNA-binding domain and the herpes simplex virus VP16 transactivation domain were fused to the amino terminus of full-length hPR (both the A and B isoforms) to produce chimeric proteins . PR dimerization was detected by the ability of cotransfected GAL4/PR and VP16/PR chimeras in COS cells to induce expression of a reporter gene under the control of GAL4-binding sites (pG5CAT) . Hormone agonist-dependent interactions were observed between the two like isoforms of PR (A-A and B-B) and between PR-A and PR-B (A-B), indicating that hormone can stimulate the formation of the three possible dimeric forms of PR within cells . In contrast, neither type I (ZK98299) nor type II (RU486, ZK112993) progestin antagonists stimulated interaction between these same hybrid PR proteins . However, activation of the VP16/PR chimera by antagonists on a progesterone response element-controlled reporter gene (DHRE-E1b-CAT) was only a fraction (4-13%) of that stimulated by agonist R5020 . One possibility for the failure to detect an induction in the two-hybrid assay is antagonist-induced repression of the activity of the VP16/PR fusion protein rather than a failure of antagonists to stimulate interaction between the hybrid proteins . To test this idea, an UP-1 carboxyl-terminal truncation mutant of PR was used to construct the two-hybrid proteins . PR-UP-1 selectively binds antagonists, but not agonists, and is fully activated in response to antagonists . Both types of progestin antagonists stimulated interactions between GAL4/PR(UP-1) and VP16/PR(UP-1) hybrid proteins, indicating that antagonists are capable of stimulating PR dimerization in cells and do not function by disrupting or preventing dimerization . To determine whether PR bound to an antagonist can dimerize in whole cells with PR bound to agonist, GAL4/PR(UP-1) was paired in the two-hybrid assay with a VP16/PR fusion protein harboring a point mutation in PR at amino acid 722 (Gly-Cys) that specifically binds progestin agonist but not antagonist . Neither R5020 nor RU486 alone stimulated interaction between these ligand-specific PR hybrid proteins . However, strong interaction was detected by addition of both agonist and antagonists, indicating the formation of mixed ligand heterodimers and that both PR partners require ligand for dimerization to occur . Based on electrophoretic gel mobility shift assays (EMSAs), these heterodimers appear to have substantially reduced DNA binding activity . Progestin antagonists inhibit agonist activation of PR at concentrations that are too low to be accounted for by a simple competition mechanism for binding to PR . We propose that antiprogestin inactivation of PR in trans by heterodimerization contributes to the biological potency of these compounds.

J Virol, 1999 Jan, 73(1), 814 - 8
Genetic selection of poliovirus 2Apro-binding peptides; Ventoso I et al.; The yeast two-hybrid system has been used to identify mammalian clones that interact with poliovirus 2A proteinase (2Apro) . Eight clones which encode previously unidentified human proteins were selected from a HeLa cell cDNA expression library . In addition, five clones encoding short peptides that interact with poliovirus 2Apro were also identified . The lengths of these peptides range from 6 to 30 amino acids, but all of them contain the Leu-X-Thr-Z motif (X represents any amino acid; Z represents a hydrophobic residue) . This sequence is invariably located just at the carboxy terminus of each peptide . This approach raises the possibility of designing substrate analogue inhibitors of 2Apro . Thus, two nonhydrolyzable peptides containing the Leu-X-Thr-Z motif prevented cleavage of eukaryotic initiation factor 4G by poliovirus 2Apro in vitro . A more general method for identifying peptides with antiproteinase activity is discussed.

J Virol, 1999 Jan, 73(1), 453 - 65
Mutations in nonconserved domains of Ty3 integrase affect multiple stages of the Ty3 life cycle; Nymark-McMahon MH et al.; Ty3, a retroviruslike element of Saccharomyces cerevisiae, transposes into positions immediately upstream of RNA polymerase III-transcribed genes . The Ty3 integrase (IN) protein is required for integration of the replicated, extrachromosomal Ty3 DNA . In retroviral IN, a conserved core region is sufficient for strand transfer activity . In this study, charged-to-alanine scanning mutagenesis was used to investigate the roles of the nonconserved amino- and carboxyl-terminal regions of Ty3 IN . Each of the 20 IN mutants was defective for transposition, but no mutant was grossly defective for capsid maturation . All mutations affecting steady-state levels of mature IN protein resulted in reduced levels of replicated DNA, even when polymerase activity was not grossly defective as measured by exogenous reverse transcriptase activity assay . Thus, IN could contribute to nonpolymerase functions required for DNA production in vivo or to the stability of the DNA product . Several mutations in the carboxyl-terminal domain resulted in relatively low levels of processed 3' ends of the replicated DNA, suggesting that this domain may be important for binding of IN to the long terminal repeat . Another class of mutants produced wild-type amounts of DNA with correctly processed 3' ends . This class could include mutants affected in nuclear entry and target association . Collectively, these mutations demonstrate that in vivo, within the preintegration complex, IN performs a central role in coordinating multiple late stages of the retrotransposition life cycle.

Genome Res, 1998 Nov, 8(11), 1202 - 15
Predicting gene regulatory elements in silico on a genomic scale; Brazma A et al.; We performed a systematic analysis of gene upstream regions in the yeast genome for occurrences of regular expression-type patterns with the goal of identifying potential regulatory elements . To achieve this goal, we have developed a new sequence pattern discovery algorithm that searches exhaustively for a priori unknown regular expression-type patterns that are over-represented in a given set of sequences . We applied the algorithm in two cases, (1) discovery of patterns in the complete set of >6000 sequences taken upstream of the putative yeast genes and (2) discovery of patterns in the regions upstream of the genes with similar expression profiles . In the first case, we looked for patterns that occur more frequently in the gene upstream regions than in the genome overall . In the second case, first we clustered the upstream regions of all the genes by similarity of their expression profiles on the basis of publicly available gene expression data and then looked for sequence patterns that are over-represented in each cluster . In both cases we considered each pattern that occurred at least in some minimum number of sequences, and rated them on the basis of their over-representation . Among the highest rating patterns, most have matches to substrings in known yeast transcription factor-binding sites . Moreover, several of them are known to be relevant to the expression of the genes from the respective clusters . Experiments on simulated data show that the majority of the discovered patterns are not expected to occur by chance.

Nat Struct Biol, 1998 Dec, 5(12), 1091 - 7
Structure of N-myristoyltransferase with bound myristoylCoA and peptide substrate analogs; Bhatnagar RS et al.; N-myristoyltransferase (Nmt) attaches myristate to the N-terminal glycine of many important eukaryotic and viral proteins . It is a target for anti-fungal and anti-viral therapy . We have determined the structure, to 2.9 A resolution, of a ternary complex of Saccharomyces cerevisiae Nmt1p with bound myristoylCoA and peptide substrate analogs . The model reveals structural features that define the enzyme's substrate specificities and regulate the ordered binding and release of substrates and products . A novel catalytic mechanism is proposed involving deprotonation of the N-terminal ammonium of a peptide substrate by the enzyme's C-terminal backbone carboxylate.

Nucleic Acids Res, 1999 Jan 1, 27(1), 153 - 5
MITOP: database for mitochondria-related proteins, genes and diseases; Scharfe C et al.; The MITOP database de/proj/medgen/mitop/ consolidates information on both nuclear- and mitochondrial-encoded genes and their proteins . The five species files- Saccharomyces cerevisiae, Mus musculus, Caenorhabditis elegans, Neurospora crassa and Homo sapiens -include annotated data derived from a variety of online resources and the literature . A wide spectrum of search facilities is given in the interelated sections 'Gene catalogues', 'Protein catalogues', 'Homologies', 'Pathways and metabolism', and 'Human disease catalogue' including extensive references and hyperlinks for each entry . Precomputed FASTA searches using all the MITOP yeast protein entries and a list of the best EST hits with graphical cluster alignments related to the yeast reference sequence are presented . The MITOP orthologue tables with cross-listing to all the protein entries for each species in the database facilitate investigations into interspecies homology . A program (MITOPROT) is available to identify mitochondrial targeting sequences and graphical depictions of several important mitochondrial processes are included . The 'Human disease catalogue' lists a total of 101 disorders related to mitochondrial protein abnormalities, sorted by clinical criteria and age of onset.

Plant Physiol, 1998 Dec, 118(4), 1473 - 80
Characterization of source- and sink-specific sucrose/H+ symporters from carrot; Shakya R et al.; To understand how sucrose (Suc) is transported from source leaves to developing tap roots of carrot (Daucus carota L.), we cloned two cDNAs (DcSUT1 and DcSUT2) for proteins with homologies to plant Suc/H+ symporters . The deduced polypeptide sequences are 52% identical and have 12 predicted membrane-spanning domains each . Transport activities were confirmed by expression of the clones in yeast cells . Both transporters had optimal activity below pH 5.0 and Michaelis constant values of 0.5 mM . Suc uptake was inhibited by protonophores, suggesting that Suc transport is linked to the proton electrochemical potential across the plasma membrane . DcSUT1 and DcSUT2 had markedly different expression patterns . Transcripts of DcSUT1 were found only in the green parts of plants, with highest levels in the lamina of source leaves, indicating that DcSUT1 is required for the loading of Suc into the phloem . In leaf lamina expression was diurnally regulated, suggesting that Suc export from the leaves is higher during the day than during the night . The mRNA of DcSUT2 was found mainly in sink organs, and no diurnal expression pattern was detected in the storage root . Here, expression was not restricted to the phloem but was much higher in storage parenchyma tissues of phloem and xylem . The close relationship of DcSUT2 with a Suc/H+ symporter from fava bean, which facilitates Suc uptake into the cotyledons of developing seeds, indicates that this carrot Suc transporter may be involved in loading Suc into storage parenchyma cells.

Immunity, 1998 Nov, 9(5), 595 - 605
Adaptor function for the Syk kinases-interacting protein 3BP2 in IL-2 gene activation; Deckert M et al.; Syk-family tyrosine kinases are essential for lymphocyte development and activation . Using a yeast two-hybrid screen to identify Syk kinases-interacting proteins (SKIPs), we isolated 3BP2, an Abl SH3-interacting protein of unknown function . 3BP2 was selectively expressed in hematopoietic/lymphoid tissues and bound via its SH2 domain activated Syk-family kinases in mammalian cells, including in antigen receptor-stimulated T cells . In addition to Zap-70, the 3BP2 SH2 domain associated in vitro with LAT, Grb2, PLCgamma1, and Cbl from activated T cell lysates . Transient 3BP2 overexpression induced transcriptional activation of the IL-2 promoter and its NFAT or AP-1 elements . This activity was dependent on the SH2 and pleckstrin-homology domains of 3BP2, and required functional Syk kinases, Ras, and calcineurin . Thus, 3BP2 is an important adaptor that may couple activated Zap-70/Syk to a LAT-containing signaling complex involved in TCR-mediated gene transcription.

J Endocrinol, 1998 Sep, 158(3), 327 - 39
Several environmental oestrogens are also anti-androgens; Sohoni P et al.; There is presently considerable interest in endocrine disruption which is a new area of endocrinology concerned with chemicals that mimic hormones, in particular sex steroids . It has been hypothesised that exposure to such chemicals may be responsible for adverse effects in both humans and wildlife . Until now, chemicals that mimic oestrogens (so-called xenoestrogens) have been the main focus of endocrine disruption research . However, recent evidence suggests that many abnormalities in the male reproductive system may be mediated via the androgen receptor . By blocking androgen action, exposure to an anti-androgen may cause changes similar to those associated with oestrogen exposure . We have used in vitro yeast-based assays to detect oestrogenic, anti-oestrogenic, androgenic and anti-androgenic activities in a variety of chemicals of current interest . We show that many of the so-called 'environmental oestrogens' also possess anti-androgenic activity . The previously reported anti-androgenic activities of vinclozolin and p,p'-1,1-dichloro-2,2-bis(p-chlorophenyl) ethylene (DDE) were confirmed . We also found that o,p'-1,1,1,-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT), bisphenol A and butyl benzyl phthalate were anti-androgenic . However, not all xenoestrogens are also anti-androgenic, because nonylphenol was found to be a weak androgen agonist . Our results demonstrate that hormone-mimicking chemicals can have multiple hormonal activities, which may make it difficult to interpret their mechanisms of action in vivo . Although not a specific objective of this study, our results also demonstrate that yeast-based assays are powerful tools with which to investigate both agonist and antagonistic hormonal activities of chemicals.

Cell, 1998 Nov 25, 95(5), 717 - 28
Dissecting the regulatory circuitry of a eukaryotic genome; Holstege FC et al.; Genome-wide expression analysis was used to identify genes whose expression depends on the functions of key components of the transcription initiation machinery in yeast . Components of the RNA polymerase II holoenzyme, the general transcription factor TFIID, and the SAGA chromatin modification complex were found to have roles in expression of distinct sets of genes . The results reveal an unanticipated level of regulation which is superimposed on that due to gene-specific transcription factors, a novel mechanism for coordinate regulation of specific sets of genes when cells encounter limiting nutrients, and evidence that the ultimate targets of signal transduction pathways can be identified within the initiation apparatus.

FEBS Lett, 1998 Nov 20, 439(3), 215 - 8
Functional identification of a fatty acid delta5 desaturase gene from Caenorhabditis elegans; Michaelson LV et al.; We have identified a cDNA from the nematode worm Caenorhabditis elegans that encodes a fatty acid delta5 desaturase . Saccharomyces cerevisiae expressing the full-length cDNA was able to convert di-homo-gamma-linolenic acid to arachidonic acid, thus confirming delta5 desaturation . The 1341 bp delta5 desaturase sequence contained an N-terminal cytochrome b5 domain and was located within a kilobase of the C . elegans delta6 desaturase on chromosome IV . With an amino acid identity of 45% it is possible that one of these genes arose from the other by gene duplication . This is the first example of a delta5 desaturase gene isolated from an animal.

Biochem Mol Biol Int, 1998 Nov, 46(4), 775 - 85
Identification and characterization of the SMT3 cDNA and gene encoding ubiquitin-like protein from Drosophila melanogaster; Huang HW et al.; A SMT3 cDNA encoding ubiquitin-like protein from Drosophila melanogaster was isolated and sequenced . Drosophila SMT3 genomic DNA was amplified by polymerase chain reaction, and its nucleotide sequence was found to be identical to that of the cDNA, indicating the absence of intron in its protein coding region . The sequence of 90 amino acids of Drosophila SMT3 exhibited 55%, 73%, 70% and 52% identity to yeast SMT3, human SMT3A, SMT3B and SMT3C protein.

Mol Cell, 1998 Nov, 2(5), 703 - 8
Coat assembly directs v-SNARE concentration into synthetic COPII vesicles; Matsuoka K et al.; COPII proteins are required to create transport vesicles and to select cargo molecules for transit from the ER . A reconstituted liposome budding reaction was used to detect the capture and concentration of membrane-associated v-SNARE molecules into synthetic COPII vesicles . A novel glutathione-phosphatidyl-ethanolamine conjugate (Glut-PE) was synthesized and incorporated into chemically defined liposomes to provide binding sites for GST hybrid proteins . Large liposomes containing bound cytoplasmic domains of the v-SNAREs, Sec22p or Bos1p, or of the ER resident proteins, Sec12p and Ufe1p, were exposed to COPII proteins and GMP-PNP . v-SNAREs but not resident proteins were concentrated in synthetic COPII vesicles generated from donor liposomes . We conclude that COPII proteins are necessary and sufficient for cargo selection and vesicle morphogenesis.

Mol Cell, 1998 Nov, 2(5), 683 - 92
yTAFII61 has a general role in RNA polymerase II transcription and is required by Gcn4p to recruit the SAGA coactivator complex; Natarajan K et al.; We obtained a recessive insertion mutation in the gene encoding yeast TBP-associated factor yTAFII61/68 that impairs Gcn4p-independent and Gcn4p-activated HIS3 transcription . This mutation also reduces transcription of seven other class II genes, thus indicating a broad role for this yTAFII in RNA polymerase II transcription . The Gcn4p activation domain interacts with multiple components of the SAGA complex in cell extracts, including the yTAFII proteins associated with SAGA, but not with two yTAFIIs restricted to TFIID . The taf61-1 mutation impairs binding of Gcn4p to SAGA/yTAFII subunits but not to components of holoenzyme mediator . Our results provide strong evidence that recruitment of SAGA, in addition to holoenzyme, is crucial for activation by Gcn4p in vivo and that yTAFII61 plays a key role in this process.

Mol Cell, 1998 Nov, 2(5), 663 - 73
Histone-like TAFs are essential for transcription in vivo; Michel B et al.; In yeast, the TBP-associated factors (TAFs) Taf17, Taf60, and Taf61(68) resemble histones H3, H4, and H2B, respectively . To analyze their roles in vivo, conditional alleles were isolated by mutagenizing their histone homology domains . Conditional alleles of TAF17 or TAF60 can be specifically suppressed by overexpression of any of the other histone-like TAFs . This and other genetic evidence supports the model of a histone octamer-like structure within TFIID . Shifting strains carrying the conditional TAF alleles to non-permissive conditions results in degradation of TFIID components and the rapid loss of mRNA production . Therefore, in contrast to previous studies in yeast that found only limited roles for TAFs in transcription, we find that the histone-like TAFs are generally required for in vivo transcription.

Mol Cell, 1998 Nov, 2(5), 653 - 61
Broad, but not universal, transcriptional requirement for yTAFII17, a histone H3-like TAFII present in TFIID and SAGA; Apone LM et al.; The RNA polymerase II general transcription factor TFIID is a multisubunit complex comprising TATA box-binding protein (TBP) and associated factors (TAFIIs) . Experiments in yeast have shown that although most TAFIIs are required for viability, many genes are transcribed normally upon inactivation of individual and even multiple yTAFIIs . Here we analyze yTAFII17, recently found to be present in both the SAGA HAT complex as well as TFIID . Functional inactivation of yTAFII17 by temperature-sensitive mutation or depletion results in loss of transcription of many, but not all, genes . The upstream activating sequence (UAS), which contains the activator binding sites, is the region that renders a gene yTAFII17 dependent . In conjunction with previous studies, our results reveal that different TAFIIs have remarkably distinct properties.

Mol Cell, 1998 Nov, 2(5), 639 - 51
Two actin-related proteins are shared functional components of the chromatin-remodeling complexes RSC and SWI/SNF; Cairns BR et al.; The yeast Saccharomyces cerevisiae contains two related chromatin-remodeling complexes, RSC and SWI/SNF, which are shown to share the actin-related proteins Arp7 and Arp9 . Depending on the genetic background tested, arp7 delta and arp9 delta mutants are either inviable or show greatly impaired growth and Swi-/Snf- mutant phenotypes . Unlike swi/snf mutants, viable arp7 delta or arp9 delta mutants have an Spt- phenotype, suggesting that RSC affects transcription . Temperature-sensitive mutations in ARP7 and ARP9 were isolated, and the amino acid changes support the structural relationship of Arp7 and Arp9 to actin . However, site-directed mutations predicted to impair ATP binding or hydrolysis did not detectably affect Arp7 or Arp9 function . Our results suggest that actin-related proteins perform important roles in chromatin-remodeling complexes by virtue of structural rather than enzymatic similarities to actin.

Mol Cell, 1998 Nov, 2(5), 593 - 603
J proteins catalytically activate Hsp70 molecules to trap a wide range of peptide sequences; Misselwitz B et al.; Proteins of the Hsp70 family of ATPases, such as BiP, function together with J proteins to bind polypeptides in numerous cellular processes . Using a solid phase binding assay, we demonstrate that a conserved segment of the J proteins, the J domain, catalytically activates BiP molecules to bind peptides in its immediate vicinity . The J domain interacts with the ATP form of BiP and stimulates hydrolysis resulting in the rapid trapping of peptides, which are then only slowly released upon nucleotide exchange . Activation by the J domain allows BiP to trap peptides or proteins that it would not bind on its own . These results explain why BiP and probably all other Hsp70s can interact with a wide range of substrates and suggest that the J partner primarily determines the substrate specificity of Hsp70s.

Mol Cell, 1998 Nov, 2(5), 549 - 58
VEGF gene delivery to muscle: potential role for vasculogenesis in adults; Springer ML et al.; Constitutive expression of VEGF after implantation of genetically engineered myoblasts into non-ischemic muscle led to an increase in vascular structures . Previously, effects of VEGF delivery to adult muscle have only been reported in ischemic tissues . The resulting vascular structures were reminiscent of those formed during embryonic vasculogenesis, rather than angiogenesis, sprouting from preexisting vessels . Initially, VEGF caused an accumulation of endothelial cells and macrophages, followed by networks of vascular channels and hemangiomas with locally high serum VEGF levels . No effects were evident in adjacent tissue or contralateral legs, where low serum VEGF was detected . These data suggest that the induction by VEGF of angiogenesis or vasculogenesis may be dose-dependent . Furthermore, VEGF expression must be carefully modulated, as overexpression is deleterious.

Proc Natl Acad Sci U S A, 1998 Dec 8, 95(25), 15112 - 7
The arabidopsis thaliana AGRAVITROPIC 1 gene encodes a component of the polar-auxin-transport efflux carrier; Chen R et al.; Auxins are plant hormones that mediate many aspects of plant growth and development . In higher plants, auxins are polarly transported from sites of synthesis in the shoot apex to their sites of action in the basal regions of shoots and in roots . Polar auxin transport is an important aspect of auxin functions and is mediated by cellular influx and efflux carriers . Little is known about the molecular identity of its regulatory component, the efflux carrier {Estelle, M . (1996) Current Biol . 6, 1589-1591} . Here we show that mutations in the Arabidopsis thaliana AGRAVITROPIC 1 (AGR1) gene involved in root gravitropism confer increased root-growth sensitivity to auxin and decreased sensitivity to ethylene and an auxin transport inhibitor, and cause retention of exogenously added auxin in root tip cells . We used positional cloning to show that AGR1 encodes a putative transmembrane protein whose amino acid sequence shares homologies with bacterial transporters . When expressed in Saccharomyces cerevisiae, AGR1 promotes an increased efflux of radiolabeled IAA from the cells and confers increased resistance to fluoro-IAA, a toxic IAA-derived compound . AGR1 transcripts were localized to the root distal elongation zone, a region undergoing a curvature response upon gravistimulation . We have identified several AGR1-related genes in Arabidopsis, suggesting a global role of this gene family in the control of auxin-regulated growth and developmental processes.

Proc Natl Acad Sci U S A, 1998 Dec 8, 95(25), 14932 - 7
Improved cervical smear assessment using antibodies against proteins that regulate DNA replication; Williams GH et al.; Carcinoma of the cervix is one of the most common malignancies . Papanicolaou (Pap) smear tests have reduced mortality by up to 70% . Nevertheless their interpretation is notoriously difficult with high false-negative rates and frequently fatal consequences . We have addressed this problem by using affinity-purified antibodies against human proteins that regulate DNA replication, namely Cdc6 and Mcm5 . These antibodies were applied to sections and smears of normal and diseased uterine cervix by using immunoperoxidase or immunofluorescence to detect abnormal precursor malignant cells . Antibodies against Cdc6 and Mcm5 stain abnormal cells in cervical smears and sections with remarkably high specificity and sensitivity . Proliferation markers Ki-67 and proliferating cell nuclear antigen are much less effective . The majority of abnormal precursor malignant cells are stained in both low-grade and high-grade squamous intraepithelial lesions . Immunostaining of cervical smears can be combined with the conventional Pap stain so that all the morphological information from the conventional method is conserved . Thus antibodies against proteins that regulate DNA replication can reduce the high false-negative rate of the Pap smear test and may facilitate mass automated screening.

Proc Natl Acad Sci U S A, 1998 Dec 8, 95(25), 14897 - 902
Identification of a proline-binding motif regulating CD2-triggered T lymphocyte activation; Nishizawa K et al.; An intracellular protein termed CD2 binding protein 2 (CD2BP2), which binds to a site containing two PPPGHR segments within the cytoplasmic region of CD2, was identified . Mutagenesis and NMR analysis demonstrated that the CD2 binding region of CD2BP2 includes a 17-aa motif (GPY{orF}xxxxM{orV}xxWxxx GYF), also found in several yeast and Caenorhabditis elegans proteins of unknown function . In Jurkat T cells, over-expression of the isolated CD2BP2 domain binding to CD2 enhances the production of interleukin 2 on crosslinking of CD2 but not the T cell receptor . Hence, a proline-binding module distinct from SH3 and WW domains regulates protein-protein interactions.

Proc Natl Acad Sci U S A, 1998 Dec 8, 95(25), 14863 - 8
Cluster analysis and display of genome-wide expression patterns; Eisen MB et al.; A system of cluster analysis for genome-wide expression data from DNA microarray hybridization is described that uses standard statistical algorithms to arrange genes according to similarity in pattern of gene expression . The output is displayed graphically, conveying the clustering and the underlying expression data simultaneously in a form intuitive for biologists . We have found in the budding yeast Saccharomyces cerevisiae that clustering gene expression data groups together efficiently genes of known similar function, and we find a similar tendency in human data . Thus patterns seen in genome-wide expression experiments can be interpreted as indications of the status of cellular processes . Also, coexpression of genes of known function with poorly characterized or novel genes may provide a simple means of gaining leads to the functions of many genes for which information is not available currently.

Proc Natl Acad Sci U S A, 1998 Dec 8, 95(25), 14787 - 92
Pontin52, an interaction partner of beta-catenin, binds to the TATA box binding protein; Bauer A et al.; beta-catenin, the vertebrate homolog of the Drosophila Armadillo protein, has been shown to have dual cellular functions, as a component of both the cadherin-catenin cell adhesion complex and the Wnt signaling pathway . At Wnt signaling, beta-catenin becomes stabilized in the cytoplasm and subsequently available for interaction with transcription factors of the lymphocyte enhancer factor-1/T-cell factor family, resulting in a nuclear localization of beta-catenin . Although beta-catenin does not bind DNA directly, its carboxyl- and amino-terminal regions exhibit a transactivating activity still not well understood molecularly . Here we report the identification of an interaction partner of beta-catenin, a nuclear protein designated Pontin52 . Pontin52 binds beta-catenin in the region of Armadillo repeats 2-5 and, more importantly, also binds the TATA box binding protein . We provide evidence for an in vivo multiprotein complex composed of Pontin52, beta-catenin, and lymphocyte enhancer factor-1/T-cell factor . Our results suggest involvement of Pontin52 in the nuclear function of beta-catenin.

Proc Natl Acad Sci U S A, 1998 Dec 8, 95(25), 14745 - 50
PAT1, a microtubule-interacting protein, recognizes the basolateral sorting signal of amyloid precursor protein; Zheng P et al.; In epithelial cells, sorting of membrane proteins to the basolateral surface depends on the presence of a basolateral sorting signal (BaSS) in their cytoplasmic domain . Amyloid precursor protein (APP), a basolateral protein implicated in the pathogenesis of Alzheimer's disease, contains a tyrosine-based BaSS, and mutation of the tyrosine residue results in nonpolarized transport of APP . Here we report identification of a protein, termed PAT1 (protein interacting with APP tail 1), that interacts with the APP-BaSS but binds poorly when the critical tyrosine is mutated and does not bind the tyrosine-based endocytic signal of APP . PAT1 shows homology to kinesin light chain, which is a component of the plus-end directed microtubule-based motor involved in transporting membrane proteins to the basolateral surface . PAT1, a cytoplasmic protein, associates with membranes, cofractionates with APP-containing vesicles, and binds microtubules in a nucleotide-sensitive manner . Cotransfection of PAT1 with a reporter protein shows that PAT1 is functionally linked with intracellular transport of APP . We propose that PAT1 is involved in the translocation of APP along microtubules toward the cell surface.

Am J Physiol, 1998 Dec, 275(6 Pt 1), L1164 - 72
CsA-sensitive purine-box transcriptional regulator in bronchial epithelial cells contains NF45, NF90, and Ku; Aoki Y et al.; Human bronchial epithelial (HBE) cells express interleukin (IL)-2 {Y . Aoki, D . Qiu, A . Uyei, and P . N . Kao . Am . J . Physiol . 272 (Lung Cell . Mol . Physiol . 16): L276-L286, 1997} . 16HBE-transformed cells contain constitutive and inducible nuclear DNA-binding activity for the purine-box/nuclear factor (NF) of activated T cell (NFAT) target DNA sequence in the human IL-2 enhancer . Transcriptional activation through the purine-box DNA sequence requires stimulation with phorbol 12-myristate 13-acetate + ionomycin, and this activation is inhibited by cyclosporin A . Immunohistochemical staining of 16HBE cells demonstrates nuclear expression of the purine-box DNA-binding proteins NF45 and NF90 and no expression of NFATp or NFATc . NF90 and NF45 associate with the DNA-dependent protein kinase catalytic subunit and the DNA-targeting subunits Ku80 and Ku70 (N . S . Ting, P . N . Kao, D . W . Chan, L . G . Lintott, and S . P . Lees-Miller . J . Biol . Chem . 273: 2136-2145, 1998) . Antibodies to Ku potently inhibit the purine-box DNA-binding complex . The purine-box transcriptional regulator in 16HBE cells likely comprises NF45, NF90, Ku80, Ku70, and the DNA-dependent protein kinase catalytic subunit.

Mol Biol Cell, 1998 Dec, 9(12), 3561 - 78
Retrograde transport from the pre-Golgi intermediate compartment and the Golgi complex is affected by the vacuolar H+-ATPase inhibitor bafilomycin A1; Palokangas H et al.; The effect of the vacuolar H+-ATPase inhibitor bafilomycin A1 (Baf A1) on the localization of pre-Golgi intermediate compartment (IC) and Golgi marker proteins was used to study the role of acidification in the function of early secretory compartments . Baf A1 inhibited both brefeldin A- and nocodazole-induced retrograde transport of Golgi proteins to the endoplasmic reticulum (ER), whereas anterograde ER-to-Golgi transport remained largely unaffected . Furthermore, p58/ERGIC-53, which normally cycles between the ER, IC, and cis-Golgi, was arrested in pre-Golgi tubules and vacuoles, and the number of p58-positive approximately 80-nm Golgi (coatomer protein I) vesicles was reduced, suggesting that the drug inhibits the retrieval of the protein from post-ER compartments . In parallel, redistribution of beta-coatomer protein from the Golgi to peripheral pre-Golgi structures took place . The small GTPase rab1p was detected in short pre-Golgi tubules in control cells and was efficiently recruited to the tubules accumulating in the presence of Baf A1 . In contrast, these tubules showed no enrichment of newly synthesized, anterogradely transported proteins, indicating that they participate in retrograde transport . These results suggest that the pre-Golgi structures contain an active H+-ATPase that regulates retrograde transport at the ER-Golgi boundary . Interestingly, although Baf A1 had distinct effects on peripheral pre-Golgi structures, only more central, p58-containing elements accumulated detectable amounts of 3-(2, 4-dinitroanilino)-3'-amino-N-methyldipropylamine (DAMP), a marker for acidic compartments, raising the possibility that the lumenal pH of the pre-Golgi structures gradually changes in parallel with their translocation to the Golgi region.

Mol Biol Cell, 1998 Dec, 9(12), 3493 - 503
Identification of potential regulatory elements for the transport of Emp24p; Nakamura N et al.; To examine the possibility of active recycling of Emp24p between the endoplasmic reticulum (ER) and the Golgi, we sought to identify transport signal(s) in the carboxyl-terminal region of Emp24p . Reporter molecules were constructed by replacing parts of a control invertase-Wbp1p chimera with those of Emp24p, and their transport rates were assessed . The transport of the reporter was found to be accelerated by the presence of the cytoplasmic domain of Emp24p . Mutational analyses revealed that the two carboxyl-terminal residues, leucine and valine (LV), were necessary and sufficient to accelerate the transport . The acceleration was sequence specific, and the terminal valine appeared to be more important . The LV residues accelerated not only the overall transport to the vacuole but also the ER to cis-Golgi transport, suggesting its function in the ER export . Hence the LV residues are a novel anterograde transport signal . The double-phenylalanine residues did not affect the transport by itself but attenuated the effect of the anterograde transport signal . On the other hand, the transmembrane domain significantly slowed down the ER to cis-Golgi transport and effectively counteracted the anterograde transport signal at this step . It may also take part in the retrieval of the protein, because the overall transport to the vacuole was more evidently slowed down . Consistently, the mutation of a conserved glutamine residue in the transmembrane domain further slowed down the transport in a step after arriving at the cis-Golgi . Taken together, the existence of the anterograde transport signal and the elements that regulate its function support the active recycling of Emp24p.

Mol Biol Cell, 1998 Dec, 9(12), 3475 - 92
Functional characterization of a Nup159p-containing nuclear pore subcomplex; Belgareh N et al.; Nup159p/Rat7p is an essential FG repeat-containing nucleoporin localized at the cytoplasmic face of the nuclear pore complex (NPC) and involved in poly(A)+ RNA export and NPC distribution . A detailed structural-functional analysis of this nucleoporin previously demonstrated that Nup159p is anchored within the NPC through its essential carboxyl-terminal domain . In this study, we demonstrate that Nup159p specifically interacts through this domain with both Nsp1p and Nup82p . Further analysis of the interactions within the Nup159p/Nsp1p/Nup82p subcomplex using the nup82Delta108 mutant strain revealed that a deletion within the carboxyl-terminal domain of Nup82p prevents its interaction with Nsp1p but does not affect the interaction between Nup159p and Nsp1p . Moreover, immunofluorescence analysis demonstrated that Nup159p is delocalized from the NPC in nup82Delta108 cells grown at 37 degrees C, a temperature at which the Nup82Delta108p mutant protein becomes degraded . This suggests that Nup82p may act as a docking site for a core complex composed of the repeat-containing nucleoporins Nup159p and Nsp1p . In vivo transport assays further revealed that nup82Delta108 and nup159-1/rat7-1 mutant strains have little if any defect in nuclear protein import and protein export . Together our data suggest that the poly(A)+ RNA export defect previously observed in nup82 mutant cells might be due to the loss from the NPCs of the repeat-containing nucleoporin Nup159p.

Mol Biol Cell, 1998 Dec, 9(12), 3455 - 73
Sec61p serves multiple roles in secretory precursor binding and translocation into the endoplasmic reticulum membrane; Pilon M et al.; The evolutionarily conserved Sec61 protein complex mediates the translocation of secretory proteins into the endoplasmic reticulum . To investigate the role of Sec61p, which is the main subunit of this complex, we generated recessive, cold-sensitive alleles of sec61 that encode stably expressed proteins with strong defects in translocation . The stage at which posttranslational translocation was blocked was probed by chemical crosslinking of radiolabeled secretory precursors added to membranes isolated from wild-type and mutant strains . Two classes of sec61 mutants were distinguished . The first class of mutants was defective in preprotein docking onto a receptor site of the translocon that included Sec61p itself . The second class of mutants allowed docking of precursors onto the translocon but was defective in the ATP-dependent release of precursors from this site that in wild-type membranes leads to pore insertion and full translocation . Only mutants of the second class were partially suppressed by overexpression of SEC63, which encodes a subunit of the Sec61 holoenzyme complex responsible for positioning Kar2p (yeast BiP) at the translocation channel . These mutants thus define two early stages of translocation that require SEC61 function before precursor protein transfer across the endoplasmic reticulum membrane.

EMBO J, 1998 Dec 1, 17(23), 7105 - 17
A map of the binding site for catalytic domain 5 in the core of a group II intron ribozyme; Konforti BB et al.; Group II introns are ribozymes with a complex tertiary architecture that is of great interest as a model for RNA folding . Domain 5 (D5) is a highly conserved region of the intron that is considered one of the most critical structures in the catalytic core . Despite its central importance, the means by which D5 interacts with other core elements is unclear . To obtain a map of potential interaction sites, dimethyl sulfate was used to footprint regions of the intron that are involved in D5 binding . These studies were complemented by measurements of D5 binding to a series of truncated intron derivatives . In this way, the minimal region of the intron required for strong D5 association was defined and the sites most likely to represent thermodynamically significant positions of tertiary contact were identified . These studies show that ground-state D5 binding is mediated by tertiary contacts to specific regions of D1, including a tetraloop receptor and an adjacent three-way junction . In contrast, D2 and D3 are not found to stabilize D5 association . These data highlight the significance of D1-D5 interactions and will facilitate the identification of specific tertiary contacts between them.

EMBO J, 1998 Dec 1, 17(23), 7078 - 90
Nhp2p and Nop10p are essential for the function of H/ACA snoRNPs; Henras A et al.; The small nucleolar ribonucleoprotein particles containing H/ACA-type snoRNAs (H/ACA snoRNPs) are crucial trans-acting factors intervening in eukaryotic ribosome biogenesis . Most of these particles generate the site-specific pseudouridylation of rRNAs while a subset are required for 18S rRNA synthesis . To understand in detail how these particles carry out these functions, all of their protein components have to be characterized . For that purpose, we have affinity-purified complexes containing epitope-tagged Gar1p protein, previously shown to be part of H/ACA snoRNPs . Under the conditions used, three polypeptides of 65, 22 and 10 kDa apparent molecular weight specifically copurify with epitope-tagged Gar1p . The 22 and 10 kDa polypeptides were identified as Nhp2p and a novel protein we termed Nop10p, respectively . Both proteins are conserved, essential and present in the dense fibrillar component of the nucleolus . Nhp2p and Nop10p are specifically associated with all H/ACA snoRNAs and are essential to the function of H/ACA snoRNPs . Cells lacking Nhp2p or Nop10p are impaired in global rRNA pseudouridylation and in the A1 and A2 cleavage steps of the pre-rRNA required for the synthesis of mature 18S rRNA . These phenotypes are probably a direct consequence of the instability of H/ACA snoRNAs and Gar1p observed in cells deprived of Nhp2p or Nop10p . Our results suggest that Nhp2p and Nop10p, together with Cbf5p, constitute the core of H/ACA snoRNPs.

EMBO J, 1998 Dec 1, 17(23), 6932 - 41
WAVE, a novel WASP-family protein involved in actin reorganization induced by Rac; Miki H et al.; Rac is a Rho-family small GTPase that induces the formation of membrane ruffles . However, it is poorly understood how Rac-induced reorganization of the actin cytoskeleton, which is essential for ruffle formation, is regulated . Here we identify a novel Wiskott-Aldrich syndrome protein (WASP)-family protein, WASP family Verprolin-homologous protein (WAVE), as a regulator of actin reorganization downstream of Rac . Ectopically expressed WAVE induces the formation of actin filament clusters that overlap with the expressed WAVE itself . In this actin clustering, profilin, a monomeric actin-binding protein that has been suggested to be involved in actin polymerization, was shown to be essential . The expression of a dominant-active Rac mutant induces the translocation of endogenous WAVE from the cytosol to membrane ruffling areas . Furthermore, the co-expression of a deltaVPH WAVE mutant that cannot induce actin reorganization specifically suppresses the ruffle formation induced by Rac, but has no effect on Cdc42-induced actin-microspike formation, a phenomenon that is also known to be dependent on rapid actin reorganization . The deltaVPH WAVE also suppresses membrane-ruffling formation induced by platelet-derived growth factor in Swiss 3T3 cells . Taken together, we conclude that WAVE plays a critical role downstream of Rac in regulating the actin cytoskeleton required for membrane ruffling.

EMBO J, 1998 Dec 1, 17(23), 6863 - 70
New COP1-binding motifs involved in ER retrieval; Cosson P et al.; Coatomer-mediated sorting of proteins is based on the physical interaction between coatomer (COP1) and targeting motifs found in the cytoplasmic domains of membrane proteins . For example, binding of COP1 to dilysine (KKXX) motifs induces specific retrieval of tagged proteins from the Golgi back to the endoplasmic reticulum (ER) . Making use of the two-hybrid system, we characterized a new sequence (deltaL) which interacts specifically with the delta-COP subunit of the COP1 complex . Transfer of deltaL to the cytoplasmic domain of a reporter membrane protein resulted in its localization in the ER, in yeast and mammalian cells . This was due to continuous retrieval of tagged proteins from the Golgi back to the ER, in a manner similar to the ER retrieval of KKXX-tagged proteins . Extensive mutagenesis of deltaL identified an aromatic residue as a critical determinant of the interaction with COP1 . Similar COP1-binding motifs containing an essential aromatic residue were identified in the cytoplasmic domain of an ER-resident protein, Sec71p, and in an ER retention motif previously characterized in the CD3epsilon chain of the T-cell receptor . These results emphasize the role of the COP1 complex in retrograde Golgi-to-ER transport and highlight its functional similarity with clathrin-adaptor complexes.

Biochemistry, 1998 Nov 10, 37(45), 15726 - 36
SH3 binding domains in the dopamine D4 receptor; Oldenhof J et al.; The dopamine D4 receptor is a G protein-coupled receptor (GPCR) that belongs to the dopamine D2-like receptor family . Functionally, the D2-like receptors are characterized by their ability to inhibit adenylyl cyclase . The dopamine D4 receptor as well as many other catecholaminergic receptors contain several putative SH3 binding domains . Most of these sites in the D4 receptor are located in a polymorphic repeat sequence and flanking sequences in the third intracellular loop . Here we demonstrate that this region of the D4 receptor can interact with a large variety of SH3 domains of different origin . The strongest interactions were seen with the SH2-SH3 adapter proteins Grb2 and Nck . The repeat sequence itself is not essential in this interaction . The data presented indicate that the different SH3 domains in the adapter proteins interact in a cooperative fashion with two distinct sites immediately upstream and downstream from the repeat sequence . Removal of all the putative SH3 binding domains in the third intracellular loop of the dopamine D4 receptor resulted in a receptor that could still bind spiperone and dopamine . Dopamine could not modulate the coupling of these mutant receptors to adenylyl cyclase and MAPK, although dopamine modulated receptor-G protein interaction appeared normal . The receptor deletion mutants show strong constitutive internalization that may account for the deficiency in functional activation of second messengers . The data indicates that the D4 receptor contains SH3 binding sites and that these sites fall within a region involved in the control of receptor internalization.

J Exp Med, 1998 Dec 7, 188(11), 1985 - 92
Accelerated neutrophil apoptosis in mice lacking A1-a, a subtype of the bcl-2-related A1 gene; Hamasaki A et al.; To elucidate the role of A1, a new member of the Bcl-2 family of apoptosis regulators active in hematopoietic cell apoptosis, we established mice lacking A1-a, a subtype of the A1 gene in mice (A1-a-/- mice) . Spontaneous apoptosis of peripheral blood neutrophils of A1-a-/- mice was enhanced compared with that of either wild-type mice or heterozygous mutants (A1-a+/- mice) . Neutrophil apoptosis inhibition induced by lipopolysaccharide treatment in vitro or transendothelial migration in vivo observed in wild-type mice was abolished in both A1-a-/- and A1-a+/- animals . On the other hand, the extent of tumor necrosis factor alpha-induced acceleration of neutrophil apoptosis did not differ among A1-a-/-, A1-a+/-, and wild-type mice . The descending order of A1 mRNA expression was wild-type, A1-a+/-, and A1-a-/- . Taken together, these results suggest that A1 is involved in inhibition of certain types of neutrophil apoptosis.

Biochem J, 1998 Dec 15, 336 ( Pt 3), 699 - 704
Identification of the separate domains in the hepatic glycogen-targeting subunit of protein phosphatase 1 that interact with phosphorylase a, glycogen and protein phosphatase 1; Armstrong CG et al.; Deletion and mutational analyses of the rat liver glycogen-targeting subunit (GL) of protein phosphatase 1 (PP1) have identified three separate domains that are responsible for binding of PP1, glycogen and phosphorylase a . The glycogen-binding domain spans the centre of GL between residues 144 and 231 and appears to be distinct from the glycogen-binding (storage) site of phosphorylase . The regulatory high-affinity binding site for phosphorylase a lies in the 16 amino acids at the C-terminus of GL . The PP1-binding domain is deduced to comprise the -RVXF- motif {Egloff, Johnson, Moorhead, Cohen and Barford (1997) EMBO J . 16, 1876-1887} located at residues 61-64 of GL and preceding lysine residues at positions 56, 57 and 59 . A possible approach for increasing glycogen synthesis in certain disorders is discussed.

Am J Hum Genet, 1998 Dec, 63(6), 1663 - 74
Functional characterization of missense mutations in ATP7B: Wilson disease mutation or normal variant?
Forbes JR, Cox DW.
Wilson disease is an autosomal recessive disorder of copper transport that causes hepatic and/or neurological disease resulting from copper accumulation in the liver and brain . The protein defective in this disorder is a putative copper-transporting P-type ATPase, ATP7B . More than 100 mutations have been identified in the ATP7B gene of patients with Wilson disease . To determine the effect of Wilson disease missense mutations on ATP7B function, we have developed a yeast complementation assay based on the ability of ATP7B to complement the high-affinity iron-uptake deficiency of the yeast mutant ccc2 . We characterized missense mutations found in the predicted membrane-spanning segments of ATP7B . Ten mutations have been made in the ATP7B cDNA by site-directed mutagenesis: five Wilson disease missense mutations, two mutations originally classified as possible disease-causing mutations, two putative ATP7B normal variants, and mutation of the cysteine-proline-cysteine (CPC) motif conserved in heavy-metal-transporting P-type ATPases . All seven putative Wilson disease mutants tested were able to at least partially complement ccc2 mutant yeast, indicating that they retain some ability to transport copper . One mutation was a temperature-sensitive mutation that was able to complement ccc2 mutant yeast at 30 degreesC but was unable to complement at 37 degreesC . Mutation of the CPC motif resulted in a nonfunctional protein, which demonstrates that this motif is essential for copper transport by ATP7B . Of the two putative ATP7B normal variants tested, one resulted in a nonfunctional protein, which suggests that it is a disease-causing mutation.

Gene, 1998 Sep 14, 217(1-2), 101 - 6
The genomic organization of Isopeptidase T-3 (ISOT-3), a new member of the ubiquitin specific protease family (UBP); Timms KM et al.; A novel Isopeptidase T gene (ISOT-3) has been identified on human mosome 3q26.2--q26.3 . gene shows 67.3% nucleotide identity and 54.8% amino acid identity to n Isopeptidase (ISOT-1) . Northern blot analysis has shown that ISOT-3 is highly essed in ovary and testes, low-level expression in six other tissues tested . In contrast, ISOT-1 is essed at high levels in brain, and there is no detectable expression in ovary . The exonic nization of these two genes highly conserved with only one variant intron position . Intron 15 in -3 is absent in ISOT-1, there is an alternate splice site at the same location . Although the --intron structure has been erved between the two genes, ISOT-3 has significantly larger intronic ons, and the overall of this gene is at least 90 kb compared to 15 kb for ISOT-1 . These data suggest that both ISOT-1 and ISOT-3 have descended from a common ancestor . In addition, the low overall sequence identity and different expression patterns may reflect differences in substrate specificity.

Plant J, 1998 Oct, 16(2), 145 - 54
Mutations in Arabidopsis thaliana genes involved in the tryptophan biosynthesis pathway affect root waving on tilted agar surfaces; Rutherford R et al.; Arabidopsis thaliana roots grow in a wavy pattern upon a slanted surface . A novel mutation in the anthranilate synthase alpha 1 (ASA1) gene, named trp5-2wvc1, and mutations in the tryptophan synthase alpha and beta 1 genes (trp3-1 and trp2-1, respectively) confer a compressed root wave phenotype on tilted agar surfaces . When trp5-2wvc1 seedlings are grown on media supplemented with anthranilate metabolites, their roots wave like wild type . Genetic and pharmacological experiments argue that the compressed root wave phenotypes of trp5-2wvc1, trp2-1 and trp3-1 seedlings are not due to reduced IAA biosynthetic potential, but rather to a deficiency in L-tryptophan (L-Trp), or in a L-Trp derivative . Although the roots of 7-day-old seedlings possess higher concentrations of free L-Trp than the shoot as a whole, trp5-2wvc1 mutants show no detectable alteration in L-Trp levels in either tissue type, suggesting that a very localized shortage of L-Trp, or of a L-Trp-derived compound, is responsible for the observed phenotype.

Biochim Biophys Acta, 1998 Nov 26, 1443(1-2), 65 - 74
Structure and expression of a laccase gene from the ligninolytic basidiomycete Ceriporiopsis subvermispora; Karahanian E et al.; A gene encoding laccase has been isolated from a genomic library of the white-rot basidiomycete Ceriporiopsis subvermispora constructed in Lambda GEM-11 . This gene (Cs-lcs1) contains an open reading frame of 2215 bp, encoding a mature protein of 499 amino acids with a 21-residue signal peptide . The protein sequence exhibits between 63 and 68% identity with laccases from other basidiomycetes and shares with all of them 10 conserved histidines and one cysteine involved in the coordination of copper atoms at the active site of the enzyme . The gene possesses 11 introns, with splicing junctions and internal lariat formation sites adhering to the GT-AG and CTRAY rules, respectively . The upstream region of Cs-lcs1 contains a TATA box, two CAAT sites, five putative metal response elements and a ACE1 element . In agreement with the presence of the latter element, transcription of Cs-lcs1 is activated by copper and silver, as shown by Northern blot and reverse transcription followed by DNA amplification analyses . Based on Southern blot analysis, Cs-lcs1 appears to be the only gene encoding laccase in C . subvermispora.

Nucleic Acids Res, 1998 Dec 15, 26(24), 5707 - 18
Crystal structure of the MATa1/MATalpha2 homeodomain heterodimer in complex with DNA containing an A-tract; Li T et al.; The crystal structure of the heterodimer formed by the DNA binding domains of the yeast mating type transcription factors, MATa1 and MATalpha2, bound to a 21 bp DNA fragment has been determined at 2.5 A resolution . The DNA fragment in the present study differs at four central base pairs from the DNA sequence used in the previously studied ternary complex . These base pair changes give rise to a (dA5).(dT5) tract without changing the overall base composition of the DNA . The resulting A-tract occurs near the center of the overall 60 degrees bend in the DNA . Comparison of the two structures shows that the structural details of the DNA bend are maintained despite the DNA sequence changes . Analysis of the A5-tract DNA subfragment shows that it contains a bend toward the minor groove centered at one end of the A-tract . The observed bend is larger than that observed in the crystal structures of A-tracts embedded in uncomplexed DNA, which are straight and have been presumed to be quite rigid . Variation of the central DNA base sequence reverses the two AT base pairs contacted in the minor groove by Arg7 of the alpha2 N-terminal arm without significantly altering the DNA binding affinity of the a1/alpha2 heterodimer . The Arg7 side chain accommodates the sequence change by forming alternate H bond interactions, in agreement with the proposal that minor groove base pair recognition is insensitive to base pair reversal . Furthermore, the minor groove spine of hydration, which stabilizes the narrowed minor groove caused by DNA bending, is conserved in both structures . We also find that many of the water-mediated hydrogen bonds between the a1 and alpha2 homeodomains and the DNA are highly conserved, indicating an important role for water in stabilization of the a1/alpha2-DNA complex.

Nucleic Acids Res, 1998 Dec 15, 26(24), 5562 - 7
In vitro assembly of an archaeal D-L-N RNA polymerase subunit complex reveals a eukaryote-like structural arrangement; Eloranta JJ et al.; Archaeal RNA polymerases (RNAPs) resemble the eukaryotic nuclear RNAPs in complexity, and many of their subunits display a high degree of sequence similarity to their eukaryotic counterparts . Here we describe specific protein-protein contacts present between individual recombinant RNAP subunits from the archaeon Methanococcus jannaschii . Subunits D and L interact specifically with each other in two-hybrid assays . D also interacts under the same conditions with the RPB11 and AC19 subunits from the yeast Saccharomyces cerevisiae, suggesting that essential elements of the binding surface between these proteins have been conserved across the archaeal/eukaryotic evolutionary domain boundary . Interactions between L and RPB3 or AC40 were, however, not detectable . Recombinant D and L subunits associate under in vitro conditions and copurify with each other during size-exclusion chromatography . Addition of an another recombinant subunit (N) to the D-L complex results in the formation of a triple complex . This D-L-N complex resembles the RPB3-RPB11-RPB10 or AC40-AC19-RPB10 complexes in eukaryotic RNAPIIand RNAPI/RNAPIII, respectively . Our data provide evidence for a close similarity in the quaternary arrangement of a subset of archaeal and eukaryotic RNA polymerase subunits and the conservation of the protein-protein contacts formed between them.

J Biol Chem, 1998 Dec 11, 273(50), 33580 - 7
Characterization of the Ah receptor-associated protein, ARA9; Carver LA et al.; The unliganded aryl hydrocarbon receptor (AHR) is found in a complex with other proteins including the 90-kDa heat shock protein (Hsp90) and a 37-kDa protein we refer to as ARA9 . We found that the three tetratricopeptide repeats found in the COOH terminus of ARA9 are necessary and sufficient for interaction with the AHR complex . Conversely, the AHR's "repressor"/Hsp90 binding domain is required for interaction with ARA9 . Because ARA9 closely resembles the 52-kDa FK506-binding protein (FKBP52), found in the unliganded glucocorticoid receptor (GR) complex, we compared the binding specificities of ARA9 and FKBP52 for AHR and GR . In co-immunoprecipitation experiments, ARA9 specifically associated with AHR-Hsp90 complex but not with GR-Hsp90 complexes . In addition, ARA9 showed a greater capacity than FKBP52 to associate with AHR-Hsp90 complexes . The biological importance of this interaction was suggested by the observation that in a yeast expression system ARA9 expression enhanced the response of AHR to the agonist beta-napthoflavone, decreasing the EC50 by greater than 5-fold and increasing the maximal response 2.5-fold . In contrast, co-expression of FKBP52 had no effect on AHR signaling . In addition, although ARA9 contains a domain similar to that found in other FK506-binding proteins, ARA9 binding to 3H-FK506 could not be detected . Finally, we have characterized the developmental and expression pattern of ARA9 in the developing mouse embryo and mapped the ARA9 locus to human chromosome 11q13.3.

Biochem Biophys Res Commun, 1998 Nov 27, 252(3), 679 - 85
Subcellular localization and protein-protein interaction regions of Ku proteins; Koike M et al.; The Ku protein is a complex of Ku70 and Ku80 subunits and is capable of binding promoters in a sequence-specific manner, although it remains unclear whether Ku is involved in transcriptional regulation . We examined the subcellular localization and determined the interaction regions of Ku . Our results indicate that heterodimers of Ku70 and Ku80 are localized in the nucleus, and that the stretches from amino acid (aa) 378 to 482 of Ku70 and from aa 374 to 502 of Ku80 are necessary for heterodimerization . These interaction regions do not contain any previously recognized protein-protein interaction motifs . To determine whether Ku contains a potential transcriptional activation domain, we examined N- and C-terminal deletion mutants of Ku70 and Ku80 for their ability to activate transcription in the GAL4-based one-hybrid system . We found that the whole Ku protein had no transcriptional activity, although the N-terminal peptide fragment of Ku70 was capable of activating transcription of the HIS3 and lacZ reporter genes in yeast cells .

J Mol Biol, 1998 Dec 11, 284(4), 963 - 73
Ku70/Ku80 protein complex inhibits the binding of nucleotide excision repair proteins on linear DNA in vitro; Frit P et al.; We have previously reported that the incision efficiency of the nucleotide excision repair (NER) reaction measured in vitro with cell-free human protein extracts was reduced by up to 80% on a linearized damaged plasmid DNA substrate when compared to supercoiled damaged DNA . The inhibition stemed from the presence of the DNA-end binding Ku70/Ku80 heterodimer which is the regulatory subunit of the DNA-dependent protein kinase (DNA-PK) . Here, the origin of the repair inhibition was assessed by a new in vitro assay in which circular or linear plasmid DNA, damaged or undamaged, was quantitatively adsorbed on sensitized microplate wells . The binding of two NER proteins, XPA and p62-TFIIH, indispensable for the incision step of the reaction, was quantified either directly in an ELISA-like reaction in the wells with specific antibodies or in Western blotting experiments on the DNA-bound fraction . We report a dramatic inhibition of XPA and p62-TFIIH association with UVC photoproducts on linear DNA . XPA and p62-TFIIH binding to DNA damage was regained when the reaction was performed with extracts lacking Ku activity (extracts from xrs6 rodent cells) whereas addition of purified human Ku complex to these extracts restored the inhibition . Despite the fact that DNA-PK was active during the NER reaction, the mechanism of inhibition relied on the sole Ku complex, since mutant protein extracts lacking the catalytic DNA-PK subunit (extracts from the human M059J glioma cells) exhibited a strong binding inhibition of XPA and p62-TFIIH proteins on linear damaged DNA, identical to the inhibition observed with the DNA-PK+ control extracts (from M059K cells) .

J Mol Biol, 1998 Dec 11, 284(4), 925 - 35
Structural and functional heterogeneity of Rap1p complexes with telomeric and UASrpg-like DNA sequences; Idrissi FZ et al.; Rap1p binds to a variety of related DNA sequences . We studied complexes of Rap1p and its DNA-binding domain with two of these sequences, the UASrpg sequence (5'-ACACCCATACATTT-3') and the Saccharomyces cerevisiae telomeric consensus (5'-ACACCCACACACCC-3') . When cloned in front of a minimal CYC1 promoter, the two sequences differed in their transcriptional potential . Whereas UASrpg or telomeric single binding sites activated transcription with approximately the same strength, adjacent UASrpg sequences showed higher synergistic activity and orientation-dependence than telomeric sequences . We also found different sequence requirements for Rap1p binding in vitro to both sequences, since a single base-pair that severely reduced binding of Rap1p to UASrpg sequences had very little effect on the telomeric sequence . The Rap1p binding domain distorted DNA molecules encompassing the UASrpg sequence or the telomeric-like sequence, as revealed by both KMnO4 hypersensitivity and by hydroxyl radical foot-printing analysis . We propose that Rap1p is able to form structurally and functionally different complexes, depending on the type of DNA sequence the complex is assembled from . This functional and structural heterogeneity may be responsible for the multiple functions that Rap1p binding sites appear to have in vivo .

J Mol Biol, 1998 Dec 11, 284(4), 859 - 65
Crystal structure of GCN4-pIQI, a trimeric coiled coil with buried polar residues; Eckert DM et al.; Coiled coils consist of two or more alpha-helices wrapped around each other with a superhelical twist . The interfaces between helices of a coiled coil are formed by hydrophobic amino acid residues packed in a "knobs-into-holes" arrangement . Most naturally occurring coiled coils, however, also contain buried polar residues, as do the cores of the majority of naturally occurring globular proteins . Two common buried polar residues in both dimeric and trimeric coiled coils are asparagine and glutamine . In dimeric coiled coils, buried asparagine, but not glutamine, residues have been shown to confer specificity of oligomerization . We have placed a glutamine residue in the otherwise hydrophobic interior of a stable trimeric coiled coil, GCN4-pII, to study the effect of this buried polar residue in a trimeric coiled-coil environment . The resulting peptide, GCN4-pIQI, is a discrete, trimeric coiled coil with a lower stability than GCN4-pII . The crystal structure determined to 1.8 A shows that GCN4-pIQI is a trimeric coiled coil with a chloride ion coordinated by one buried glutamine residue from each monomer .

Biochemistry, 1998 Dec 1, 37(48), 17024 - 9
Autophosphorylation and protein kinase activity of p21-activated protein kinase gamma-PAK are differentially affected by magnesium and manganese; Tuazon PT et al.; To examine the requirements for activation of the p21-activated protein kinase gamma-PAK (Pak2, PAK I) from rabbit reticulocytes by Cdc42(GTPgammaS), autophosphorylation with ATP(Mg) or ATP(Mn) and its effects on protein kinase activity were examined . Autophosphorylation with ATP(Mg) alone was minimal with negligible protein kinase activity; the rate of autophosphorylation was increased 3-4-fold upon binding of Cdc42(GTPgammaS), resulting in a 3-fold stimulation of protein kinase activity with peptide and protein substrates . The rate of autophosphorylation with ATP(Mn) was 4.7-fold faster than with ATP(Mg) alone and was stimulated 2-fold by Cdc42(GTPgammaS) . However, gamma-PAK autophosphorylated with ATP(Mn) in the presence or absence of Cdc42(GTPgammaS) did not phosphorylate peptide or protein substrates in the presence of ATP(Mn), indicating that gamma-PAK can utilize ATP(Mn) for autophosphorylation but not for phosphorylation of exogenous substrates . Tryptic phosphopeptide maps of gamma-PAK autophosphorylated with ATP(Mg) alone showed 3 phosphopeptides, while with Cdc42(GTPgammaS) a total of 9 major phosphopeptides was observed . When gamma-PAK was autophosphorylated with ATP(Mn) in the presence or absence of Cdc42(GTPgammaS), 7 major phosphopeptides were observed, which were identical to peptides obtained with Cdc42(GTPgammaS) and ATP(Mg) . Utilizing a recombinant mutant of gamma-PAK with alanine replacing threonine 402 in the catalytic region (T402A), it was determined that the two additional phosphopeptides observed in active PAK (peptides 7 and 8) were due to phosphorylation of threonine 402 . These results show that Mn sustains autophosphorylation on serine but does not support autophosphorylation of threonine 402, which is required for activity toward exogenous substrates, or phosphorylation of these substrates.

Semin Cell Dev Biol, 1998 Oct, 9(5), 493 - 501
Protein and lipid sorting between the endoplasmic reticulum and the Golgi complex; Nickel W et al.; Vesicular traffic within the early secretory pathway is mediated by COPI- and COPII-coated vesicles . While COPII-coated vesicles appear to be involved exclusively in the export of secretory proteins and lipids from the endoplasmic reticulum (ER), COPI-coated vesicles seem to function in both anterograde and retrograde transport between the ER-Golgi intermediate compartment (IC) and the Golgi as well as in intra-Golgi transport . Here, we focus on (i) the mechanisms how these transport carriers are formed from a given donor membrane; and (ii) the possible mechanisms involved in sorting of proteins and lipids into such transport vesicles .

Appl Environ Microbiol, 1998 Dec, 64(12), 4834 - 41
Characterization of the critical amino acids of an Aspergillus parasiticus cytochrome P-450 monooxygenase encoded by ordA that is involved in the biosynthesis of aflatoxins B1, G1, B2, and G2; Yu J et al.; The conversion of O-methylsterigmatocystin (OMST) and dihydro-O-methylsterigmatocystin to aflatoxins B1, G1, B2, and G2 requires a cytochrome P-450 type of oxidoreductase activity . ordA, a gene adjacent to the omtA gene, was identified in the aflatoxin-biosynthetic pathway gene cluster by chromosomal walking in Aspergillus parasiticus . The ordA gene was a homolog of the Aspergillus flavus ord1 gene, which is involved in the conversion of OMST to aflatoxin B1 . Complementation of A . parasiticus SRRC 2043, an OMST-accumulating strain, with the ordA gene restored the ability to produce aflatoxins B1, G1, B2, and G2 . The ordA gene placed under the control of the GAL1 promoter converted exogenously supplied OMST to aflatoxin B1 in Saccharomyces cerevisiae . In contrast, the ordA gene homolog in A . parasiticus SRRC 2043, ordA1, was not able to carry out the same conversion in the yeast system . Sequence analysis revealed that the ordA1 gene had three point mutations which resulted in three amino acid changes (His-400-->Leu-400, Ala-143-->Ser-143, and Ile-528-->Tyr-528) . Site-directed mutagenesis studies showed that the change of His-400 to Leu-400 resulted in a loss of the monooxygenase activity and that Ala-143 played a significant role in the catalytic conversion . In contrast, Ile-528 was not associated with the enzymatic activity . The involvement of the ordA gene in the synthesis of aflatoxins G1, and G2 in A . parasiticus suggests that enzymes required for the formation of aflatoxins G1 and G2 are not present in A . flavus . The results showed that in addition to the conserved heme-binding and redox reaction domains encoded by ordA, other seemingly domain-unrelated amino acid residues are critical for cytochrome P-450 catalytic activity . The ordA gene has been assigned to a new cytochrome P-450 gene family named CYP64 by The Cytochrome P450 Nomenclature Committee.

J Nat Prod, 1998 Nov, 61(11), 1374 - 8
Acanthosterol sulfates A-J: ten new antifungal steroidal sulfates from a marine sponge Acanthodendrilla sp; Tsukamoto S et al.; Ten new steroidal sulfates, acanthosterol sulfates A-J (1-10), have been isolated from a marine sponge, Acanthodendrilla sp., collected in western Japan . Acanthosterol sulfates I and J (9 and 10) showed antifungal activity against the yeast Saccharomyces cerevisiae A364A and its mutants at 0.1 mg/disk.

J Cell Biol, 1998 Nov 30, 143(5), 1215 - 25
Unc-45 mutations in Caenorhabditis elegans implicate a CRO1/She4p-like domain in myosin assembly; Barral JM et al.; The Caenorhabditis elegans unc-45 locus has been proposed to encode a protein machine for myosin assembly . The UNC-45 protein is predicted to contain an NH2-terminal domain with three tetratricopeptide repeat motifs, a unique central region, and a COOH-terminal domain homologous to CRO1 and She4p . CRO1 and She4p are fungal proteins required for the segregation of other molecules in budding, endocytosis, and septation . Three mutations that lead to temperature-sensitive (ts) alleles have been localized to conserved residues within the CRO1/She4p-like domain, and two lethal alleles were found to result from stop codon mutations in the central region that would prevent translation of the COOH-terminal domain . Electron microscopy shows that thick filament accumulation in vivo is decreased by approximately 50% in the CB286 ts mutant grown at the restrictive temperature . The thick filaments that assemble have abnormal structure . Immunofluorescence and immunoelectron microscopy show that myosins A and B are scrambled, in contrast to their assembly into distinct regions at the permissive temperature and in wild type . This abnormal structure correlates with the high degree of instability of the filaments in vitro as reflected by their extremely low yields and shortened lengths upon isolation . These results implicate the UNC-45 CRO1/She4p-like region in the assembly of myosin isoforms in C . elegans and suggest a possible common mechanism for the function of this UCS (UNC-45/CRO1/She4p) protein family.

Acta Anat (Basel), 1998, 162(2-3), 151 - 6
Serine/threonine protein phosphatases type 1, 2A and 2C in vertebrate retinae; Selke D et al.; A number of retinal proteins are phosphorylated by a variety of kinases, resulting in well-known regulatory effects . The identity and role of corresponding phosphatases is less clear . We simultaneously measured the activity of serine/ threonine protein phosphatases type 1, 2A and 2C in bovine retinae . The enzymes were classified according to substrate specificity, divalent cation requirement and the effect of phosphatase subtype-specific inhibitors . The total- and specific activity of phosphatase type 2A was prevalent . Type 2C was 10-fold less abundant . 80% of type 1 and 50% of type 2A and type 2C, respectively, were soluble . An economic purification scheme was developed . We demonstrated the presence of phosphatase isozymes 2Calpha and 2Cbeta in bovine rod outer segments by enzymatic analysis as well as immunological techniques . The results suggest a yet unknown role of phosphatase type 2C in phototransduction . On the other hand, the immense amount of protein phosphatases found to be soluble - therefore not associated with rod outer segment membranes - points towards participation of these enzymes in the process of visual transduction not considered thus far.

Genes Dev, 1998 Nov 15, 12(22), 3613 - 24
Formation and specification of ventral neuroblasts is controlled by vnd in Drosophila neurogenesis; Chu H et al.; During Drosophila neural development, neuroblasts delaminate from the neuroectoderm of each hemisegment in a stereotypic orthogonal array of five rows and three columns (ventral, intermediate, and dorsal) . Prevailing evidence indicates that the individual neuroblast fate is determined by the domain-specific expression of genes along the dorsoventral and anteroposterior axis . Here, we analyze the role of Vnd, a NK-2 homeodomain protein, expressed initially in the ventral neuroectoderm adjacent to the ventral midline, in the dorsoventral patterning of the neuroectoderm and the neuroblasts . We show that in vnd null mutants most ventral neuroblasts do not form and the few that form do not develop ventral fates, but instead develop intermediate-like fates . Furthermore, we demonstrate that Vnd influences the gene expression patterns in the ventral proneural clusters and neuroectoderm, and that its action in neuroblast formation includes, but is not exclusive to the activation of proneural AS-C genes . Through the use of GAL4/UAS gene-expression system we show that ectopic Vnd expression can promote ventral-like fates in intermediate and dorsal neuroblasts and can suppress certain normal characteristics of the intermediate and dorsal neuroectoderm . Our results are discussed in the context of the current evidence in dorsoventral patterning in the Drosophila neuroectoderm.

Genes Dev, 1998 Nov 15, 12(22), 3551 - 63
Synaptonemal complex morphogenesis and sister-chromatid cohesion require Mek1-dependent phosphorylation of a meiotic chromosomal protein; Bailis JM et al.; Development of yeast meiotic chromosome cores into full-length synaptonemal complexes requires the MEK1 gene product, a meiosis-specific protein kinase homolog . The Mek1 protein associates with meiotic chromosomes and colocalizes with the Red1 protein, which is a component of meiotic chromosome cores . Mek1 and Red1 interact physically in meiotic cells, as demonstrated by coimmunoprecipitation and the two-hybrid protein system . Hop1, another protein associated with meiotic chromosome cores, also interacts with Mek1 but only in the presence of Red1 . Red1 displays Mek1-dependent phosphorylation, both in vitro and in vivo, and Mek1 kinase activity is necessary for Mek1 function in vivo . Fluorescent in situ hybridization analysis indicates that Mek1-mediated phosphorylation of Red1 is required for meiotic sister-chromatid cohesion, raising the possibility that cohesion is regulated by protein phosphorylation.

Genes Dev, 1998 Nov 15, 12(22), 3482 - 7
Allosteric interactions between capping enzyme subunits and the RNA polymerase II carboxy-terminal domain; Cho EJ et al.; mRNA capping is a cotranscriptional event mediated by the association of capping enzyme with the phosphorylated carboxy-terminal domain (CTD) of RNA polymerase II . In the yeast Saccharomyces cerevisiae, capping enzyme is composed of two subunits, the mRNA 5'-triphosphatase (Cet1) and the mRNA guanylyltransferase (Ceg1) . Here we map interactions between Ceg1, Cet1, and the CTD . Although the guanylyltransferase subunit can bind alone to the CTD, it cannot be guanylylated unless the triphosphatase subunit is also present . Therefore, the yeast mRNA guanylyltransferase is regulated by allosteric interactions with both the triphosphatase and CTD.

Lipids, 1998 Oct, 33(10), 1043 - 9
A semiautomated enzymatic method for determination of nonesterified fatty acid concentration in milk and plasma; Christmass MA et al.; An enzymatic assay for the determination of nonesterified fatty acid concentrations in milk and plasma is described . The procedure is semiautomated for use with a plate luminometer or plate spectrophotometer and enables routine batch processing of large numbers of small samples (< or =5 microL) . Following the activation of nonesterified fatty acids (NEFA) by acylCoA synthetase, the current assay utilizes UDP-glucose pyrophosphorylase to link inorganic pyrophosphate to the production of NADH through the reactions catalyzed by phosphoglucomutase and glucose-6-phosphate 1-dehydrogenase . With this assay sequence the formation of NADH from NEFA is complete within 50 min at 37 degrees C . Enzymatic spectrophotometric techniques were unsuitable for NEFA determination in human milk due to the opacity of the sample . The use of the NADH-luciferase system has overcome this problem, allowing the enzymatic determination of NEFA in human milk . Sample collection and treatment procedures for milk and plasma have been developed to prevent enzymatic lipolysis and to limit interference from enzymes present in milk . The recovery of palmitic acid added to milk and plasma samples was 94.9+/-2.9 and 100+/-4.5%, respectively . There was no difference (P = 0.13) in plasma NEFA concentrations determined by the current method and a commercially available enzymatic spectrophotometric technique (Wako NEFA-C kit) . Plasma NEFA concentrations determined by gas chromatography were 28% higher compared to both the Wako NEFA-C kit and the current method.

Science, 1998 Nov 27, 282(5394), 1721 - 4
Control of cyclin ubiquitination by CDK-regulated binding of Hct1 to the anaphase promoting complex; Zachariae W et al.; Proteolysis of mitotic cyclins depends on a multisubunit ubiquitin-protein ligase, the anaphase promoting complex (APC) . Proteolysis commences during anaphase, persisting throughout G1 until it is terminated by cyclin-dependent kinases (CDKs) as cells enter S phase . Proteolysis of mitotic cyclins in yeast was shown to require association of the APC with the substrate-specific activator Hct1 (also called Cdh1) . Phosphorylation of Hct1 by CDKs blocked the Hct1-APC interaction . The mutual inhibition between APC and CDKs explains how cells suppress mitotic CDK activity during G1 and then establish a period with elevated kinase activity from S phase until anaphase.

Neuroreport, 1998 Oct 5, 9(14), 3259 - 63
Expression of copper trafficking genes in the mouse brain; Nishihara E et al.; Copper homeostasis in the brain must be strictly maintained, since copper is an essential trace element and is potentially toxic . To understand the mechanism of copper homeostasis in the brain, we cloned several mouse homologues of copper trafficking genes and performed in situ hybridization histochemistry . mCTR1, mATX1, and mATP7a were highly expressed in the choroid plexus, indicating that the choroid plexus uses the trafficking pathway from uptake to efflux to transport copper to the cerebrospinal fluids . We suggest that these genes may regulate copper concentration in the brain through the choroid plexus.

Proteins, 1998 Nov 15, 33(3), 383 - 95
Structural model for family 32 of glycosyl-hydrolase enzymes; Pons T et al.; A structural model is presented for family 32 of the glycosyl-hydrolase enzymes based on the beta-propeller fold . The model is derived from the common prediction of two different threading methods, TOPITS and THREADER . In addition, we used a correlated mutation analysis and prediction of active-site residues to corroborate the proposed model . Physical techniques (circular dichroism and differential scanning calorimetry) confirmed two aspects of the prediction, the proposed all-beta fold and the multi-domain structure . The most reliable three-dimensional model was obtained using the structure of neuraminidase (1nscA) as template . The analysis of the position of the active site residues in this model is compatible with the catalytic mechanism proposed by Reddy and Maley (J . Biol . Chem . 271:13953-13958, 1996), which includes three conserved residues, Asp, Glu, and Cys . Based on this analysis, we propose the participation of one more conserved residue (Asp 162) in the catalytic mechanism . The model will facilitate further studies of the physical and biochemical characteristics of family 32 of the glycosyl-hydrolases.

J Biol Chem, 1998 Dec 4, 273(49), 32870 - 7
A ribosomal protein is required for translational regulation of GCN4 mRNA . Evidence for involvement of the ribosome in eIF2 recycling; Mueller PP et al.; In amino acid-starved yeast cells, inhibition of the guanine nucleotide exchange factor eIF2B by phosphorylated translation initiation factor 2 results in increased translation of GCN4 mRNA . We isolated a suppressor of a mutant eIF2B . The suppressor prevents efficient GCN4 mRNA translation due to inactivation of the small ribosomal subunit protein Rps31 and results in low amounts of mutant 40 S ribosomal subunits . Deletion of one of two genes encoding ribosomal protein Rps17 also reduces the amounts of 40 S subunits but does not suppress eIF2B mutations or prevent efficient GCN4 translation . Our findings show that Rps31-deficient ribosomes are altered in a way that decreases the eIF2B requirement and that the small ribosomal subunit mediates the effects of low eIF2B activity on cell viability and translational regulation in response to eIF2 phosphorylation.

J Biol Chem, 1998 Dec 4, 273(49), 32848 - 56
Endoplasmic reticulum degradation of a mutated ATP-binding cassette transporter Pdr5 proceeds in a concerted action of Sec61 and the proteasome; Plemper RK et al.; Degradation of misfolded or tightly regulated proteins in the endoplasmic reticulum (ER) is performed by the cytosolic ubiquitin-proteasome system and therefore requires their prior transport back to the cytosol . Here, we report on the extraction and degradation mechanism of a polytopic membrane protein . Rapid proteasomal degradation of a mutated form of the ATP-binding cassette transporter Pdr5 retained in the ER is initialized at the lumenal face of the ER membrane . Using different antibodies directed against the cytosolic tails or a lumenal loop of the transmembrane protein, it could be demonstrated that the turnover of Pdr5* demands the concerted action of both the Sec61 translocon and the ubiquitin-proteasome system . We observed a stabilization of the entire molecule within the ER membrane in yeast mutants characterized by a reduced translocation capacity or by functionally attenuated proteasomes . Moreover, no degradation intermediates were detected in any of the mutants that impede degradation of Pdr5* . Therefore, initial steps are rate-limiting for cleavage and mutations that impede downstream events prevent initiation of the process . Our data suggest that ER degradation is a mechanistically highly integrated process, requiring the combined operation of components of the degradation system acting at the lumenal face of the ER membrane, the Sec61 translocon, and the ubiquitin-proteasome system.

J Biol Chem, 1998 Dec 4, 273(49), 32500 - 5
The inhibitory upstream open reading frame from mammalian S-adenosylmethionine decarboxylase mRNA has a strict sequence specificity in critical positions; Mize GJ et al.; The upstream open reading frame (uORF) in the 5' leader of the mammalian mRNA encoding S-adenosylmethionine decarboxylase (AdoMetDC) serves as a negative regulatory element by suppressing translation of the associated downstream cistron . Certain changes in the amino acid sequence of the hexapeptide (sequence MAGDIS) encoded by the uORF destroy suppressive activity, implying specific interaction with a cellular target . In this paper, we examine the extent of alterations that can be tolerated in this uORF . The mammalian AdoMetDC uORF inhibits downstream translation when placed into the 5' leader of a yeast mRNA with characteristics resembling those in mammalian cells, suggesting that the encoded peptide has a similar target across species . Using yeast for the initial screen, we tested the specificity of the critical three codons at the 3' end of the uORF by saturation mutagenesis . Altered uORFs selected from the primary yeast screen were then retested in mammalian cells . The requirements at codons 4 and 5 were quite stringent; only aspartic acid at codon 4 yielded a fully suppressive peptide, and only valine could substitute productively for isoleucine at codon 5 . The specificity at codon 6 was much looser, with many substitutions retaining suppressive activity in both yeast and mammalian cells.

J Biol Chem, 1998 Dec 4, 273(49), 32421 - 9
The Orc4p and Orc5p subunits of the Xenopus and human origin recognition complex are related to Orc1p and Cdc6p; Tugal T et al.; The location of origins of DNA replication within the Saccharomyces cerevisiae genome is primarily determined by the origin recognition complex (ORC) interacting with specific DNA sequences . The analogous situation in vertebrate cells is far less clear, although ORC subunits have been identified in several vertebrate organisms including Xenopus laevis . Monoclonal antibodies were raised against Xenopus Orc1p and used for single-step immunoaffinity purification of the entire ORC from an egg extract . Six polypeptides ( approximately 110, 68, 64, 48, 43, and 27 kDa) copurified with Xenopus Orc1p . Protein sequencing also showed the 64-kDa protein to be the previously identified Xenopus Orc2p . Microsequencing of the 43- and 48-kDa proteins that copurified with Orc1p and Orc2p led to their identification as the Orc4p and Orc5p subunits, respectively . Peptide sequences from the 43-kDa protein also allowed the isolation of cDNAs encoding the Xenopus, mouse, and human ORC4 subunits . Human ORC5 was also cloned; its sequence displayed extensive homology to both Drosophila and yeast ORC5 . Surprisingly, comparison of the amino acid sequences of Orc1p, Orc4p, and Orc5p suggests that they are structurally related to each other and to the replication initiation protein, Cdc6p . Finally, we present the sequence of the putative Xenopus and human Orc3p.

J Biol Chem, 1998 Dec 4, 273(49), 32388 - 92
Gcn5p, a transcription-related histone acetyltransferase, acetylates nucleosomes and folded nucleosomal arrays in the absence of other protein subunits; Tse C et al.; Gcn5p is the catalytic subunit of several type A histone acetyltransferases (HATs) . Previous studies performed under a limited range of solution conditions have found that nucleosome core particles and nucleosomal arrays can be acetylated by Gcn5p only when it is complexed with other proteins, e.g . Gcn5-Ada, HAT-A2, and SAGA . Here we demonstrate that when assayed in buffer containing optimum concentrations of either NaCl or MgCl2, purified yeast recombinant Gcn5p (rGcn5p) efficiently acetylates both nucleosome core particles and nucleosomal arrays . Furthermore, under conditions where nucleosomal arrays are extensively folded, rGcn5p acetylates folded arrays approximately 40% faster than nucleosome core particles . Finally, rGcn5p polyacetylates the N termini of free histone H3 but only monoacetylates H3 in nucleosomes and nucleosomal arrays . These results demonstrate both that rGcn5p in and of itself is catalytically active when assayed under optimal solution conditions and that this enzyme prefers folded nucleosomal arrays as a substrate . They further suggest that the structure of the histone H3 N terminus, and concomitantly the accessibility of the H3 acetylation sites, changes upon assembly into nucleosomes and nucleosomal arrays.

J Biol Chem, 1998 Dec 4, 273(49), 32377 - 9
CREB is a regulatory target for the protein kinase Akt/PKB; Du K et al.; The nuclear factor CREB stimulates the expression of cellular genes following its protein kinase A-mediated phosphorylation at Ser-133 . Ser-133 phosphorylation, in turn, activates target gene expression by promoting recruitment of the co-activator CBP . Recent studies showing that CREB and its paralog CREM are required for survival of certain cell types prompted us to examine whether CREB is a nuclear target for activation via the growth factor-dependent Ser/Thr kinase Akt/PKB . When overexpressed in serum-stimulated cells, Akt/PKB potently induced Ser-133 phosphorylation of CREB and promoted recruitment of CBP . Correspondingly, Akt/PKB stimulated target gene expression via CREB in a phospho(Ser-133)-dependent manner . Akt/PKB induced CREB activity only in response to serum stimulation, and this effect was suppressed by the phosphatidylinositol 3-kinase inhibitor LY 294002 . Our results support the notion that Akt/PKB promotes cell survival, at least in part, by stimulating the expression of cellular genes via the CREB/CBP nuclear transduction pathway.

Nucleic Acids Res, 1998 Dec 1, 26(23), 5365 - 71
Processing of telomeric DNA ends requires the passage of a replication fork; Dionne I et al.; During telomere replication in yeast, chromosome ends acquire a long single-stranded extension of the strand making the 3' end . Previous work showed that these 3' tails are generated late in S-phase, when conventional replication is virtually complete . In addition, the extensions were also observed in cells that lacked telomerase . Therefore, a model was proposed that predicted an activity that recessed the 5' ends at yeast telomeres after conventional replication was complete . Here, we demonstrate that this processing activity is dependent on the passage of a replication fork through yeast telomeres . A non-replicating linear plasmid with telomeres at each end does not acquire single-stranded extensions, while an identical construct containing an origin of replication does . Thus, the processing activity could be associated with the enzymes at the replication fork itself, or the passage of the fork through the telomeric sequences allows a transient access for the activity to the telomeres . We therefore propose that there is a mechanistic link between the conventional replication machinery and telomere maintenance.

Nucleic Acids Res, 1998 Dec 1, 26(23), 5333 - 42
Double-strand break repair in Ku86- and XRCC4-deficient cells; Kabotyanski EB et al.; The Ku86 and XRCC4 proteins perform critical but poorly understood functions in the repair of DNA double-strand breaks . Both Ku 86- and XRCC4-deficient cells exhibit profound radiosensitivity and severe defects in V(D)J recombination, including excessive deletions at recombinant junctions . Previous workers have suggested that these phenomena may reflect defects in joining of the broken DNA ends or in protection of the ends from nucleases . However, end joining in XRCC4-deficient cells has not been examined . Here we show that joining of both matched and mismatched DNA ends occurs efficiently in XRCC4-deficient cells . Furthermore, analysis of junctions shows that XRCC4 is not required to protect the ends from degradation . However, nucleotide sequence analysis of junctions derived from joining of mismatched DNA ends in XRCC4-deficient cells revealed a strong preference for a junction containing a 7 nt homology . Similar results were obtained in Ku86-deficient cells . These data suggest that in the absence of XRCC4 or Ku86, joining is assisted by base pairing interactions, supporting the hypothesis that these proteins may participate in aligning or stabilizing intermediates in end joining.

Nucleic Acids Res, 1998 Dec 1, 26(23), 5243 - 50
Characterization and selectivity of catalytic antibodies from human serum with RNase activity; Vlassov A et al.; IgG purified from sera of several patients with systemic lupus erythematosus and hepatitis B are shown to present RNA hydrolyzing activities that are different from the weak RNase A-type activities found in the sera of healthy donors . Further investigation brings evidence for two intrinsic activities, one observed in low salt conditions and another specifically stimulated by Mg2+ions and distinguishable from human sera RNases . Cleavage of RNA substrates by the latter activity is not sequence-specific but sensitive to both subtle conformational and/or drastic folding changes, as evidenced by comparative analysis of couples of structurally well-studied RNA substrates . These include yeast tRNAAsp and its in vitro transcript and human mitochondrial tRNALys-derived in vitro transcripts . The discovery of catalytic antibodies with RNase activities is a first step towards creation of a new generation of tools for the investigation of RNA structure.

Proc Natl Acad Sci U S A, 1998 Nov 24, 95(24), 14302 - 7
Autoregulation at the level of mRNA 3' end formation of the suppressor of forked gene of Drosophila melanogaster is conserved in Drosophila virilis; Audibert A et al.; The Drosophila melanogaster Suppressor of forked {Su(f)} protein shares homology with the yeast RNA14 protein and the 77-kDa subunit of human cleavage stimulation factor, which are proteins involved in mRNA 3' end formation . This suggests a role for Su(f) in mRNA 3' end formation in Drosophila . The su(f) gene produces three transcripts; two of them are polyadenylated at the end of the transcription unit, and one is a truncated transcript, polyadenylated in intron 4 . Using temperature-sensitive su(f) mutants, we show that accumulation of the truncated transcript requires wild-type Su(f) protein . This suggests that the Su(f) protein autoregulates negatively its accumulation by stimulating 3' end formation of the truncated su(f) RNA . Cloning of su(f) from Drosophila virilis and analysis of its RNA profile suggest that su(f) autoregulation is conserved in this species . Sequence comparison between su(f) from both species allows us to point out three conserved regions in intron 4 downstream of the truncated RNA poly(A) site . These conserved regions include the GU-rich downstream sequence involved in poly(A) site definition . Using transgenes truncated within intron 4, we show that sequence up to the conserved GU-rich domain is sufficient for production of the truncated RNA and for regulation of this production by su(f) . Our results indicate a role of su(f) in the regulation of poly(A) site utilization and an important role of the GU-rich sequence for this regulation to occur.

Proc Natl Acad Sci U S A, 1998 Nov 24, 95(24), 14278 - 83
Physical interaction between components of DNA mismatch repair and nucleotide excision repair; Bertrand P et al.; Nucleotide excision repair (NER) and DNA mismatch repair are required for some common processes although the biochemical basis for this requirement is unknown . Saccharomyces cerevisiae RAD14 was identified in a two-hybrid screen using MSH2 as "bait," and pairwise interactions between MSH2 and RAD1, RAD2, RAD3, RAD10, RAD14, and RAD25 subsequently were demonstrated by two-hybrid analysis . MSH2 coimmunoprecipitated specifically with epitope-tagged versions of RAD2, RAD10, RAD14, and RAD25 . MSH2 and RAD10 were found to interact in msh3 msh6 and mlh1 pms1 double mutants, suggesting a direct interaction with MSH2 . Mutations in MSH2 increased the UV sensitivity of NER-deficient yeast strains, and msh2 mutations were epistatic to the mutator phenotype observed in NER-deficient strains . These data suggest that MSH2 and possibly other components of DNA mismatch repair exist in a complex with NER proteins, providing a biochemical and genetical basis for these proteins to function in common processes.

Proc Natl Acad Sci U S A, 1998 Nov 24, 95(24), 14272 - 7
An artificial cell-cycle inhibitor isolated from a combinatorial library; Cohen BA et al.; Understanding the genetic networks that operate inside cells will require the dissection of interactions among network members . Here we describe a peptide aptamer isolated from a combinatorial library that distinguishes among such interactions . This aptamer binds to cyclin-dependent kinase 2 (Cdk2) and inhibits its kinase activity . In contrast to naturally occurring inhibitors, such as p21(Cip1), which inhibit the activity of Cdk2 on all its substrates, inhibition by pep8 has distinct substrate specificity . We show that the aptamer binds to Cdk2 at or near its active site and that its mode of inhibition is competitive . Expression of pep8 in human cells retards their progression through the G1 phase of the cell cycle . Our results suggest that the aptamer inhibits cell-cycle progression by blocking the activity of Cdk2 on substrates needed for the G1-to-S transition . This work demonstrates the feasibility of selection of artificial proteins to perform functions not developed during evolution . The ability to select proteins that block interactions between a gene product and some partners but not others should make sophisticated genetic manipulations possible in human cells and other currently intractable systems.

Proc Natl Acad Sci U S A, 1998 Nov 24, 95(24), 14076 - 81
The orientation of the AP-1 heterodimer on DNA strongly affects transcriptional potency; Chytil M et al.; Activation of gene transcription in eukaryotes requires the cooperative assembly of an initiation complex containing many protein subunits . The necessity that these components contact each other and the promoter/enhancer in defined ways suggests that their spatial arrangement might influence the activation response . Indeed, growing evidence indicates that DNA architecture can profoundly affect transcriptional potency . Much less is known about the influence of protein architecture on transcriptional activation . Here, we examine the architectural dependence of activator function through the analysis of matched pairs of AP-1*DNA complexes differing only in their orientation . Mutation of a critical Arg residue in the basic-leucine zipper domain of either Fos or Jun yielded single point-mutant heterodimers that bind DNA in a single defined orientation, as determined directly by native chemical ligation/affinity cleavage; by contrast, the corresponding wild-type protein binds DNA as a roughly equal mixture of two isomeric orientations, which are related by subunit interchange . The stereochemistry of the point-mutant heterodimers could be switched by inversion of a C*G base pair in the center of the AP-1 site, thus providing access to both fixed orientational isomers . Yeast reporter gene assays consistently revealed that one orientational isomer activates transcription at least 10-fold more strongly than the other . These results suggest that protein architecture, especially the spatial relationship of the activation domain to the promoter, can exert a powerful influence on activator potency.

Proc Natl Acad Sci U S A, 1998 Nov 24, 95(24), 14066 - 70
DNA end-joining catalyzed by human cell-free extracts; Baumann P et al.; Mammalian cells defective in DNA end-joining are highly sensitive to ionizing radiation and are immunodeficient because of a failure to complete V(D)J recombination . By using cell-free extracts prepared from human lymphoblastoid cell lines, an in vitro system for end-joining has been developed . Intermolecular ligation was found to be accurate and to depend on DNA ligase IV/Xrcc4 and requires Ku70, Ku86, and DNA-PKcs, the three subunits of the DNA-activated protein kinase DNA-PK . Because these activities are involved in the cellular resistance to x-irradiation and V(D)J recombination, the development of this in vitro system provides an important advance in the study of the mechanism of DNA end-joining in human cells.

Biochem Biophys Res Commun, 1998 Nov 18, 252(2), 529 - 33
cDNA cloning and characterization of a constitutively expressed isoform of the human peroxin Pex11p; Abe I et al.; We cloned a human cDNA encoding an isoform of the peroxin Pex11p, termed Pex11pbeta, by screening of human liver cDNA library using as a probe human EST-derived, approximately 300 bp-long nucleotides showing homology to PEX11 from Candida boidinii and Saccharomyces cerevisiae . PEX11beta encoded a protein comprising 259 amino acids, with two putative transmembrane segments, showing approximately 40% identity to inducible Pex11palpha, at the amino acid sequence level . Pex11pbeta was found to be a peroxisomal protein, as assessed by colocalization with acyl-CoA oxidase, an enzyme catalyzing the first step of peroxisomal beta-oxidation system, in Pex11pbeta-expressing Chinese hamster ovary cells . PEX11beta was not induced in rats by treatment of clofibrate, a peroxisome proliferator, in contrast to PEX11alpha .

Biochem Biophys Res Commun, 1998 Nov 18, 252(2), 524 - 8
Intracellular association of FGF-2 with the ribosomal protein L6/TAXREB107; Shen B et al.; By using the yeast two-hybrid system, we identified the ribosomal protein L6/TAXREB107 as an intracellular partner for FGF-2 . L6/TAXREB107 also mediates the DNA binding of the HTLV-1 transactivator Tax . In vitro binding experiments indicated that both the high-molecular-weight forms (HMW) and the 18-kDa form of FGF-2 bind to L6/TAXREB107 . Deletion analysis suggested that L6/TAXREB107 has two binding sites for HMW FGF-2 and one binding site for 18-kDa FGF-2, implying that the unique N-terminal extension of the HMW FGF-2 is one of the binding domains for L6/TAXREB107 . Transfection assays showed that high expression of either HMW or 18 kDa FGF-2 stimulates Tax-mediated transactivation in NIH 3T3 cells . This result suggests a possible role of FGF-2 in Tax-mediated HTLV-1 transformation as well as FGF-2 binding to ribosomes and/or their precursors .

Biochem Biophys Res Commun, 1998 Nov 18, 252(2), 285 - 9
Phosphoinositide-specific phospholipase C interacts with phosphatidylinositol kinase homolog TOR2; Lin H et al.; The Saccharomyces cerevisiae PLC1 gene encodes a homolog of the delta isoform of mammalian phosphoinositide-specific phospholipase C . Cells deleted for PLC1 gene (plc1Delta) are viable but display several phenotypes, including temperature and osmotic sensitivity and defects in utilization of carbon sources other than glucose . We have used the two hybrid screen to identify Plc1p-interacting proteins . One of the identified proteins was Tor2p, a putative phosphatidylinositol (PtdIns) kinase involved in regulation of protein synthesis, cell cycle progression and organization of the actin cytoskeleton . This interaction was confirmed biochemically by coprecipitation of Plc1p and Tor2p . The results suggest that Tor2p, as a PtdIns kinase, produces phosphorylated PtdIns, which is then hydrolyzed by the associated Plc1p . The proximity of Tor2p to Plc1p may therefore result in a regulated spatial and temporal coupling of synthesis and hydrolysis of phosphorylated forms of PtdIns .

Arch Biochem Biophys, 1998 Dec 1, 360(1), 99 - 104
Partial characterization of the active site human platelet cAMP phosphodiesterase, PDE3A, by site-directed mutagenesis; Cheung PP et al.; Phosphodiesterases (PDE) are important for the downregulation of the intracellular level of the second messenger cyclic adenosine monophosphate (cAMP) by hydrolyzing cAMP to 5'AMP . Previous studies from our laboratory suggested that the human platelet PDE3A active site has two essential histidine residues and one cysteine residue . We therefore decided to begin mutating histidines and cysteines in the conserved domain of PDE3A . A truncated but catalytically active recombinant PDE3A protein was expressed in yeast cells . A six-histidine tag was engineered to the carboxyl end to facilitate protein purification by nickel column . This cDNA construct, PDE3ADelta1, was used for the mutant construction . Mutations were introduced by mutant oligonucleotides during PCR or DNA replication and were confirmed by sequencing . The mutant cDNAs were expressed in PDE-deficient yeast host Saccharomyces cerevisiae strain GL62 . The expression levels of the recombinant PDE mutant proteins were monitored by Western blotting using a rabbit anti-platelet PDE3A polyclonal antibody . The kcat, KM, and IC50 for cyclic guanosine monophosphate (cGMP) and IC50 for milrinone were determined for each mutant . Two highly conserved histidines and four conserved cysteines were each mutated to alanine . C816 is present in the 44-amino acid insert unique to PDE3 . The mutant C816A is poorly expressed (>2%) and probably is not folded appropriately . H840 is the second histidine in the second motif for metal binding, HDXXH . The mutation H840A, although expressed moderately well, has undetectable activity and is probably responsible for binding a bivalent cation, e.g., Mn2+, essential for catalysis . H869 is a highly conserved amino acid not in a metal-binding motif . H869A is well expressed with a normal kcat . However, the Km for cAMP and the IC50 for cGMP are each fourfold greater in the mutant than those in the wild-type recombinant, suggesting that this histidine is in the inhibitory binding site . Three well-conserved cysteines were mutated, but C942A, C945A, and C1013A all had kinetic values similar to the wild type . The results have identified a histidine at the active site essential for catalysis and a second histidine important for inhibitor binding and have further supported the essential nature of the unique 44-amino acid insert .

Arch Biochem Biophys, 1998 Dec 1, 360(1), 62 - 74
Cloning and functional expression of a cDNA encoding geranylgeranyl diphosphate synthase from Taxus canadensis and assessment of the role of this prenyltransferase in cells induced for taxol production; Hefner J et al.; Geranylgeranyl diphosphate synthase supplies the essential acyclic precursor for Taxol biosynthesis in methyl jasmonate-induced Taxus canadensis suspension cell cultures . A cDNA encoding this prenyltransferase was cloned from an induced T . canadensis cell library . The recombinant enzyme expressed in yeast was confirmed by radiochromatographic analysis to produce geranylgeranyl diphosphate from farnesyl diphosphate and {4-14C}isopentenyl diphosphate and was subjected to preliminary kinetic characterization . The deduced amino acid sequence of this gymnosperm geranylgeranyl diphosphate synthase (393 residues) resembles those of geranylgeranyl diphosphate synthases of angiosperm origin, except for the 90-100 N-terminal residues that correspond to the plastidial transit peptide . The full-length preprotein (42.6 kDa) and two truncated versions, corresponding to putative "mature proteins" from which the transit peptide was deleted, were transformed into a yeast mutant defective for the beta-subunit of type II geranylgeranyl transferase . Under conditions of regulated expression, both the full-length construct and the longest of the truncations (at Phe 99) were able to complement the mutant . However, when these two constructs were overexpressed in a wild-type yeast strain, they were apparently toxic, most probably due to depletion of endogenous farnesyl diphosphate as the cosubstrate for the geranylgeranyl diphosphate synthase reaction . In vitro activity of the corresponding recombinant enzymes paralleled the expression level of the constructs as determined by SDS-PAGE analysis of the appropriate proteins of predicted size, and was correlated with toxicity in the wild-type yeast strain and with ability to complement the mutant strain . Results from the analysis of geranylgeranyl diphosphate synthase activity levels and measurement of the corresponding steady-state mRNA levels during the time course of Taxol production in induced T . canadensis suspension cell cultures, and comparison to similar data for activity and message levels for taxadiene synthase, the committed step of the pathway, indicated that for each enzyme both the level of corresponding message and catalytic activity rapidly increased after methyl jasmonate induction .

Arch Biochem Biophys, 1998 Dec 1, 360(1), 10 - 4
Stabilization against thermal inactivation promoted by sugars on enzyme structure and function: why is trehalose more effective than other sugars?
Sola-Penna M, Meyer-Fernandes JR.
Trehalose has been described to act as the best stabilizer of structure and function of several macromolecules . Although other sugars also stabilize macromolecules, none of them are as effective as trehalose . The extraordinary effect of trehalose has been attributed to several of its properties such as making hydrogen bonds with membranes or the ability to modify the solvation layer of proteins . However, the explanations always result in a question: Why is trehalose more effective than other sugars? Here, we show that trehalose has a larger hydrated volume than other related sugars . According to our results, trehalose occupies at least 2.5 times larger volume than sucrose, maltose, glucose, and fructose . We correlate this property with the ability to protect the structure and function of enzymes against thermal inactivation . When the concentrations of all sugars were corrected by the percentage of the occupied volume, they presented the same effectiveness . Our results suggest that because of this larger hydrated volume, trehalose can substitute more water molecules in the solution, and this property is very close to its effectiveness . Finally, these data drive us to conclude that the higher size exclusion effect is responsible for the difference in efficiency of protection against thermal inactivation of enzymes .

Pharmacogenetics, 1998 Oct, 8(5), 423 - 32
Metabolism of dexfenfluramine in human liver microsomes and by recombinant enzymes: role of CYP2D6 and 1A2; Haritos VS et al.; Dexfenfluramine has been widely used as an appetite suppressant in the treatment of obesity . It was recently shown that the apparent non-renal clearance of dexfenfluramine was significantly lower in poor metabolizers than in extensive metabolisers of debrisoquine which suggested the involvement of the polymorphically expressed enzyme, CYP2D6, in dexfenfluramine metabolism . In this study, human liver microsomes and yeast-expressed recombinant enzymes were used to examine dexfenfluramine metabolism in vitro . In human liver microsomes, the major product of dexfenfluramine was nordexfenfluramine with lesser amounts of a novel metabolite, N-hydroxynordexfenfluramine, and ketone and alcohol derivatives being formed . Eadie-Hofstee plots (v against v/{s}) of nordexfenfluramine formation between 1 and 1000 microM substrate concentration were biphasic in three of four liver microsome samples examined, with mean Km values of 3 and 569 microM for the high and low affinity enzymes, respectively . At a substrate concentration (0.5 microM) around the known therapeutic plasma concentration, there was negligible inhibition of microsomal dexfenfluramine N-dealkylation by sulphaphenazole and ketoconazole, but between 33 and 100% inhibition by quinidine, and 0-58% inhibition by 7,8-naphthoflavone in seven liver samples . In human liver microsomes, there was also a significant correlation (rs= 0.79, n = 10, P < 0.01) between dextromethorphan O-demethylation and dexfenfluramine (at 1 microM) N-dealkylation activities . Dexfenfluramine was a specific inhibitor (IC50 46 microM) of CYP2D6-mediated dextromethorphan O-demethylation in human liver microsomes but did not appreciably inhibit six other cytochrome P450 isoform-selective activities for CYP1A2, 2A6, 2C9, 2C19, 2E1 and 3A activities in human liver microsomes . Yeast-expressed recombinant human CYP2D6 metabolized dexfenfluramine with high affinity (Km 1.6 microM, Vmax 0.18 nmol min(-1) nmol P450(-1)) to nordexfenfluramine which was the sole product observed . Recombinant CYP1A2 was a lower affinity enzyme (Km 301 microM, Vmax 1.12 nmol min(-1) nmol P450(-1)) and produced nordexfenfluramine with small amounts of N-hydroxynordexfenfluramine . This is the first detailed study to examine the in-vitro metabolism of dexfenfluramine in human liver microsomes and by recombinant human P450s . We were able to identify CYP2D6 (high affinity) and CYP1A2 (low affinity) as the major enzymes catalysing the N-dealkylation of dexfenfluramine in human liver microsomes.

Photochem Photobiol, 1998 Nov, 68(5), 749 - 53
The N-terminal amino acid sequences of the firefly luciferase are important for the stability of the enzyme; Sung D et al.; The structural and catalytic role of the N-terminal amino acid sequence of Photinus pyralis luciferase was investigated by site-directed mutagenesis . The firefly luciferase activity of a series of deletion and site-directed mutants in the amino-terminal region was investigated in vitro and in vivo . The mutant luciferases were produced either by in vitro transcription and translation or by expressing the cDNA encoding firefly luciferase in the yeast Saccharomyces cerevisiae as a fusion protein to the galactose DNA binding domain protein . Deleting the N-terminal amino acid residues from 3 to 10 dramatically reduced the luciferase activity to less than 1% of the wild-type activity . A marked decrease in the activity (to < 5%) was also observed with the Lys8Glu mutant . The Gly10Arg and Pro11Ala mutants showed 20-30% activity compared to the wild type . On the other hand, mutant Asn6Lys retained the wild-type activity level . Randomizing 3-11 N-terminal amino acids also showed a marked decrease in activity (< 1%) . The mutant luciferases with extremely low levels of enzymatic activity were thermally unstable . These mutational and stability data correlate with the crystal structure of firefly luciferase in which specific amino acids in the N-terminal region form hydrogen bonds to the amino acids in the neighboring beta-stranded sheet . Because the N-terminal region is not part of the active site, the present results suggest that the highly conserved N-terminal amino acid sequences of the firefly luciferase are important for stabilizing the protein in its proper conformation for optimal enzyme activity.

Haematologica, 1998 Sep, 83(9), 771 - 7
Expression of cell cycle regulatory genes in chronic myelogenous leukemia; Iolascon A et al.; BACKGROUND AND OBJECTIVE: Cell cycle regulatory genes are frequently altered in a variety of malignancies . The structure and pattern of expression of eight genes involved in cell division cycle control were studied in leukemic cell samples prepared from bone marrow of patients affected by chronic myelogenous leukemia . DESIGN AND METHODS: Ten cell preparations were obtained from patients in the chronic phase, five from those in myeloid blast crisis and five from those in the lymphoid acute phase . Moreover, bone marrow CD34+ cells, purified from healthy subjects and patients with chronic myelogenous leukemia (both during chronic and acute phases), were analyzed . The investigated genes were RB1, p53 and six cyclin-dependent kinase inhibitor genes (p15INK4B, p16INK4A, p18INK4C, p21WAF1/CIP1, p27Kip1, p57Kip2) . RESULTS: We found that none of these genes is structurally altered in either the chronic or acute phases, with the single exception of the p16INK4A gene, which was homozygously deleted in 1 case of lymphoid evolution . p57Kip2 expression is down-regulated during the evolution towards the blast crisis both in malignant and CD34+ cells . In addition, a significant up-regulation of p15INK4B gene expression is observable during the development of the acute phase of malignancy . INTERPRETATIONS AND CONCLUSIONS: The transcriptional modulation of some cyclin-dependent kinase inhibitors might contribute to the fatal blast crisis of chronic myelogenous leukemia.

Biochim Biophys Acta, 1998 Nov 19, 1448(1), 109 - 14
SCL binds the human homologue of DRG in vivo; Zhao XF et al.; A suspected oncoprotein, human development regulated GTP-binding protein (DRG) has never been identified though homologues were found in mouse, Xenopus, Drosophila, yeast and Halobacteria . During a search for SCL binding partners using the yeast 2-hybrid system, we isolated two independent cDNA clones (clone L51 and clone V3) of the human DRG homologue from human fetal liver and human thymus cDNA libraries . Only one amino acid difference was found between human and mouse DRG proteins . Although a human DRG has been previously deposited in the SWISS-PROT Database, we believe that we have cloned the bona fide human DRG based on the highly conserved primary amino acid structure between our cloned human homologue and the mouse DRG.

Int J Oncol, 1998 Dec, 13(6), 1275 - 80
The replication inhibition activity of the WT1 tumor suppressor protein resides in its N-terminal 298 amino acid region, and does not require specific binding of the protein to the replication origin sequence; Murthy N et al.; Using the simian virus 40 replication origin as the model, it was reported previously that the Wilms' tumor suppressor protein WT1 can inhibit DNA replication . In the present study, we found that a hybrid protein (termed GAL4-WT1AE Z) consisting of the DNA-binding domain of the yeast transcription factor GAL4 fused in-frame with the N-terminal 298 amino acid (aa) transcriptional regulatory region of WT1 retained the ability to inhibit replication . The hybrid protein and the full-length WT1 inhibited replication without regard to the presence or absence of their binding sites in the replication origin region, indicating that inhibition of replication by the proteins does not require their specific binding to the origin region . The inhibition efficiency of the hybrid protein was the same as that of WT1, indicating that the replication inhibition activity resides in the N-terminal 298 aa region, and that the C-terminal zinc finger-containing DNA-binding domain of WT1 is functionally dispensable for this effect.

Nature, 1998 Nov 12, 396(6707), 184 - 6
Histone acetyltransferase activity of CBP is controlled by cycle-dependent kinases and oncoprotein E1A; Ait-Si-Ali S et al.; Transforming viral proteins such as E1A force cells through the restriction point of the cell cycle into S phase by forming complexes with two cellular proteins: the retinoblastoma protein (Rb), a transcriptional co-repressor, and CBP/p300, a transcriptional co-activator . These two proteins locally influence chromatin structure: Rb recruits a histone deacetylase, whereas CBP is a histone acetyltransferase . Progression through the restriction point is triggered by phosphorylation of Rb, leading to disruption of Rb-associated repressive complexes and allowing the activation of S-phase genes . Here we show that CBP, like Rb, is controlled by phosphorylation at the G1/S boundary, increasing its histone acetyltransferase activity . This enzymatic activation is mimicked by E1A.

FEBS Lett, 1998 Oct 23, 437(3), 229 - 32
Protein phosphatase type 2C active at physiological Mg2+: stimulation by unsaturated fatty acids; Klumpp S et al.; Type 2C serine/threonine protein phosphatases (PP2C) so far require unphysiologically large amounts of Mg2+ ions for activity . Activators and inhibitors are not available, targeting subunits unknown . Studying the regulation of PP2C isozymes in bovine retinae, we found that the activity of PP2C increased specifically by the addition of mono- and polyunsaturated fatty acids . Activation was most pronounced at low Mg2+ levels (10-fold stimulation of PP2Calpha by 0.5 mM arachidonic acid at 0.7 mM Mg2+) . Sensitivity of PP2Cbeta was 30-50% less, revealing for the first time enzymatic differences among the PP2C isozymes . Combining unsaturated fatty acids with physiological Mg2+ concentrations resulted in PP2C activity that by far exceeded the dephosphorylation rates obtained otherwise . This suggests that PP2C activity has been severely underestimated in the past . In the presence of fatty acids, Ca2+ ions became inhibitory in the micromolar range . We conclude that unsaturated fatty acids may play a role in the regulation of PP2C activity.

FEBS Lett, 1998 Oct 23, 437(3), 172 - 6
Selective suppression of stress-activated protein kinase pathway by protein phosphatase 2C in mammalian cells; Hanada M et al.; Protein phosphatase 2Calpha (PP2Calpha) or PP2Cbeta-1 expressed in COS7 cells suppressed anisomycin- and NaCl-enhanced phosphorylations of p38 co-expressed in the cells . PP2Calpha or PP2Cbeta-1 expression also suppressed both basal and stress-enhanced phosphorylations of MKK3b and MKK6b, which are upstream protein kinases of p38, and of MKK4, which is one of the major upstream protein kinases of JNK . Basal activity of MKK7, another upstream protein kinase of JNK, was also suppressed by PP2Calpha or PP2Cbeta-1 expression . However, basal as well as serum-activated phosphorylation of MKK1alpha, an upstream protein kinase of ERKs, was not affected by PP2Cbeta or PP2Cbeta-1 . A catalytically inactive mutant of PP2Cbeta-1 further enhanced the NaCl-stimulated phosphorylations of MMK3b, MKK4 and MKK6b, suggesting that this mutant PP2Cbeta-1 works as a dominant negative form . These results suggest that PP2C selectively inhibits the SAPK pathways through suppression of MKK3b, MKK4, MKK6b and MKK7 activities in mammalian cells.

Oncogene, 1998 Nov 12, 17(19), 2531 - 4
BRCA2 associates with acetyltransferase activity when bound to P/CAF; Fuks F et al.; Predisposition to hereditary breast cancer has been attributed in part to inherited mutations in the BRCA2 gene . The large protein it encodes is still poorly characterized with respect to functions . We have previously shown that BRCA2 has transcriptional activation potential conferred by its amino-terminal third exon . Here, we show that BRCA2 interacts with a transcriptional co-activator protein, P/CAF, which possesses histone acetyltransferase activity . The interaction with P/CAF is demonstrated in vitro as well as in vivo and is shown to be mediated by residues 290-453 of BRCA2 . Consistent with the binding to an acetyltransferase, BRCA2 is shown to associate with acetyltransferase activity in nuclear extracts . Contrary to a recent report, we find no evidence in support of an intrinsic HAT activity in BRCA2 amino-terminus . Our results further substantiate the notion that BRCA2 has transcriptional activation function and suggest that one mechanism by which BRCA2 regulates transcription may be through the recruitment of histone-modifying activity of the P/CAF co-activator.

J Biol Chem, 1998 Nov 27, 273(48), 32360 - 8
Interactions of p62(dok) with p210(bcr-abl) and Bcr-Abl-associated proteins; Bhat A et al.; A 62-kDa Ras GTPase-activating protein (RasGAP)-associated protein is tyrosine-phosphorylated under a variety of circumstances including growth factor stimulation and in cells transformed by activated tyrosine kinases . A cDNA for p62(dok), reported to be the RasGAP-associated 62-kDa protein, was recently cloned from Abl-transformed cells . In this study, the interactions of p62(dok) with Bcr-Abl and associated proteins were examined . In 32D myeloid cells and Rat-1 fibroblasts transformed by p210(bcr-abl), p62(dok) is tyrosine-phosphorylated and co-immunoprecipitates with Bcr-Abl, RasGAP, and CrkL, a Src homology 2 (SH2) and SH3 domain-containing adaptor protein . Tyrosine-phosphorylated p62(dok) from cells expressing p210(bcr-abl) bound directly to the SH2 domains of Abl and CrkL in a gel overlay assay . Previous work has shown that an SH2 domain deletion mutant of Bcr-Abl is defective in transforming fibroblasts but remains capable of inducing myeloid growth factor independence . In both fibroblasts and myeloid cells expressing this mutant, p62(dok) is underphosphorylated as compared with cells expressing full-length p210(bcr-abl) but remains capable of associating with Bcr-Abl . However, in a gel overlay assay, p62(dok) from cells expressing the SH2 domain deletion was incapable of associating directly with SH2 domains of Abl and CrkL . Interestingly, no direct binding between Bcr-Abl and p62(dok) could be demonstrated in a yeast two-hybrid assay . These data suggest that indirect interactions mediate the interaction between Bcr-Abl and p62(dok) and that the SH2 domain of Bcr-Abl is required for hyperphosphorylation of p62(dok) . Further, hyperphosphorylation of p62(dok) correlates with the ability of Bcr-Abl to transform fibroblasts but not with the induction of growth factor independence in myeloid cells.

J Biol Chem, 1998 Nov 27, 273(48), 32080 - 7
Characterization of a p53-related activation domain in Adr1p that is sufficient for ADR1-dependent gene expression; Young ET et al.; The yeast transcriptional activator Adr1p controls expression of the glucose-repressible alcohol dehydrogenase gene (ADH2), genes involved in glycerol metabolism, and genes required for peroxisome biogenesis and function . Previous data suggested that promoter-specific activation domains might contribute to expression of the different types of ADR1-dependent genes . By using gene fusions encoding the Gal4p DNA binding domain and portions of Adr1p, we identified a single, strong acidic activation domain spanning amino acids 420-462 of Adr1p . Both acidic and hydrophobic amino acids within this activation domain were important for its function . The critical hydrophobic residues are in a motif previously identified in p53 and related acidic activators . A mini-Adr1 protein consisting of the DNA binding domain of Adr1p fused to this 42-residue activation domain carried out all of the known functions of wild-type ADR1 . It conferred stringent glucose repression on the ADH2 locus and on UAS1-containing reporter genes . The putative inhibitory region of Adr1p encompassing the protein kinase A phosphorylation site at Ser-230 is thus not essential for glucose repression mediated by ADR1 . Mini-ADR1 allowed efficient derepression of gene expression . In addition it complemented an ADR1-null allele for growth on glycerol and oleate media, indicating efficient activation of genes required for glycerol metabolism and peroxisome biogenesis . Thus, a single activation domain can activate all ADR1-dependent promoters.

J Biol Chem, 1998 Nov 27, 273(48), 31992 - 9
Replication factor C disengages from proliferating cell nuclear antigen (PCNA) upon sliding clamp formation, and PCNA itself tethers DNA polymerase delta to DNA; Podust VN et al.; Replication factor C (RF-C) and proliferating cell nuclear antigen (PCNA) assemble a complex, called sliding clamp, onto DNA . The clamp in turn loads DNA polymerases (pol) delta and epsilon to form the corresponding holoenzymes, which play an essential role in replication of eukaryotic chromosomal DNA and in several DNA repair pathways . To determine the fate of RF-C after loading of PCNA onto DNA, we tagged the RF-C subunit p37 with a protein kinase A recognition motif, so that the recombinant five-subunit RF-C complex could be 32P-labeled and quantitatively detected in femtomolar amounts . Nonspecific binding of RF-C to DNA was minimized by replacing the p140 subunit with an N-terminally truncated p140 subunit lacking the previously identified nonspecific DNA binding domain . Neither of these modifications impaired the clamp loading activity of the recombinant RF-C . Using gel filtration techniques, we demonstrated that RF-C dissociated from the DNA after clamp loading or pol delta holoenzyme assembly, while PCNA or PCNA.pol delta complex remained bound to DNA . PCNA catalytically loaded onto the template-primer was sufficient by itself to tether pol delta and stimulate DNA replication . The readdition of RF-C to the isolated PCNA.DNA complex did not further stimulate pol delta DNA synthesis . We conclude that pol delta holoenzyme consists of PCNA and pol delta core and that RF-C serves only to load PCNA clamp.

EMBO J, 1998 Nov 16, 17(22), 6678 - 88
Control of Swe1p degradation by the morphogenesis checkpoint; Sia RA et al.; In the budding yeast Saccharomyces cerevisiae, a cell cycle checkpoint coordinates mitosis with bud formation . Perturbations that transiently depolarize the actin cytoskeleton cause delays in bud formation, and a 'morphogenesis checkpoint' detects the actin perturbation and imposes a G2 delay through inhibition of the cyclin-dependent kinase, Cdc28p . The tyrosine kinase Swe1p, homologous to wee1 in fission yeast, is required for the checkpoint-mediated G2 delay . In this report, we show that Swe1p stability is regulated both during the normal cell cycle and in response to the checkpoint . Swe1p is stable during G1 and accumulates to a peak at the end of S phase or in early G2, when it becomes unstable and is degraded rapidly . Destabilization of Swe1p in G2 and M phase depends on the activity of Cdc28p in complexes with B-type cyclins . Several different perturbations of actin organization all prevent Swe1p degradation, leading to the persistence or further accumulation of Swe1p, and cell cycle delay in G2.

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